Extraction Of Extracellular Polymeric Substances (EPS) And Study On Its Interaction With Cr(?)

With the rapid development of industrial technology, a lot of heavy metals discharge into the environment, which make the pollution of heavy metal become a major environmental problem, especially the pollution of Cr(VI). Extracellular polymers substance (EPS) secreted by microorganisms contains a lot of negative charged groups, for example,-COOH,-NH2,-OH,-SH and PO43- etc. These groups can reacte with heavy metal ions, then as a kind of bioremediation reagent attracted more and more attention. The mechanism that EPS participate in Cr(VI) reduction has not been elucidated, so this study expect to provide theoretical basis to understand it.S. oneidensis MR-1 and mixed bacteria were used as the study subjects. First of the study is to selecte an appropriate EPS extraction method for MR-1. We explore the characteristic of Cr(VI) reduction by MR-1 and its interaction with EPS. Also the influence of Cr(VI) on EPS and community structure of mixed bacteria was investigated. The main contents of this study are as following:(1) The efficiency of EPS extraction methods:centrifugation (control), heating (40,45, 50 and 60?), H2SO4, EDTA and NaOH method were compared from the viewpoint of whether extraction method damage to bacteria, the content of EPS and whether EPS were contaminated. The result indicated that the centrifugation method lead to little cell lysis during extraction but failed to obtain most of the EPS. SEM demonstrated serious cracking in the cells after heating at 45,50 and 60? extraction, whereas the sample with heating at 40? did not exhibit serious damage. The contents of proteins and polysaccharides produced by heating treatment at 40? were 7.12 and 1.60 mg g-1 dry cell, respectively, which was much higher than control, so 40? is the idea heating extraction tempreture. The result of FTIR revealed that H2SO4 reagent can remain in EPS sample, even damaged to the functional groups of EPS component. EDTA extraction did not result in cell lysis, but EDTA can complex with EPS and formed EDTA-EPS complexes. EDTA extraction would be an ideal method if aim of the experiment is just to remove EPS. The content of nucleic acid in NaOH method is high, which indicated that the material within cells released significantly.(2) The partical size, Zeta potential, contact angle and activity of MR-1 after EPS extraction by heating method (40?) and MR-1 without EPS extraction were compared. Zeta potential of MR-1 with EPS was lower than that of MR-1 after EPS extraction. The contact angle of MR-1 with EPS was 86°, and it decreased to 60° after EPS extraction. The result indicated that EPS of MR-1 contained more hydrophobic composition than hydrophilic composition. The ETSA of MR-1 after EPS extraction was lower than control, and the content of N-acetylglucosamine was higher than control. These two results indicated that heating method at 40? decreased the activity of cell membrane. The extraction tempreture of heating method should be futher optimize in the following experiments.(3) The rate of Cr(VI) reduction by S. oneidensis MR-1 was reduced with the increase of Cr(VI) concentration. The rate of Cr(VI) reduction by bacterium MR-1 with EPS was faster than that of MR-1 after EPS extraction. In a certain concentration range, the higher the concentration of sodium fumarate was, the faster the rate of Cr(VI) reduction was, which indicated that EPS and sodium fumarate promoted Cr(VI) reduction. With the increase of Cr(VI) concentration, the content of proteins in EPS improved. SEM demonstrated the morphology of bacteria under 50 and 100 mg L-1 initial Cr(VI) concentration was like that of control. This result indicated that the Cr(VI) resistance of MR-1 was high. But when the concentration of Cr(VI) increased to 200 mg L-1, the morphology of bacterium changed obviously, indicating high concentration of Cr(VI) damaged to bacterium. The intensity of the following functional groups on bacterial surfaces:-OH, C-H, amide I, amide II, PO2-, C-O and C-O-C increased with the enhancement of the concentration of Cr(VI). These functional groups may connected with Cr(VI) reduction.(4) The rate of Cr(VI) reduction by mixed bacteria under aerobic and anaerobic conditions were compared. The rate of Cr(VI) reduction under anaerobic was slower than that under aerobic before 36 h. After 48 h, the rate of Cr(VI) reduction under anaerobic was faster than that under aerobic obviously. The EPS content of mixed bacteria without the addition of Cr(VI) was much higher than that of mixed bacteria with Cr(VI) addition. Analysis of community structure showed that the OTU value of original sample declined distinctly after domestication under different conditions. The number of common OTU of original sample, aerobic without Cr(VI), aerobic with Cr(VI), anaerobic without Cr(VI) and anaerobic with Cr(VI) was 4, while the particular OTU value was 509,6,6,12 and 0, respectively. The result indicated that the diversity of microbial community changed obviously. The analysis of principal component showed that the samples with Cr(VI) was far from the samples without Cr(VI) in the PC1 axis direction. The samples under aerobic and anaerobic conditions also separated in the PC1 and PC2 axis direction, but the distances were relatively close. The analysis of community composition indicated that Citrobacter was the dominant population of the mixed bacteria under the condition without Cr(VI). Under the condition with Cr(VI), the relative abundance of Citrobacter decreased obviously, while the relative abundance of Enterobacter increased. The results indicated that most bacteria of Citrobacter had weak resistance to Cr(VI), but most bacteria of Enterobacter had strong resistance to Cr(VI).