Study On The Bioaccumulation Of Cobalt From Rhodopseudomonas Palustris By Proteomic Analysis

Excess of cobalt often cause serious effects on the growth of crops and human health. Rhodopseudomonas palustris is a non-sulfur purple photosynthetic bacterium, which is of characterized by widespread distribution, low cost of culture and environmental adaptability. Besides, it exhibits higher tolerance and bioaccumulation capacities to different metals ions. Therefore, it has broad application prospects and important significance to study the bioaccumulation conditions and mechanism of cobalt. Optimized bioaccumulation conditions of cobalt were investigated by Rhodopseudomonas palustris. Then, protein expression in the process of bioaccumulation as well as certain related proteins function was analysed by using proteomics and real time fluorescent quantitative polymerase chain reaction methods. These results preliminary revealed the bioaccumulation mechanism of cobalt by Rhodopseudomonas palustris. These results are as follows:1. Effect of four different metabolic pathways was studied on cobalt bioaccumulation, anaerobic growth in the light, anaerobic growth in the dark, aerobic growth in the light and aerobic growth in the dark. We found that the cobalt removal efficiency of aerobic growth in the back was higher than other conditions and the value even reached 89.4% at initial cobalt concentration of 80 mg/L. Optimization of pH and temperature of cobalt bioaccumulation was 6.5, 30°C, respectively. Under the optimum condition, cobalt removal efficiency was 84.9% and bioaccumulation capacity was 287.18 mg/g at initial cobalt concentration of 160 mg/L. The pseudo-second-order kinetic models were better fit for the removal process, with a correlation coefficient R2=0.9508-0.9913. In the coexisting ion experiment, Cu²⁺, Zn²⁺, Cd²⁺, Mn²⁺ and Fe²⁺ except Ni²⁺ was not much pronounced influenced the removal efficiency of cobalt.2. The results of cell compartmentalization showed that cobalt was distributed on the cell surface, periplasmic space, membrane and cytoplasm and bioaccumulation capacity was 4.99, 49.82, 24.43, 7.69 mg/g, respectively. Cobalt was mainly distributed in the periplasmic space and cell membraneof Rhodopseudomonas palustris, which accounted for 85.32% of the total amount of cobalt. The nature of cellular cobalt was ascertained using X-ray diffraction analysis of powdered bacterial biomass. The control biomass, lacking any distinct peak, which was characterized amorphous nature. However, Co²⁺-loaded biomass had several distinct peaks, which indicated the cobalt crystals were deposited in the cells. In comparison with XRD standards in the database, the data of Co²⁺-loaded biomass corresponded to cobalt phosphate octahydrate?JCPDS # 01-0121?3. Two-dimensional electrophoresis technology was established to separate the proteins of Rhodopseudomonas palustris and obtained the protein map with high resolution. Through the PDQuest software analysis, we found that there are 36 protein spots was significantly different expression. Among them, 25 proteins were identified by MALDI-TOF-MS-MS, of which 19 proteins were up-regulated and 6 proteins were down regulated. After using NCBI protein database for matching and GO functional annotation analysis, the identified proteins were carried out to subdivide into seven groups based on their functional categories, including protein synthesis, protein proteolysis, stress protein, carbohydrate metabolism,amino acid metabolism, transport protein and other proteins. It was implied that the process of bioaccumulation cobalt was a complex network regulation system, which was related to the expression of multiple proteins in the cell. Methionine adenosyltransferase?METK? played an important role in detoxification.Phosphate ABC transporter substrate-binding protein?PSTS? was widely up-regulated. Combined with X-ray diffraction and cell compartmentalization, PSTS may be bioaccumulate cobalt ions by regulation of phosphate in the cell periplasm.4. The mRNA of cobalt-free control cells and cobalt-enrichment cells was extracted and cDNA was obtained by reverse transcription technique. Choosing SYBR Green I as the fluorescent dye, it was found that expression quantity of METK and PSTS was increased 4.03-fold and 12.99-fold in the presence of 80 mg/L cobalt normalized to the expression of a 16 S rRNA gene, respectively.It was revealed that real time fluorescent quantitative PCR data had a positive correlation between the content of the proteins and their mRNA levels, so the METK and PSTS protein played significant role in the mechanism of bioaccumulation cobalt.