| Purple sulfur bacterium was one group of anoxygen photosynthetic bacteria living in the unique habitats and emerged on earth about3.5Ga years ago. It was an ideal model organism to study the origin of life, the role of the carbon, nitrogen and sulfur in material recycling, as well as anoxygenic photosynthesis mechanism. It played an important role in the removal of organic matter, nitrogen and phosphorus and the toxic sulfide in sewage. Compared with purple non-sulfur bacteria, its research was far less depth and extensive because of its special habitats, difficulty to obtain pure cultures and its low biomass.Okenone carotenoid is one of the most ancient biomarkers discovered so far, and has been as an effective tool for the study of earth’s evolution and environmental adaptation mechanism. Now15kinds of purple sulfur bacteria were found that they could product Okenone carotenoid. A halophilic purple sulfur bacteria Marichromatium sp.283-1was obtained by our group which contained the unique photosynthetic pigment Okenone from Qingdao Dongfeng saltworks. In this research, the biological characteristics, osmotolerant mechanism, the photosynthetic pigment composition analysis, effect of environmental factors on the synthesis of photosynthetic pigments and the separation, identification, preparation and physical and chemical properties, production optimization of the major carotenoid Okenone from the halophilic purple sulfur bacteria Marichromatium sp.283-1were studied.The main results were as follows:1. Results of biological characteristics showed that the strain283-1was sensitive to oxygen, and could grow at these condition of the highest15%NaCl,1.8g/LNa2S.9H2O,7.7g/L NaNO2, pH6-9, temperature from15to45℃, light intensity within the range of100～8000Lux. Therefore, it was anaerobic, halophilic, with tolerance of a high concentrations of sulphur, salt, alkali, NaNO2and fixing carbon. Carbon, nitrogen and salinity, pH, light, temperature had an effect on its morphology. On high salt stress conditions cell morphological diversity, increase volume, specific surface area decreased; in the alkaline environment cells was stick and a bigger volume. Under low temperature stress, the cells are rod-shaped, bigger volume, more intracellular accumulation of sulfur particles, under high temperature stress, the cells are long rod, bigger volume, more intracellular sulfur particles.2.Marichromatiun sp.283-1was a moderate halophilic photosynthetic purple sulfur bacterium and its osmotolerant property and mechanism were studied. The results shown that strain283-1could maximally tolerate2.3mol/L NaCl in improved purple sulfur bacterium medium. The growth of283-1specifically depended on anion Na+and anion Cl-was more suitable provide intracellular osmotic pressure than anion Br-.283-1accumulated betaine to equilibrate extracellulary osmotic pressure, while betaine, which was most important compatible solutes, increased with increasing of the NaCl concentration, reaching156.4mg/gby dry weight under a2.0mol/L NaCl concentration. Exogenous betaine was sufficient to promote the growth of283-1under high salinities and enhance its salinity tolerance.3.1n order to improve the biomass, medium composition of the strain283-1was optimized. Effects of the six factors CH3COONa, NaHCO3, NH4C1, Na2S.9H2O, MgSO4and Inorganic salt on the biomass were evaluated by using single-factor test, Plackett-Burman design, the steepest ascent experiment and central composite design combined with response surface analysis in this work. Results showed that sodium acetate and sodium sulfide were most significant factors and the optimal media were obtained. With the optimized media, the biomass improved6times compared with the control and the value was in agreement with the predicted.4. Pigments were extracted from cell pellets by acetone and methanol and its comprehensive analysis was carried out using UV-VIS absorption spectrophotometry, thin layer chromatography (TLC), HPLC and mass spectrometry (MS). The results were shown that under light and anaerobic conditions, eight kinds of photosynthetic pigments were obtained including four bacterochlorophylls, BChl aGG, BChl aDHGG, BChl aTHGG BChl aP and BPhe, and three carotenoids OH-Rg-ketoI the Rg-ketoⅢ and Okenone. BChlap and okenone were main components and their contents were39.6%and47.4%, respectively. And we developed two types of photopigment fingerprintings by TLC and HPLC. The fingerprintings analyses are suitable for rapid determination of photopigments of purple sulfur bacteria and have important application in control of regulation mechanism for photopigment synthesis.5. Effects of the environmental factors of light, temperature and salinity on pigment synthesis were studied using developed photopigment fingerprintings of TLC and HPLC. The results showed that different light intensity mainly affected the content of carotenoids and bacteriochlorophyll, while temperature had great influence on the pigment components. At20℃, accumulation of carotenoids only was Okenone, and TLC fingerprinting was not detected the other two carotenoids. At30℃and2000Lux contents of bacterochlorophyll and carotenoids were the highest. At45℃and5000Lux, contents of bacteriochlorophyll and carotenoids is the lowest. The salinity had almost effect on the pigment components, but a greater impact on the relative content of each component. At11%NaCl, accumulation of BChl aP and BPhe increased significantly, and especially more than70%increase of BChl aP. However, the accumulation of OH-Rg-ketoI was down more than40%. At1%NaCl, the relative content BPhe and Okenone both increased significantly.6.On the basis of photosynthetic pigment composition analysis, we obtain the purified Okenone carotenoid by the acetone methanol extract, saponification, TLC and HPLC analysis. Effect of light (including ultraviolet), temperature, pH, metal ions, oxidizing agents, reducing agents, free radicals, and food additives and so on was researched on Okenone stability. The results showed that under the dark or1000Lux, and4℃,30℃and50℃conditions, Okenone had good stability, high lightfastness and heat capacity. Strong acids and bases destroyed Okenone largely. Food additives and metal ion effected little on Okenone stability, while Fe3+, Mg2+, Al3+and Cu2+, especially Fe3+effected the stability. Therefore Okenone should be stored to avoid of contacting with these substances. The shape and fine structure of Okenone carotenoid absorption spectral was influenced by the polarity of organic solvent, the fine structure weakened or disappeared and the absorption peaks smoothed when it was in the polarity solvent. Okenone solution had a strong absorption of ultraviolet radiation, so it could be by sacrificing itself to play a protective role.[DPPH·] as a radical detection reagent could be used as determination of ability of okenone clearing [DPPH·] radical scavenging, the results showed that Okenone had the anti-oxidation biological activity and EC50=0.022mg/mL.7. Based on the optimized biomass, effects of five kinds of inorganic salts and trace elements on Okenone production was researched by the plackett Burman design and the response surface analysis. The results showed that trace elements, sodium bicarbonate and potassium dihydrogen phosphate had the significant impact on Okenone production. The optimal medium was obtained by the Box-Benhnken design. In optimal medium, the Okenone production was from1.454mg/L to2.926mg/L, and an increase of101.2%. |
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