Investigation on the feeding strategy for the fed-batch fermentation of Halobacterium salinarum and the bacterial pigment production for the scavenging of free radicals

• Main Authors: Jhe-GengHsu, 許哲耕
• Other Authors: Mei-Jywan Syu
• Format: Others
• Language: zh-TW
• Published: 2014
• Online Access: http://ndltd.ncl.edu.tw/handle/4y5uh3

碩士 === 國立成功大學 === 化學工程學系 === 102 === Investigation on the feeding strategy for the fed-batch fermentation of Halobacterium salinarum and the bacterial pigment production for the scavenging of free radicals Author: Jhe- Geng Hsu Advisor: Mei- Jywan Syu Department of Chemistry engineering, National Cheng Kung University SUMMARY Halobacterium salinarum is an ancient Halobacteriaceae which is able to grow at a high salt environment. It can secrete bacterial pigments which is quite potential with antioxidant application. The purpose of this study is to investigate a efficient way to culture H. salinarum. First, I culture H. salinarum in batch system to make sure which temperature and medium composition can let H. salinarum grows best. Then, I culture H. salinarum in fed-batch system with condition which makes H. salinarum grows best and investigate the best feeding way. In order to have a better H. salinarum saturated cell density, the culturing was set at 37 oC with 0.1 g/L glucose, 0.5 g/L Na-glutamate, 3 g/L Na3-citrite, 7.5 g/L casamino acid, 3 g/L yeast extract, and 200 g/L sodium chloride in medium. H. salinarum grows well during the initial volume test of 1 L with the above conditions by feeding medium containing 0.1 g/L glucose, 0.5 g/L Na-glutamate, and 200 g/L sodium chloride. And so does the free radical scavenging test of the pigment extracted from this kind of culture. Keywords: Halobacterium salinarum, fed-batch fermentation, free radical scavenging, antioxidant INTRODUCTION Halobacterium salinarum is an ancient Halobacteriaceae which is able to grow at a high salt environment. H. salinarum can secrete bacterial pigments with antioxidant function. Many reactions in daily life, like biological aging and food spoilage, are inseparable from oxidation. Therefore, it is quite potential to extract the secreted orange pigment as antioxidants from H. salinarum fermentation. H. salinarum is a rod shape halophilic archaea. It needs at least 1.5 M sodium chloride for culturing H. salinarum and the optimal sodium chloride concentration is 2.0 M ~5.2 M. The result of H. salinarum culturing and the quality of pigments H. salinarum secreted may be influence by many conditions such as temperature, light, salt concentration, and organic nutrient concentration. This study cultures H. salinarum and changes culturing temperature, salt concentration, and organic nutrient concentration to make sure which condition can let H. salinarum grows best and secretes pigment which has highest free radical scavenging effect. MATERIALS AND METHODS The strains of the extremophile microorganisms H. salinarum was bought from bioresource collection and research center (BCRC), Hsinchu, Taiwan. The culture medium contained sodium chloride, magnesium sulfate, potassium chloride, sodium citrate, yeast extract, casamino acid, Iron(II) chloride, Manganese chloride and Na-glutamate. The pigments were extracted from H. salinarum by methol: acetone (2:7) and used in free radical scavenging activity test. The pigment was measured by 1, 1-diphenyl-2-picryl-hydrazil (DPPH), solution of DPPH (0.3 mM) in methanol was prepared. 0.75 mL of pigment solution was added to 0.3 mL DPPH solution. he mixture was shaken vigorously and stand at room temperature for 30 min. Then the absorbance was measured at 517 nm by using UV-Visible spectrophotometer. The DPPH scavenging effect was calculated by equation: DPPH scavenging effect (%) = ((A_0-A))/A_0 ×100% RESULTS AND DISCUSSION The effective methods of cultivating H. salinarum were investigated in this study. The first step is to explore the effect of a variety of medium ingredients and the incubation temperature on its batch culture, trying to sum up a relatively good fermentation environment for the further fed-batch experiments. They are to seek a better H. salinarum saturation concentration for the free radical scavenging test, which 2,2'-diphenyl-1- picrylhydrazyl radical (DPPH •) provides a source of free radicals, by its secreted pigment. The result of H. salinarum culture and affect by all conditions are listed in table 1. Table 1. Results of culturing H. salinarum with different condition 　 high concentration medium concentration low concentration yeast extract 5 g/L grows better (3 g/L) casamino acid grows better (7.5 g/L) 5 g/L no obvious change (2 g/L) sodium citrate no obvious change (5 g/L) 3 g/L no obvious change (1 g/L) Na-glutamate grows worse (3 g/L) 1 g/L grows better (0.5 g/L) glucose pigment changed (1.0 g/L) pigment changed (0.5 g/L) initial grow rate increase (0.1 g/L ) sodium chloride 250 g/L grows better (200 g/L) grows worse (100g/L ) In order to have a better H. salinarum saturated cell density, the culturing was set at 37 oC with 0.1 g/L glucose, 0.5 g/L Na-glutamate, 3 g/L Na3-citrite, 7.5 g/L casamino acid, 3 g/L yeast extract, and 200 g/L sodium chloride in medium. H. salinarum grows well during the initial volume test of 1 L with the above conditions by feeding medium containing 0.1 g/L glucose, 0.5 g/L Na-glutamate, and 200 g/L sodium chloride. And so does the free radical scavenging test of the pigment extracted from this kind of culture. After set best batch culture condition, this study use this condition to culture H. salinarum by fed-batch culture and try different feeding way to expect the increase of H. salinarum broth saturation concentration and free radical scavenging effect of the pigment which it secrets. The result of fed-batch culture shows that H. salinarum can grows better when the culture is starting from initial volume 1 L and stop feeding 24 hour for each feeding 12 hour. CONCLUSION H. salinarum grows best in batch culture when the medium composed with 0.1 g/L glucose, 0.5 g/L Na-glutamate, 3 g/L Na3-citrite, 7.5 g/L casamino acid, 3 g/L yeast extract, and 200 g/L sodium chloride. When culture H. salinarum in fed-batch system with above condition, the better way to let H. salinarum grows better is starting from initial volume 1 L and stop feeding 24 hour for each feeding 12 hour.