Isolation, Morphological And Biochemical Lab Report

Isolation, Morphological, and Biochemical Characterization. A high-throughput screening of cartenoid-producing microorganisms resulted in 25 astaxanthin-producing bacterial strains from marine water samples that were collected from the Pacific coast of Japan (unpublished results). Among the isolates, a novel, highly selective astaxanthin-producing marine bacterium, labeled strain (N-5), was isolated based on its ability to produce orange pigment on NA plate after 2 days of cultivation at 30 °C. Strain N-5 formed colonies that were medium in size (1.0−2.0 mm in diameter), circular, shiny, and orange (Figure 1B). Unless otherwise specified, all characteristics described hereafter are those of the cells of N-5 grown on NA at 30 °C for 2 days.
The strain exhibited positive reactions for catalase, oxidase, βglucosidase, and esculin hydrolysis. Strain N-5 was negative in glucose acidification, O−F test, indole production, arginine dihydrolase, urease, gelatin hydrolysis, lysine decarboxylase, and tryptophan deaminase. In assimilation tests using API 20 NE among 12 carbon sources tested, growth of strain N-5 was observed only in the presence of glucose, maltose, and malate (Table 1). In addition, biochemical identification and metabolic characterization of strain N-5 were performed using the Biolog identification system.35 Since strain N-5 was found to be a Gram-negative and oxidase positive, GN2 microplate was used. Table S1 shows a summary of the Biolog results of using Table 1. Physiological Characteristics of Brevundimonas Strain N-5different carbon sources (95 substrates) by strain N-5. Results showed that strain N-5 is capable of utilizing 29 out of the total 95 as sole carbon sources (Table S1). Strain N-5 was identified as Brevundimonas vesicularis with high similarity of 0.83 and 100% probability, according to the Biolog identification system (carbon source utilization
Astaxanthin and its hydroxylated derivatives were purified from the carotenoids extract of N-5 using an open chromatography column of silica gel 60 coupled with a mobile phase of hexane−acetone (7:3, v/v). Using HPLC method C, a first fraction (1) was eluted at 16.66 min and gave a [M+ + 1] ion peak at m/z 629, corresponding to 2,2′-dihydroxyastaxanthin. A second fraction (2) was eluted at 20.85 min and gave a [M+ + 1] ion peak at m/z 613, corresponding to 2′hydroxyastaxanthin. Finally, a third fraction (3) that represents astaxanthin was eluted at 24.61 min and gave a [M+ + 1] ion peak at m/z