Isolation of pseudoalteromonas agarovorans and optimization of saccharification

Abstract

To obtain the strain for effective saccharification of alginate, a number of marine bacteria were isolated from seawater in Korea. Among ten strains, the strain CHO-33 was selected, because it exhibited the high saccharification yield. In order to identify the isolated strain CHO-33, the biochemical and physiological characteristics were determined. The cell was round-shaped, Gram-negative, motile, and strictly aerobic. Growth occurs in media containing 1-8% of NaCl and temperature ranging from 5-38oC. The strain CHO-33 could utilize D-glucose, maltose, and D-mannitol but it does not utilize D-fructose, mannose, sucrose, and glycogen. In addition, this strain was sensitivity to carbenicillin, tetracycline, ampicillin, and streptomycin. In order to perform the phylogenetic analysis, the 16s rDNA of the strain cho-33 was amplified by PCR. The lengths of the almost complete 16 rDNA gene sequences of strain CHO-33 was 1,439 bp. The 16s rDNA gene sequences of the related taxa were obtained from GeneBank. Phylogenetic analysis based on 16S rDNA gene sequences indicated that the strains CHO-33 is similar to the type strains of belong to Pseudoalteromonas sp., indicating that the strain CHO-33 is member of the genus Pseudoalteromonas The strain CHO-33 was found to form a coherent cluster with the type strain of Pseudoalteromonas agarovorans (98.4%), P. atlantica X82134 (97.3%), P. espejiana X82143 (97.1%), P carrageenovora X82136 (96.8%), P. tetraodonis X82139 (96.8%), P. issachenkonii AF316144 (95.0%), P. undina X82140 (96.7%), P. elyakovii AF082562 (98.3%), P. distincta AF082564 (98.1%), P. haloplanktis X67024 (97.1%), P. nigrifaciens X82146 (97.6%), and P. antartica X98336(94.3%). Therefore, the strain CHO-33 was determined to be Pseudoalteromonas agarovorans CHO-33, a taxon that is physiologically, chemotaxonomically, and phylogenetically distinct from the related strains. Environmental factors effecting saccharification from alginate using Pseudoalteromonas agarovorans CHO-33 were investigated in flask cultures. The cell concentrations increased from 0.2 to 0.7 OD at 660 nm when the algitation rate increased from 0 to 180 rpm. On the other hand, the maximum concentration of sugar was obtained at 6.8 g/L after 3 days of culture at 15 rpm. After 2 days of preculture at 29℃, the sugar concentration peaked at 9.55 g/L after 3 days of culture. When 30 g/L of NaCl was used, the maximum concentration of sugar, 10.12 g/L, was obtained after 3 days of culture. Yeast extract and peptone were the best nitrogen source for effective saccharification. Especially, the sugar concentration was 10.8 g/L after 3 days of culture using a mixture of 1.0 g/L of yeast extract and 1.0 g/L of peptone. Under optimum conditions of culture and media using Pseudoalteromonas agarovorans CHO-33, scale-up for effective saccharification from alginate was carried out in 5 L flasks. The cell concentration after 2 days of culture was 0.2 to 0.3 OD at 660 nm and showed no further increase after 3 day of culture. The sugar concentrations from alginate were increased with increasing culture time to 11.95 g/L after 2 days of culture. The rate of saccharification was 0.25 g sugar/hr, which was about 16.0 fold higher than that of S. maltophilia.