Screening And Identification For Agarase-Producing Marine Bacteria And Optimization Of Its Fermentation Conditions

A preliminary screening for agar-degrading bacteria from the coastal waters of Xiamen and the surface of laver yielded, more than ten isolated with promising agarolytic activity. To investigate the phylogenetic position of these strains, the 16S rDNA sequences were cloned, sequenced and compared with those of related strains. Physiology and biochemistry were also performed.Clone 16S rDNA sequence for molecular identification in these strains. Then enzyme production conditions of one strain which produced more agarase enzyme were optimized. Results are as follows, (1) agar degrading bacteria isolated from the colony morphology can be divided into two categories, the bacterias isolated from the seawater samples secreted extracellular enzymes to make agar plates soft, dip in or around the colonies clear zone, the bacterias isolated from leachate of seaweed had an phenomena that the agar surface was gelatinizated around the bacterial colonies. (2) physical and chemical properties of agar degradation analysis showed that, five strains, QM1, QM4, QM5, QM8 and QM9, could ferment glucose and produce acid; QM5 and QM8 could produce gas; five strains, QM1, QM4, QM5, QM8 and QM9, could ferment sugar and produce acid, but no gas. only QM8 can ferment lactose and produce acid. Anaerobic and aerobic results showed that, QM1, QM2 and QM4 were aerobic; QM5 and QM8 anaerobic bacteria. (3) a length of 1.5 kb fragment was obtained by the PCR of 16S rDNA. And the sequencing results were compared with the Genbank database. It was confirm that the strains were classified as members of genera of Vibrio, Pseudomonas and Proteobacteria. Respectively, QM1 and QM11 were identified as Vibrio sp. CF4-11 and Vibrio shilonii strain SW-2. QM4 as an uncultured marine bacteria. While QM2, QM5, QM8 and QM9 as Pseudomonas sp. LB-2, Gamma proteobacterium B12, Pseudomonas sp. Hyss58 and Pseudomonas stutzeri. (4) on which a strain(QM1) of the best high agarase enzyme production conditions were optimized, including fermentation time, temperature, pH, speed, agar concentration, NaCl concentration on enzyme production. After optimization of fermentation, the crude enzyme activity of the 3.4 U/ml; optimal conditions for enzyme production temperature of 28°C, pH 7.5, speed 150 r/m, agar concentration of 0.20%, NaCl concentration of 2.0%, fermentation 36 h.All the results indicated that there were rich resources of agar degrading bacterias on Xiamen coast. It is the basis for vitro agar enzyme expression, activity of the enzyme test and development and utilization of the agar.