Screening And Identification Of Marine Agar-degrading Bacteria And Studies On Their Agarases

The macroalgae in the ocean are important biomasses. As a representative of marine macroalgae, red algae synthesize a great diversity of polysaccharides, which constitute their cell walls. Agar is an important polysaccharide, which consists the cell walls of red algae. In this paper, we carried on the study of isolating agar-degrading bacteria from red algae and the agarases secreted by them.First, we isolated the agar-degrading bacteria from the surface of Gracilaria blodgettii samples collected from Hainan, China. We isolated 20 strains in total from the two Gracilaria blodgettii samples,14 strains belong to Gammaproteobacteria in Proteobacteria and most of the rest 6 strains belong to Flavobacteria in Bacteroidetes. Among the isolated agar-degrading bacteria, strains 2P4,000 and 038 showed highest 16S rDNA gene sequence similarity 95% to Marinomonas pontica. Thus strains 2P4, 000 and 038 were considered as novel species of genus Marinomonas tentatively. Strain 017 showed highest 16S rDNA gene sequence similarity 92.98% to Teredinibacter turnerae and was considered as representing a novel genus in Gammaproteobacteria tentatively. Strain B011 shared highest sequence similarity 92.78% with Pseudofulvibacter geojedonensis in 16S rDNA gene and was considered as representing a novel genus within Flavobacteriaceae tentatively. Strain 0182 was closest to Crocinitomix catalasitica and they shared 90.03%16S rDNA gene sequence similarity. Thus strain 0182 was considered as representing a novel genus within Cryomorphaceae tentatively. Strain D/C003 showed highest 16S rDNA gene sequence similarity 91.48% to Reichenbachiella agariperforans and was considered as representing a novel genus within Flammeovirgaceae. Second, we isolated an agar-degrading strain, designated Z1, from the surface of Gelidium amansii Lamx. sample collected from Rongcheng Shandong, China. The polyphasic taxonomy study of strain Z1 was performed and then it was considered as representing a novel genus within Gammaproteobacteria. Strain Z1 was Gram-stain-negative bacterium and formed pale-yellow, smooth, circular colonies on marine agar culture media. Optimal growth was observed at 28-30 ℃, pH 7-7.5 and in the presence of 1-3 g/100mL NaCl.Except for agarase activities, strain Z1 also showed amylase, cellulase and lipase activities, but alginate lyase and urease activities were not detected. Finally, we isolated an agar-degrading strain, designated Q1, from the surface of Porphyra yezoensis sample collected from Weihai Shandong, China. The polyphasic taxonomy study of strain Q1 was performed and then it was considered as representing a novel species of genus Catenovulum within Alteromonadaceae. Strain Q1 was Gram-stain-negative, facultatively anaerobic bacterium and formed pale-white colonies, liquefying agar. Optimal growth was observed at 30-33℃, pH 7-7.5 and in the presence of 1-3 g/100mL NaCl. Other than agarase activities, strain Q1 showed amylase and alginate lyase activities as well. In addition, we optimized the fermentation conditions of agarase production, using the methods of single factor experiment, PBD significant factors selection trial and BBD response surface analysis trial. The agarase activity after optimization reached 1.64 U/mL, which was 3.2 times the original activity. The corresponding fermentation conditions were 0.17 g/100mL agar,0.99 g/100mL NaCl, pH 8.48,0.5 g/100mL yeast extract powder,0.7 g/100mL txyptone,28℃,120 mL culture media amount,2% inoculum size and 48 h.Furthermore, the genomes of strain Z1, JL1 (the reference strain of strain Zl) and Q1 were sequenced. The full genome sequences of Z1 and JL1 were 3.9 MB and 3.8 MB respectively. On the basis of the analysis and annotations of protein sequences, we inferred that strains Z1 and JL1 produced a common a-agarase. The full genome sequences of Q1 were 4.3 MB and twelve genes encoding agarase were predicted. The predicted and encoded agarases contained both a-agarase and β-agarase. Besides, a gene encoding κ-carrageenase and two genes encoding alginate lyase were predicted.