Library Establishment Of Nata De Coco-producing Strains And Identification Of A Proposed New Species

The application of bacterial cellulose in food industry is named Nata De Coco. Hainan province is the foundation of the production Nata De Coco, has wide variety of strain sources, wide variety of germplasm resource, but they don't pay any attention on the information of strains biological characteristics, and few people do medium optimization for their variety strains. Which is a limiting factor of Nata De Coco produce industry. Coupled with the low-cost production of Nata De Coco from Vietnam, Thailand and so on, they are cheaper in Nata De Coco producing, this had a huge impact of Nata De Coco produce industry, many companies have give up the coconut production business, the strains were often abandoned, which is a waste of germplasm, which makes the cellulose-producing strains collection, separation, identification and protection of germplasm even more urgent.In this study,18batch isolate were collected from Hainan province, agencies and organizations.144isolated strains were purified, with the strains preserved by laboratory, the total strain number are158. Among them,93strains were identified by molecular biology and morphological characteristics. According to the shapes of the bacteria, they can be divided into five types, according to the characters transparency, uplift and viscosity of colony can be divided into five types, according to16S rDNA, dnaK, groEL and rpoB gene analyze, they all belongs to a genus Komagataeibacter,79of them are K. europaeus strain, K. hansenii strain, K. nataicola strains, and14of them are unknown species.The pair-wise alignment of dnaK. gene of14unknown species results show that, the similarity of its dnaK gene were higher than99.0%, the threshold value to distinguish different species, indicating that the14strains belonging to the same unknown species. WE2, WE3and WE7were isolated from metamorphic coconut milk, the production of WE7is higher among them, so choose it to identity by polyphasic taxonomy method.The morphological characteristics of WE7:We can see obvious colony in the NCW medium after two days growth, the colony is neat, surface is smooth, colony slightly protuberant, middle granule.coryneform, gram negative bacteria, aerobic, oxidase negative, catalase positive, no flagella, no motility, the diameter is about0.5?rn, the length is about1.6to1.6?rn, single, pairs or in chain.The physiological characteristics of it:The growth pH range is3.00-6.50, the growth temperature range is15-37?. Do not produce water soluble pigment, cannot produce ?-pyrone compounds from d-glucose, can produce2-KGA,5-KGA and gluconic acid from d-glucose. The major fatty acid type is C18:1(52.28%), the secondary fatty acid type is C17:0(9.91%), C16:0(9.05%) and C18:0(5.04%) major types of ubiquinone Q10.Molecular sequence analysis:The length of16S rDNA gene was1362bp. The hightest similarity of16S rDNA gene were with Gluconacetobacter entanii LTH4560T, the similarity value was99.56%. But the dnaK gene, groEL and rpoB gene can do identify work within the genus, such as the least value anong a species of dnaK gene is99.0%, groEL gene is at99.2%, rpoB gene is98.8%. And the similarity of WE7in three genetic similarity were significantly lower than the threshold value. The genomic DNA (G+C)%is63.2%.Compare with the closest strain with WE7:WE7could utilize D-mannitol, D-gluconate and glycerol as the sole carbon source and can grow without acetic acid and alcohol. Those are the features which could distinguish it and G. entanii LTH4560T which has the closest relationship to it. Unlike K. hansenii LMG1527T, WE7could utilize D-fructose, sucrose, D-mannitol, D-galacturonic acid and produce ethanol. WE7could tolerate3%acetic acid in the case of the presence of3%ethanol, but K. maltaceti LMG1529T coule tolerate2%acetic acid only. There are some similar characteristics between WE7and Komagataeibacter genus strains, such as having no motility, no flagella, does not produce a water-soluble dye, can not use D-glucose production ?-pyrone compound, but the Gluconacetobacter genus strains is different.Comprehensive the polyphasic taxonomy result, the strain WE7is proposed to be a new species, and named as Komagataeibacter hanunivense sp. nov. WE7.These strains form a small but distinctive library, the library contains158strains,9known species, they are K. nataicola, K. europaeus, K. hansenii, K. rhaeticus, K. sucrofermentans, K. swingsii, K. xylinus, K. maltaceti, G. diazotrophicus and a novel species, which laying a solid foundation on the genetic improvement of excellent strains, the conservation of genetic resource, and the study of strain genetic diversity.