Isolation And Identification Of A Strain Natural Algae-bacteria Symbiont And Stydy On Their Symbiotic Functions

Microalgae were widely applied in energy,environment treatment,as well as food and medicine industry.However,large scale cultivation and collection of microalgae were the major problems restricting the development of algae application.In natural water,there were complex interactions between algae and microorganism.Based on interactions between algae and microorganism,Oswald and Gotaas firstly put forward the concept of algae-bacteria symbiotic system.With the characteristics of fast growth,strong adaptation,easy cultivation,algae can release a variety of extracellular metabolites,such as lipids,carbohydrates,amino acids and so on.Some organic matters can not be utilized by algae.Microorganism had advantages on decomposition of organic matter and supplied the carbon dioxide,inorganic nitrogen and phosphorus compounds by metabolism as carbon source and nutrition for algae photosynthesis.In this paper,we investigated the symbiontic functions of a natural strain of algae-bacteria symbionts to enhance production of algae and reduce the cost in collection.(1)Bacteria were isolated by the method of solid plate coating from a natural algae-bacterium symbionts(TY01)preserved in the lab and identified by the methods of gram staining and 16S rDNA sequences.In co-culture,we investigated the effects of bacteria in phycosphere on algal concentration,dry weight,chlorophyll a,indoleacetic acid concentration and algae on bacterial growth.Four strains(BW,BY,BX,BR)were separated and identified as Ochrobactrum sp.(gram negative),Pseudomonas sp.(gram negative),Microbacterium sp.(gram positive)and Nocardioides sp.(gram positive)respectively based on 16S rDNA sequences.After 14 days co-culture,strain BX can significantly increased 81.48%of algal cells concentration than that of the control(only algae in medium).After 24 days co-culture,strains of BX and BY had the function of improving algae dry weight by 22.1%and 22.1%respectively.Algal chlorophyll a content and content of chlorophyll a single cells were reduced by strains of BX,BR and BY.IAA concentration was detected to 1.04 mg·L-1 when strain BX co-culture with algae.Bacteria concentration of strain BX decreased 91.72%than initial concentration when cultured in BG11 medium.However,it increased 70.07%than initial concentration when co-culture with algae.We concluded that microbacterium secreted IAA to enhance the algal growth and acquire nutrient from algae.(2)Total DNA were extracted by the method of CTAB.PCR technology was applied for amplifying 18S rDNA and ITS.Combined with morphological characteristics,the algae was identified as Parachlorella kessleri.Common binary fission and special trinary fission were observed in microscopic algae images.The max concentration of UGA was 2.60×107 cells mL-1 in BG11 medium and 2.85×107 cells mL-1 in BG12 medium(which is same as BG11 medium except the nitrogen source instead by 1.22 g/L sodium nitrite)culturing for 20 days.Specific activity of nitrite reductase was 154.57 nkat·mg-1 in BG11 medium and 16.56 nkat·mg-1 in BG12 medium.The fastest rate of nitrite degradation reached to 22 mg·L-1·day-1 sodium nitrite in a 12 day culture period assay and the corresponding biomass was 2.61 × 107 cells mL-1 in 12 days.In the first 8 days,the trend of content of chlorophyll a in each cell indicated the proper multiplied time was four days.These results revealed nitrite was the preferred nitrogen source for algae growth while nitrate was the better nitrogen source for nitrite reductase production.Specific activity of NiR and rate of nitrite degradation exhibited a good nitrite degradation capability by the strain of UGA.(3)Algal flocculating activity,lipid content and components of fatty acids were determined in co-cultivation.When pH was 11,flocculating activity of algae in co-cultivation was 80.3%,more 18.6%than that in pure cultivation.The optimized pH was 11;CaC12 concentration was 10 mM;FeC13 was 1.2 mM.The maximum flocculating activity was 96.51%.Lipid content of algae in co-cultivation significantly increased to 45.76%,higher 10.1%than that in pure cultivation.C16 and C18 as the major fatty acid compositions in total fatty acids were 76.14%and 88.70%respectively.2 kinds of fatty acids(C16:3 and C20:1)absent in pure algae were detected in co-cultivation.Contents of C16:3 and C20:1 were 1.86%and 0.31%respectively.The research revealed that Microbacterium sp.have significance in promoting algal biomass productivity,increasing flocculating activity and enhancing lipid productivity.