Study On The Mechanism Of Interaction Between Two Microalgae Strains And Their Associated Bacteria In Phycosphere

Microalgae,defined as a polyphyletic group of unicellular photosynthetic eukaryotes,are responsible for half photosynthesis and carbon dioxide fixation on Earth.Because of the small difference in morphological structure,how to accurately and quickly identify the microalgae in the environment is critical for the start of algal research.Bacteria can use the organic carbon source derived by microalgae and drive the global biogeochemical cycles.Therefore,the interactions between bacteria and microalgae are very important in the aquatic ecosystem.Besides,these interactions such as symbiosis,parasitism,competition,etc.,also involve the exchanges of various metabolites.Because these metabolic exchanges are very complicated,the exchanges of nutrients between microalgae and bacteria have always been the focus of water ecology research.In addition,signals exchanges also play a key role in the bacteria-microalgae interaction,but there is still not clear report on the types of signals and their role in this inter-kingdom interactions.In this study,we optimized the rapid identification method of microalgae to quickly and accurately identify the microalgae isolated in the aquatic environment.After that,we used two model systems of microalgae and associated bacteria to explore the nutrient exchanges and signal communication in the interaction of bacteria and algae.In the freshwater system,the microbiome isolated from TaiHu lake were compared with the microbiome in Chlorella phycosphere.Transcriptome and nitrogen metabolism substrate analysis was used to demonstrate the effects of the isolated Rhizobium on the growth and biofuel accumulation of Chlorella.These effects were also proved by culturing at different nitrogen concentrations.This study guides whether the natural phycosphere bacteria should be removed in algal cultivation since the effects on oil accumulation.In the marine ecosystem,the adhesion of the bacteria to the diatom was explored in our study,and the effects of the bacterially derived quorum-sensing signals on bacterial motility and attachment were also studied.This is the first study pointed out the effect of quorum sensing signal systems on the colonization of bacteria to diatom in the natural ecological environment.The specific results are as follows:1.The direct PCR method optimized in this study can directly perform PCR amplification on freshwater algae cultures and resuspended marine microalgae in distilled water.Algal culture is directly added into the PCR system as a DNA template without any pretreatment.This method is the most simplified direct PCR method by far.The quality of PCR products is like that of conventional PCR and colony PCR.Furthermore,the products are suitable for normal DNA barcode identification analysis and multi-site DNA barcodes analysis.2.Rhizobium radiobacter TH729 isolated from Chlorella promoted the growth of C.variabilis F275 with an increase of 27%in growth rate.Analysis of the nitrogen concentration of the substrate showed that the nitrate content of axenic F275 culture decreased by 12.33%compared with blank control,while the nitrogen concentration of both axenic TH729 and the co-cultured increased by 5.48%and 16.43%,respectively.Compared with axenic Chlorella culture,co-culture showed a significant increase in nitratenitrogen content of 32%.Our study has shown that R.radiobacter TH729 can fix nitrogen and increase the nitrogen concentration in the co-culture system.3.C.variabilis F275 showed expression changes of 449 genes in co-culture with R.radiobacter TH729.Among them,262 genes were down-regulated.NR and NRT genes related to nitrogen metabolism pathways were significantly down-regulated.Also,many key genes related to fatty acid oxidation such as long-chain fatty acyl-CoA synthetase gene and acetyl-CoA oxidase gene were significantly up-regulated.Axenic culture of C.variabilis F275 with different nitrogen concentrations showed that the biofuel production and productivity decreased with the increase of nitrogen concentration.Compared with the culture at the nitrogen concentration of 0.37 mM,the biofuel production and productivity of C.variabilis F275 decreased by 62.5%and 45.5%,respectively when at a nitrogen concentration of 11.76 mM.Therefore,it has proved that the nitrogen derived by R.radiobacter TH729 is not conducive to the accumulation of biofuel in the cultivation of C.variabilis F275.4.The bacteria S.pseudonitzschiae F5 and Phaeobacter sp.F10 isolated from A.glacialis A3 adhered to the TEP produced by A3.When F5 was co-cultured with A3,the growth rate of A3 increased by 27.6%.Bacterial genome analysis showed that both F5 and F10 have homologous flagellar synthesis genes,pili synthesis genes,extracellular polysaccharide synthesis genes and quorum sensing genes.It indicated that both bacteria are mobile,adhesive and capable of synthesizing quorum-sensing signal molecules.Both of F5 and F10 produced three different quorum-sensing signal molecules:3-oxo-C10-HSL,3oxo-C16-HSL and 3-oxo-C16:1-HSL which was identified by HPLC-MS/MS.All three signaling molecules can significantly inhibit the motility of these two bacteria,while only 3-oxo-C16:1-HSL can significantly increase the biofilm formation of the two bacteria.The above results showed that the concentration of organic matters is higher in the region of TEP or phycosphere which is produced by microalgae.As a result,these organic matters attract bacteria by chemotaxis which may cause a high density of bacteria in these regions.After that,the quorum sensing promotes biofilm formation and decrease the motility of bacteria and thus keep the bacteria stably living in nutrient-rich areas than bulk water.5.The bacteria in Roseobacter clade usually have the quorum sensing system and classified into different types by its luxR/I genes.F5 has two sets of luxR/I genes.One set homologous to the only luxR/I gene pair of F10,belonging to the B group in the previous classification.The other set of luxR/I of F5 is specific for S.pseudonitzschiae.Most of the Roseobacters have quorum sensing system,motility,and adhesion ability.Furthermore,many of them are isolated from the surface of microalgae,macroalgae,and other marine animals,suggesting that this group has tight relations with their inter-kingdom hosts.