| Microalgae are a group of autotrophic microorganisms with photosynthetic capability.They can synthesize polysaccharide,lipid,protein and other organic substances by fixing carbon dioxide through photosynthesis.As the new generation of biomass resources,microalgae have great potential to produce fine chemicals,bulk chemicals and energy in the future.Compared with traditional fossil fuels,the absolute advantages of microalgae biodiesel are the reproducibility and sustainability.To produce biodiesel,microalgae should be the better resource because they grow faster than traditional oil crops and could adapt to a variety of soil type that would not compete for the farmland.Cultivation of microalgae is affected by many factors.Under the premise of obtaining more stable culture and higher yield,how to reduce the cost has always been the most concerned problem in the field of industrial application.Open pond is the most regular and cost-effective cultivation systems in the industry.However,the microalgae cultured in the open ponds will inevitably be invaded by other microorganisms,which may lead to yield decline or pond crash.In view of the close relationship between microalgae and the surrounding microorganisms,how to efficiently utilize these microorganisms during the microalgae cultivation has attracted lots of attention.To obtain stable microalgae-bacteria systems during the open cultivation,microalgae growth-promotion bacteria(MGPB)were isolated from stable microalgae culture,the interaction mechanisms of microalgae-MGPB were deeply studied,and microalgae-MGPB co-culture were further applied into open cultivation systems in this paper.The main contents and results are as follows:1.Analysis of the bacterial community around the Chlorella sorokiniana,which had been cultured in open pond for two months.Results of metagenomic sequencing showed that the main phyla around the C.sorokiniana were Bacteroidetes,Proteobacteria and Firmicutes.The core genera were heavily involved in material metabolism in the phycosphere.Based on that,the key to maintain the population dominant of MGPBs in the microalgae culture is the selection of MGPBs,which could be closely associated with the extracellular exudates of C.sorokiniana.In this work,13 bacterial strains were isolated from the open pond culture of C.sorokiniana,and they came from six different genera.After the bacteria were co-cultured with C.sorokiniana in erlenmeyer flasks,strain C-10(Achromobacter sp.)and C-13(Rhizobium sp.)promoted the growth of C.sorokiniana by 31.79%and 79.29%.Meanwhile,the two strains could survive accompanying with the growth of C.sorokiniana.So Achromobacter sp.and Rhizobium sp.were selected as MGPBs to build the microalgae-bacteria co-culture systems in future works.2.The selected MGPBs strain C-10 and C-13 were co-cultured with C.sorokiniana in closed photobioreactors,respectively.Strain C-10 promoted the growth of C.sorokiniana by 13.80%and did not affect the lipid content.C-13 could not only promote the growth of C.sorokiniana by 15.88%,but also increase the lipid content by 7.45%.Scanning electron microscope showed that C.sorokiniana secreted sticky extracellular polymeric substances(EPS)during the growth and the MGPBs could adhere to the surface of microalgal cells or disperse in the culture.The MGPB could grow in the algal culture by using the oxygen and extracellular polysaccharide produced by C.sorokiniana.Strain C-13 had nitrogen fixation capability and could supply nitrogen for C.sorokiniana growth only when the nitrogen in the culture was extremely insufficient.3.The types of Acyl-homoserine lactones(AHLs)secreted by the MGPBs were detected by HPLC-MS/MS.The main AHLs secreted by strain C-10 was C14-HSL,and strain C-13 could secrete C8-HLS and C14-HSL.To study the signal communication mechanism between microalgae and the MGPBs,C8-HSL and C14-HSL of different concentration were added into the culture medium of C.sorokiniana.The results showed that both C8-HSL and C14-HSL could promote the growth of C.sorokiniana,and C14-HSL performed better,which could increase the biomass of C.sorokiniana by 13.86%.C8-HSL and C14-HSL of 50 nmol L-1 could increase the lipid content of C.sorokiniana by 11.31%and 45.80%.The photosynthetic capability of C.sorokiniana could also be affected by AHLs.For C8-HSL,the lower concentration in the culture could improve the maximum photosynthetic rate of C.sorokiniana.For C14-HSL,it could improve the maximum photosynthetic rate of C.sorokiniana under tested concentration.The light compensation point could also be improved by C8-HSL and C14-HSL,and the C.sorokiniana need higher light intensity to accumulate organic matter.C8-HSL and C14-HSL of 50 nmol L-1 could increase the cell viability of C.sorokiniana.The stress response of C.sorokiniana to AHLs was investigated by detecting the activity of antioxidant enzymes.After C8-HSL was added to the culture,stress response occurred and the reactive oxygen species increased in the algal cells,so the activities of superoxide dismutase(SOD)and peroxidase(POD)increased.As the concentration of C8-HSL increased in the culture,the stress response of algal cells gradually weakened.C14-HSL under tested concentration would not increase the reactive oxygen species of C.sorokiniana.Transcriptome analysis showed that addition of C8-HSL and C14-HSL could affect the intracellular transcription level of C.sorokiniana.Compared with the control group,356 and 397 genes were significantly regulated by C8-HSL and C14-HSL,respectively.The gene clusters related to lipid metabolism and photosynthesis were up-regulated or down-regulated by C8-HSL and C14-HSL.In this way,C8-HSL and C14-HSL could affect the intracellular lipid content and photosynthesis of C.sorokiniana.4.To explore the performance of MGPBs in the open culture,the MGPBs were co-cultured with C.sorokiniana in open cultivation systems indoors and outdoors.Under stable cultivation environment indoor,strain C-10 could not adapt to the changes of bacterial composition to keep dominant population in the culture and was replaced by other bacteria in niche,and lost the microalgae growth-promotion capability.Strain C-13 could grow fast in the microalgae culture to dominate in bacterial community,so it could promote C.sorokiniana growth by 13.76%in the open culture.The outdoor open cultivation showed that both of the selected MGPBs lost the microalgae growth-promotion capability if the bacteria were added to the culture only once at the beginning.Because it was difficult for the selected MGPBs to dominate in bacterial community by themselves under fluctuating culture conditions.If strain C-13 was added to the culture at regular intervals,it could keep the population dominance in the bacterial community and promote the growth of C.sorokiniana by 13.29%.The four batches of microalga-MGPB co-culture in the outdoor systems resulted that environmental changes such as temperature,light intensity and weather would affect the biomass of C.sorokiniana,while the microalgae growth-promotion capability of strain C-13 was stable during the open culture.Therefore,it is possible to apply strain C-13 to the industrial open cultivation of C.sorokiniana.Bacterial agents,containing 50 g L-1 of trehalose as protectant,were prepared by freeze-drying technology and well revival rate was obtained. |
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