High-efficient Bioconversion Of Nitrate With Coproduction Of High-value Biomass By Chlorella Pyrenoidosa

Chlorella pyrenoidosa is an unicellular green algae,which has the characteristics of fast growth,strong environmental adaptability and metabolic diversity,and is superior in quick transformation of nitrate into high-value nitrogen-containing compounds,like protein and chlorophyll.Nowadays,the rapid development of industrial society not only accelerates the serious water pollution caused by NO₃^-,but also increases the demands for protein.In this paper,C.pyrenoidosa was mixotrophically cultured in the modified basal medium containing high nitrate nitrogen to investigate the feasibility of NO₃^-assimilation with co-production of high-value biomass for achieving the deep purification and resource utilization of wastewater.Firstly,the Erlenmeyer flasks culture conditions of C.pyrenoidosa were systematically optimized using consumption of NO₃^-as the index.Then,the operation parameters of the photo fermenter culture system were further optimized to validate and enhanced the efficiency of NO₃^-consumption rate with co-production of high walue biomass by C.pyrenoidosa.Finally,the cryopreservation technology for C.pyrenoidosa was established to meet the developing needs of microalgae biomass products and the preservation effects were systematically evaluated.The main experimental results were as follows:1.The appropriate increase of initial inoculum density and decrease of initial sodium nitrate concentration could promote the rapid consumption of NO₃^-by C.pyrenoidosa.When fed-batch strategy was carried out,the highest biomass yield?35.95 g/L?,average NO₃^-assimilation rate?2.06 g/L/d?and algal protein content?42.44%of the dry weight?were obtained after 6 days of continuous mixotrophic cultivation under the conditions of initial sodium nitrate of 15 g/L and initial inoculation density of 1×10⁸ cells/m L.Increasing light intensity could promote the cell growth of C.pyrenoidosa,but no significant differences was observed on the consumption rate of NO₃^-.By using a staged increase of light intensity?the light intensity was stepwise increased from 120 to 210?mol/m²/s while adding glucose into the system?as illumination mode,the specific growth rate of cells could be significantly promoted to the highest(0.65 d^–1),and compared with the constant illumination condition,the total chlorophyll content and total carotenoid content in the cells were increased by 8.19%and 102.78%respectively.2.The effects of initial glucose concentration,sodium nitrate concentration and control level of p H value on the growth and consumption of NO₃^-with proteins co-production of C.pyrenoidosa were investigated in a 5 L photo fermenter.The optimum culture conditions were determined as the initial glucose concentration of 5 g/L,Na NO₃ concentration of 10 g/L,automatical addition of 0.5?1.0 mol/L HNO₃ for nitrogen supplementation and p H control?p H 6.5?.A phased glucose feeding strategy was adopted to control the glucose concentration at 10?20 g/L in the fed-batch cultivation mode.The biomass productivity,average consumption rate of NO₃^-and protein productivity of C.pyrenoidosa were 7.88 g/L/d,5.15g/L/d and 4.51 g/L/d respectively after continuous fermentation for 60 h.3.The effects of C.pyrenoidosa growth stage and light quality on the cell growth,the consumption rate of NO₃^-with co-producing of proteins were systematically investigated in a5 L photo fermenter.The results showed that rapid growth and proliferation were performed when C.pyrenoidosa in the end of logarithmic phase and the later stage of stability was inoculated into new medium,and there was no significant difference in cell biomass yield and specific growth rate.However,the levels of supplemental glucose exerted significant effects on cell growth,consumption rate of NO₃^-,and protein productivity during fermentation?p<0.05?.Using a natural white light?R:G:B=18.9:76.1:5.1?in a two-stage repeated fed-batch culture for 180 h,the highest biomass yield?7.22 g/L/d?of C.pyrenoidosa was achieved at the end of the cultivation,with the average NO₃^-consumption rate of 3.88 g/L/d and 2.86 g/L/d respectively.Using a mixed light?cold white light and warm white light,R:G:B=14.6:83.0:2.4?in a four-stage repeated fed-batch culture for 276 h,the average NO₃^-consumption rate of C.pyrenoidosa were 3.69 g/L/d,3.10 g/L/d,2.29 g/L/d and 1.94 g/L/d respectively,and the maximum protein content reached 55.64%at 264 h.In the second stage of two cultivation processes,the average consumption rate of NO₃^-and PO₄^3-and protein productivity under mixed white light were respectively increased by 8.39%,30.0%and 22.71%compared to natural white light.4.The concentrated algae paste of C.pyrenoidosa(8×10¹⁰cells/m L)obtained after pretreatment?repeated washing and centrifugation?,was resuspended in BG11 medium to obtain algae fluid?1×10⁸cells/m L?.Then,the concentrated algae paste and algae fluid were preserved after pasteurization or not to storage at room temperature of 18?25?or refrigerated temperature of 4?,respectively.And the cell activity and biomass quality of concentrated algae paste and algae fluid were systematically evaluated during 15 days of continuous storage.The best method to select was to store fresh products at 4?after pretreatment.In this case,after 15 days of storage,the relative cell activity,the loss rate of the content of chlorophyll,carotenoid and protein of C.pyrenoidosa algae fluid were 61.36%,100%,100%and 100%,respectively.And the cell activity,the loss rate of total content of chlorophyll,carotenoid and protein of concentrated algae paste were 48.26%,97.20%,100%,and 98.20%,respectively.