Optimization Of Heterotrophy-Photoinduction Culture Of Chlorella Pyrenoidosa

Chlorella is rich in proteins,pigments,polysaccharides,polyunsaturated fatty acids,vitamins and minerals and other nutrients,so it has a wide range of application value in food health care,feed and medicine and other fields,and its development has a very broad market application prospect.How to increase the density of Chlorella culture and obtain high value-added products is the key technical problem of large-scale culture.The heterotrophic culture of Chlorella with organic carbon sources can improve the biomass yield,but heterotrophic culture is not conducive to the accumulation of high value-added products such as protein and pigment which need light induction conditions.High biomass of microalgae and high yield of value-added products could be obtained by the method of Heterotrophy-Photoinduction two-stage series culture.In this study,the heterotrophic and photoinduction methods were used to culture Chlorella pyrenoidosa,and the culture conditions were optimized for heterotrophic and photoinduced autotrophic stages,including carbon source,nitrogen source,inoculation amount,rotational speed,dilution ratio,light intensity,and ventilation rate.In order to lay the foundation for large-scale cultivation of high quality Chlorella pyrenoidosa,a culture mode which can simultaneously obtain high biomass,high lutein and protein content of Chlorella.The main research contents and results are as follows:1.The effects of glucose concentration,nitrogen source type and concentration,inoculation amount and rotation speed on the growth of Chlorella were investigated by single factor experiment.It was found that the concentration of glucose at 0-50 g/L could promote the growth of Chlorella,and the highest cell density was obtained at 10g/L.High concentration of glucose prolonged the growth retardation period of Chlorella.The inorganic nitrogen source of potassium nitrate was more favorable to the growth of Chlorella than the organic nitrogen source of urea and mixed nitrogen,and the optimal concentration of potassium nitrate was 0.5 g/L.The growth retardation period of Chlorella was significantly shortened by increasing the inoculation amount,and the cell density reached the maximum when the inoculation amount was 10%.Chlorella cell density increased with increasing rotation speed and reached the maximum at 180 rpm.Orthogonal experiment was further used to optimize the concentration of glucose and KNO₃ in heterotrophic culture as well as the inoculation level.It was found that when the concentration of glucose was 7.5 g/L,the concentration of potassium nitrate was 0.25 g/L,and the inoculation amount was10%,Chlorella reached the maximum cell density and chlorophyll content.3.01×10⁸cells/m L and 28.60 mg/L,respectively2.In order to further improve the cell density and nutrient content of Chlorella,the feeding conditions of heterotrophic culture stage were optimized.By measuring the content changes of glucose and NO₃-N in Chlorella culture medium and combining with the cell growth curve,the optimal feeding time was 54 h,and the optimal feeding amount of glucose and potassium nitrate was 2 g/L and 0.1 g/L,respectively.According to the effects of fed-batch and semi-continuous feeding on the growth and nutrient accumulation of Chlorella,a new feeding strategy was designed,that is,three times of fed-batch followed by two times of semi-continuous feeding.Lutein,protein,total fat and dry weight obtained by this method were 10.05 mg/L,57.63 mg/L,32.80%and 4.35 g/L,respectively,which were significantly higher than those obtained by only semi-continuous culture and fed-batch culture3.To investigate the effects of dilution ratio,light intensity,ventilation and other culture conditions on the growth and protein accumulation of Chlorella in light-induced stage.It was found that when the dilution factor was 1-100 times,the protein content firstly increased and then decreased,and the protein content reached the highest at the dilution factor of 5 times and the dry weight reached the highest at the dilution factor of 1 times.The protein content of Chlorella increased with the increase of light intensity,but the dry weight of Chlorella decreased with the increase of light intensity.When the light intensity was 10000 Lux,the dry weight of Chlorella was very low,which brought difficulties to the harvest.Therefore,the condition of high light intensity was not considered.When ventilation was 1-4 vvm,protein content firstly increased and then decreased,and reached the maximum at 2 vvm.The change trend of dry weight was the same as that of protein.The optimal conditions were determined as follows:dilution factor 2,illumination intensity 3000 Lux,ventilation volume 1.8 vvm,and the highest yield of protein was 111.05 mg/L.4.The cell dry weight was 1.09 g/L,and the protein content was 102.57 mg/L.Compared with the vial culture,the difference was within 20%,which was in line with the error of the amplification culture.It was found that blue light,red light and mixed light were not good light sources for the growth of Chlorella pyrenoidosa.Red and blue light even inhibit the growth of Chlorella.The optimum light quality for protein accumulation was determined to be white light.