Studies To Increase The Protein Content Of Chlorella Under Heterotrophic Cultivation

Chlorella is a unicellular green microalga.Chlorella biomass had various of bioactive compounds such as proteins,polyunsaturated fatty acids,pigments and carbohydrates.Thus,exploiting Chlorella biomass has been one of the most hot topics for microalgae.Chlorella strains can use light energy to grow by photosynthesis.However,some Chlorella strains can utilize organic carbon sources to grow by heterotrophic cultivation under dark conditions.Notably,the protein content of Chlorella by heterotrophic culture was lower than that by autotrophic culture.To address this issue,this project firstly screened the best ratio of carbon and nitrogen in the medium to promote the microalgae growth and bioactive biosynthesis of Chlorella sp.MBFJNU-17.Then,different regulation stage was developed to increase the growth rate and the metabolism switch for improving protein content of Chlorella sp.MBFJNU-17.Lastly,the developed strategy was further verified using a 10 L fermenter.The main contents and results of this project were as follows:(1)16 kinds of different media with different ratios of carbon and nitrogen sources were selected to evaluate the growth and protein content of Chlorella sp.MBFJNU-17 by heterotrophic culture.The results showed that the medium with low carbon and high nitrogen concentrations was beneficial to promote the protein biosynthesis of Chlorella.Microalgae achieved the highest protein content(54.66 ± 1.47%)in the glucose concentration of 20 g/L and urea concentration of 8 g/L.However,the low microalga biomass was obtained by this microalga with this medium.When the urea content increased to 12 g/L,the maximum chlorophyll content of microalgae was 1.60 ± 0.06%.With the increase of glucose concentration to 80 g/L and urea concentration to 12 g/L,the maximum biomass and protein content of microalgae were 17.23 ± 0.85 g/L and 42.53 ± 4.15%,respectively.When glucose concentration was 80 g/L and urea concentration was 1 g/L,the biomass,and the contents of protein and chlorophyll of Chlorella sp.MBFJNU-17 distinctly decreased,while the highest contents of carbohydrate(51.23 ± 2.35%)and oil(18.73 ±1.73%)attained by microalga.Microalgae did not completely consume the glucose and urea substrates when the glucose concentration was more than 40 g/L and urea concentration was higher than 4 g/L.Therefore,Chlorella powder with different biochemical composition can be obtained by changing the concentration of carbon and nitrogen sources in the medium.(2)Two-stage regulation strategy was developed for heterotrophic Chlorella sp.MBFJNU-17 to produce high protein content.The first stage was the nitrogen starvation culture.In the low nitrogen medium,heterotrophic Chlorella reached a certain biomass,but its protein content was low.The second stage was the over compensation stage with nitrogen to induce the protein biosynthesis by Chlorella sp.MBFJNU-17.In this case,three different schemes were established to evaluate the ability of heterotrophic Chlorella sp.MBFJNU-17 for protein content.These three strategies were:(A)the microalga cells from the first stage were centrifugated and then transferred into the fresh media with different concentrations of nitrogen source.(B)The microalga cells from the first stage were further cultivated when the original medium was supplemented with corresponding carbon source and nitrogen source.(C)The microalgae cells from the first stage was centrifugated and transferred into the media containing only carbon source and nitrogen source.In the second stage,the concentration of urea(1 g/L,2 g/L,3 g/L and 4 g/L)was optimized.The results showed that when the urea concentration was 3g/l,the protein content of Chlorella by strategy A was increased from 21.07 ± 1.83% to 56.02 ± 2.22%,while the carbohydrate content decreased from 57.42 ± 0.06% to 6.85 ± 0.60%.For the strategy B,when the urea concentration was 3g/L,the protein content of Chlorella sp.MBFJNU-17 reached 50.97 ±1.77% and carbohydrate decreased to 7.61 ± 0.81%.As for,strategy C,the highest protein content of Chlorella was only 30.15 ± 3.93% when the concentration of urea was 4 g/L.Based on these results,it was better to adopt strategy B for the two stage regulation culture;in this case,the optimal urea concentration was 3 g/L.(3)The biomass and biochemical composition of Chlorella sp.MBFJNU-17 in the two-stage regulation culture and autotrophic culture mode were determined.The results showed that heterotrophic Chlorella sp.MBFJNU-17 exhibited the switch between carbohydrate and protein during the two-stage regulation culture.By transmission electron microscopy,a large number of starch granules of Chlorella sp.MBFJNU-17 cells were clearly seen in the first stage.After the second stage of culture,the starch in the cells had been transformed into protein.Finally,the protein content of Chlorella sp.MBFJNU-17 reached the value for the autotrophic culture.The composition and relative content of fatty acids and amino acids were not significantly different from those of Chlorella.Similarly,the developed two-stage regulation was also suitable for improving the protein content of heterotrophic Chlorella F9.(4)Two mechanisms for luxury absorption and over compensation were used to regulate the fermentation of Chlorella sp.MBFJNU-17 in a 10 L fermenter,by determined the changes of protein content.The increment in the protein content of Chlorella was not obvious by the luxury absorption.Differently,the protein content of Chlorella sp.MBFJNU-17 was decreased in the nitrogen starvation;when the microalgae cells were transferred into the high nitrogen stage,the protein content was increased and then decreased.The highest protein content of Chlorella sp.MBFJNU-17 by the nitrogenstarvation to high-nitrogen induce strategy was 36.81±0.28% during fermentation,the protein content was 24.9±0.89% after fermentation.The protein content of Chlorella sp.MBFJNU-17 by the low-nitrogen to high-nitrogen induce strategy was increased to45.28±0.32%.The protein content of microalga maintained above 40%.The protein content of Chlorella was 43.26±0.32% after fermentation.Therefore,the regulation of heterotrophic Chlorella by low nitrogen and rich nitrogen stage was the promising approach to improve its protein content.In this paper,the effects of different combinations of carbon and nitrogen concentrations on the physiology and biochemistry of Chlorella were analyzed,and the strategy of increasing the protein content of heterotrophic Chlorella by stages was established.At the same time,the feasibility of the strategy was verified in a 10 L fermenter.