Technology Study On Heterotrophic Cultivation Of Chlorella Using Sucrose

Chlorella pyrenoidosa is a kind of spherical unicellular green alga,which contains abundant proteins,lipids,vitamins,as well as other bioactives.However,the light attenuation leads to poor growth of C.pyrenoidosa in photoautotrophic culture.Heterotrophic cultivation can avoid those problems mentioned above.But the carbon sources such as glucose are expensive,while the relatively cheap sucrose is difficult to be utilized by C.pyrenoidosa under heterotrophic cultivation.Therefore,it results in the high cost of heterotrophic cultivating C.pyrenoidosa in commercial production.In this study,the mechanism of C.pyrenoidosa using sucrose was preliminarily investigated,and the reason why C.pyrenoidosa was difficult to utilize sucrose was found.In addition,the effective technology of heterotrophic cultivating C.pyrenoidosa using sucrose was developed by co-culture C.pyrenoidosa with other microorganisms.(1)C.pyrenoidosa was heterotrophic and mixotrophic cultured using sucrose as the sole carbon source.C.pyrenoidosa cannot survive at heterotrophic culture mode.And even under mixotrophic culture,the growth of C.pyrenoidosa was equivalent to that in autotrophic culture.At the same time,no sucrose was utilized by algal cells,and no monosaccharides were detected in the medium.So C.pyrenoidosa lacks both extracellular sucrose hydrolase and sucrose transporter activity that cannot grow using sucrose as the sole carbon source.(2)Sucrose can be extracellularly hydrolyzed by Rhodotorula glutinis.In addition,the hydrolysis rate of sucrose was faster than the utilization rate of hydrolyzed monosaccharide,which leads to abundant monosaccharides accumulation in the medium.Based on the unbalancedness,C.pyrenoidosa was co-cultured with R.glutinis using sucrose as the sole carbon source.The highest C.pyrenoidosa cell density of 111.48×106 cells/mL was obtained at the initial ratio of alga/yeast=20:1,and the highest final cell number proportion of algae was 45%.Moreover,the lipid content reached 30%,which was higher than that obtained in glucose contained system.(3)Furthermore,the most commonly used Saccharomyces cerevisiae which has the same secretory activity of sucrose hydrolase,was immobilized by cell immobilization technology.Then,C.pyrenoidosa was heterotrophicly cultured using sucrose as the sole carbon source by co-culture with immobilized S.cerevisiae.In the cultures with 150 IYB in 100 mL culture,the highest final biomass concentration and lipid content of 2.08 g/L and 29%were obtained,respectively.More importantly,pure algal suspension was efficiently obtained in this system as that the immobilized S.cerevisiae can be easily separated through simple filtration.In addition,the immobilized yeast beads can be repeatedly used for at least three times,which can further reduce culture cost.At the same time,according to the optimization conditions of the shaker culture,the pilot-scale culture of C.pyrenoidosa was operated in the air lift reactor.In this system,the lipid content of C.pyrenoidosa reached 37%,which was significantly higher than that of flask culture.(4)A symbiotic yeast which can enhance C.pyrenoidosa growth using sucrose as the sole carbon source at heterotrophic cultivation was accidentally found in a contaminated algal suspension.This yeast was isolated and identified as Cryptococcus sp.After that,a co-culture system was developed by mix culture of C.pyrenoidosa and Cryptococcus sp.using sucrose as the sole carbon source.In this system,the highest C.pyrenoidosa cell density of 118.8×106 cells/mL was obtained at the initial ratio of alga/yeast=30:1.Unlike our previous study,the final cell number proportion of algae was much higher than that co-cultured with R.glutinis as the highest algal number proportion reached 83.8%and it was more than 77%in all cultures.Furthermore,the protein and lipid content was also greatly enhanced,which reached 51.9%and 27.1%,respectively.