Alleviating Oxygen Stress,Contamination-Free And Lipid Accumulation During Cultivation Of Chlorella Vulgaris

Freshwater green algal Chlorella vulgaris is an industrially important microalgal species,which has been widely used in healthy food,animal feed and cosmetics.It is of great potential as a promising feedstock for biodiesel production,which has attracted lots of attention recently.However,the contamination especially from flagellate,coupled with oxygen stress may lead to low cell productivity.It will consume massive energy to solve and has become one of the major concerns in industrial farming of C.vulgaris and other freshwater microalgae.There is a need to improve LP and biomass productivity,with strategies of free-contamination in unsterile medium excluding deoxidization equipment by optimizing culture condition.Effect of triple stress on LC was studied and all these data will provide theoretical and technical support.（1）160 mM NaHCO₃ at pH 9.5 could inhibit flagellate effectively.Result shows that all four salts could inhibit flagellate and the effect degree rank was NaHCO₃﹥KHCO₃﹥NaCl﹥KCl.Experimetal methods conclude DNA gel electrophoresis,trypan blue identification plus morphological observation.The inhibition effect of 160 mM NaHCO₃ was enhanced with increasing pH and it is hard to find flagellate at pH 9.5.One possible mechanism of inhibitory effect is inoc stress lead to membrane disruption.Another possible mechanism is damage of RVI.（2）NaHCO₃ and NaCl were chosen to investigate effects on C.vulgaris.Results show that NaHCO₃ can stabilize medium pH.NaHCO₃ will enhance cell growth from concentration of 0 mM to 80 mM by offering more carbon source.High concentration such as 160 mM will decrease cell density comparing with160 mM NaCl.We found that single cell weight and cell volume in NaHCO₃was increased with cell aggregation.Effect of NaHCO₃ on cell growth is much more complex than NaCl.However,the mechanism of stress is not clear in flask experiment.（3）In order to investigate the different effects of NaHCO₃ and NaCl on C.vulgaris,experiments were operated in 3 L PBR with parameters controlled strictly.The results show that DCW_maxax in 160 mM NaCl was greater than 160mM NaHCO₃ at same pH,and the favorable pH was 8.5 in NaHCO₃ instead of7.5 in NaCl.This is mainly because the high[dCO₂]that NaHCO₃ brought to.Furthermore,NaHCO₃ can stimulate lipid accumulation more than NaCl did.The highest LC and LP were both obtained in 160 mM NaHCO₃ at pH 9.5,with494 mg?g^-11 and 44.5 mg?L^-1?d^-11 respectively.The reason for lipid stimulating by DIC could be explained by high[CO₃^2-]at high pH.Aggregation needs nitrogen and this is a physiological response to mitigate high concentration of DIC in 160mM NaHCO₃ medium.No flagellate was found in unsterile medium with 160mM NaHCO₃.（4）Harmful effect of oxygen stress was discussed coupled with mitigation mechanism by adding NaHCO₃.Results indicate that oxygen stress aroused by high[dO₂]has negative effect on cell growth as well as lipid production,and the effect is positively associated.The negative effect in medium with 400%air saturated of dO₂ would be reduced by adding 160 mM NaHCO₃,LP and biomass productivity have increased 139%and 98%,respectively.The mechanism of this phenomenon would attribute to two aspects.One is the enhancement of ROS enzyme eliminating system.The other is the activation of CCM,which increase photosynthesis and inhibit photo-respiration.（5）Effect of triple stress on lipid accumulation of C.vulgaris was studied.Results indicate that on the basis of two stress under pH 9.5+160 mM NaHCO₃ or pH 9.5+160 mM NaCl,the supplement of nitrogen stress would form triple stress,which will induce lipid accumulation to some extent.The highest LC was obtained under pH 9.5+160 mM NaHCO₃+nitrogen limitation condition with 510 mg?g^-1.Cells will decompose chlorophyll for nitrogen source when extracellular nitrogen was depleted,leading to low photosynthetic efficiency and low cell growth rate.Adding 160 mM NaHCO₃ is an effective method for safe cultivation in unsterile medium especially at pH 9.5.