The Effects Of Organic Nutrition On Biochemical Characteristics Of Chlorella Pyrenoidosa

Chlorella pyrenoidosa with comprehensive nutrition is the ideal human health food.It is also widely used in aquaculture and livestock feed additives,and has huge commercial value.C.pyrenoidosa grows fast under mixotrophic condition,is not easy to be contaminated by bacteria.Therefore,C.pyrenoidosa can be cultivated under mixotrophic condition to obtain the algal product.Domestic,municipal and industrial waste waters are rich in organic matter,and C.pyrenoidosa is also an ideal microorganism in the treatment of organic wastewater.At present,the studies on micro-algal biotechnology focus on screening the algal strains with the high lipid,protein and hydrogen productivity,and utilizing organic wastewater more efficiently to obtain high algal yield,while the effects of organic matter,especially single organic matter on algal biochemical composition are ignored.Therefore,this study on effect of organic carbon source on biochemical composition has an important significance in microalgal biotechnologcial application.In addition,some lakes,rivers and ponds are rich in organic compounds,and some algae lived in these environments are mixotrophy.The study can also provide valuable information on algal mixctrophic growth in nature.There were some literatures on the effects of glucose on microalgal growth and biochemical composition.However,the researches about other sugars were rare.Glucose,galactose,fructose,sucrose,maltose,lactose and starch are common organic carbon sources in the mixotrophic culture medium and in nature.Our data showed that monosaccharides exerted stronger stimulative effects on the algal growth compared with disaccharides and starch.For the tested monosaccharides,glucose was the best carbon source for the mixotrophic growth of C.pyrenoidosa,followed by galactose and fructose.For the tested disaccharides,maltose was the best carbon source for the mixotrophic growth,followed by sucrose and lactose.These data showed the differentially stimulative effect of starch and different sugars on the growth of C.pyrenoidosa.After 10-day culture,addition of 1.0 and 3.0g L^-1 glucose and 3.0g L^-1 galactose led to the significant decrease of lipid content,while 0.5 and 5.0g L^-1 glucose,0.5^-1.0 and 5.0 g L^-1 galactose,and 0.5-5.Og L^-1 fructose,disaccharide and starch did not significantly affect the lipid content.The protein contents of mixotrophic cells with addition of 0.5-5.0g L^-1 glucose and galactose?23.8-47.4%and 25.9-50.8%,respectively?were significantly lower than that of autotrophic cells?65.6%?,and the inhibitory effects of the two sugars on protein content tended to increase with the increasing sugar concentration.Also,addition of 3.0 and 5.0g L^-1 fructose resulted in significant decrease of protein content,although 0.5 and 1.0g L^-1 fructose did not significantly affect protein content.Consistent with the lipid content,the protein content was not significantly affected by addition of 0.5-5.0g L^-1 disaccharide and starch.Corresponded to the decrease of protein content,the carbohydrate contents of mixotrophic cells with addition of 0.5-5.0g L^-1 glucose and galactose?37.6-67.8%and 35.5-62.9%,respectively?were significantly higher than that of autotrophic cells?18.5%?,and addition of 3.0 and 5.0 g L^-1 fructose also leaded to significant increase of carbohydrate content.Like lipid and protein contents,the carbohydrate content was not significantly affected by addition of 0.5-5.0 g L^-1 disaccharide and starch.The fast growth can lead to nitrogen limitation that influenced the microalgal biochemical composition.Both the nitrogen limitation and the types of sugar might induce the biochemical change,and the real reason led to this result needed to be proved.Compared with normal inorganic nitrogen,inorganic nitrogen limitation led to a significant decrease of protein content and the significant increase of carbohydrate content in autotrophic cells,while did not resulted in significant change of lipid content.Similar to the autotrophic cells,inorganic nitrogen limitation simultaneously led to no significant change of lipid content,significant decrease of protein content and significant increase of carbohydrate content in the mixotrophic cells with addition of fructose,three disaccharides and starch.However,inconsistent with the biochemical change of the autotrophic cells,inorganic nitrogen limitation led to no significant change of carbohydrate content,increase of lipid content and significant decrease of protein in the mixotrophic cells with addition glucose and galactose.Consistent with normal,compared with control,there was no significant difference in lipid,protein and carbohydrate contents among the mixotrophic cells with addition of three disaccharides and starch with inorganic nitrogen limitation condition.There was no significant difference in lipid content,but the decrease of protein content and increase of carbohydrate content were observed with addition of glucose and galactose.Compared with nitrogen limitation,the lipid content of control decreased,and increased with addition of glucose,while the lipid content did not have signficant change with addition of sucrose and starch under nitrogen deficiency.The protein content of control and with addition of glucose,sucrose and starch all decreased significantly under nitrogen deficiency.Under nitrogen deficiency,the significant increase of carbohydrate content occurred in the mixotrophic cells with addition of sucrose and starch and the autotrophic cells,while no significant change of carbohydrate content occurred in the mixotrophic cells with glucose.Under nitrogen deficiency,there was no significant change of lipid with addition of sucrose and starch comparing with control,while a increase with addition of glucose occurred.The protein content were decreased with the addition of glucose and starch,while it had no significant change with addition of sucrose.The carbohydrate content was increased with addition of glucose,while it had no significant change with addition of sucrose and starch.These indicated that the nitrogen limitation was indeed a factor influenced the microalgae biochemical composition.It needed more experiments to further prove whether the type of sugar was also a factor that influenced the cellular biochemical composition.The nitrogen limitation did not appear in the initial growth phase?3 days?and the exponential growth phase?6 days?.The experiment of 3 days and 6 days was carried out to verify the issue that the type of organic carbon was a factor that influenced the cellular biochemical composition.The variation pattern of biochemical composition affected by addition of different sugars and starch was similar on day 3,day 6 and day 10.During incubation of day 3 to day 10,addition of glucose and galactose significantly decreased the lipid content.Also,addition of three monosaccharides significantly decreased the protein content,and significantly affected the carbohydrate content.The effects of disaccharides and starch on biochemical composition were not so remarkable as three monosaccharides during growth.These results showed that the type of organic carbon was a factor that influenced the microalgal biochemical composition.This finding suggested that disaccharides and starch had the biotechnological potential to increase the growth of C.pyrenoidosa while maintaining a high protein content in the microalgal biomass.The effects of amino acid on microalgae growth and biochemical composition were rarely reported.Serine,alanine,cysteine,glutamic acid and glutamine were used to explore the effects in this study.High concentrations of organic nitrogen(1.0 and 3.0 g L^-1)inhibited the growth of C.pyrenoidosa and low concentration of organic nitrogen(0.05-0.3g L^-1)promoted the growth.Theoretically,sufficient nitrogen promotes the protein synthesis,but compared with the control,the 5 tested organic nitrogen(0.05g L^-1,culturing for 10 days)did not affect the biochemical composition significantly.Inorganic nitrogen may hinder the organic nitrogen utilization,resulting in the above results.The experimental results with low inorganic nitrogen concentration was carried out to verify this issue.Compared with normal inorganic nitrogen,there was no significant change in lipid content,and protein content was decreased,while carbohydrate content was increased under the low inorganic nitrogen condition.These results showed that for C.pyrenoidosa,the organic nitrogen?amino acids?can not replace the inorganic nitrogen source.The bulk of organic carbon exists in soil,lakes,rivers and ponds,and the algae growing in soil or aquatic environment enriched with organic carbon are mixotrophic rather than autotrophic.It is important to study the biochemical composition changes of C.pyrenoidosa in the presence of other pollutants under autotrophic and mixotrophic conditions.The paraquat was choses as a represent pollutant.At the same time,the effects of paraquat on pigment synthesis,antioxidant systems and related gene expression abundance in C.pyrenoidosa were observed.Cultured for 3 days,paraquat inhibited the growth of C.pyrenoidosa under both autotrophic conditions and mixotrophic condition.C.pyrenoidosa exhibited markedly different growth responses towards paraquat at different concentrations under autotrophic and mixotrophic conditions.At 0.8 ?M paraquat,the inhibition percentage of growth rate under mixotrophic condition was lower compared with autotrophic condition,while it was higher at 1.8 and 3.4 ?M paraquat.This indicated that the differential susceptibility of cell growth to paraquat toxicity occurred between autotrophically grown cells and mixotrophically grown cells.Under autotrophic condition,compared with the control,paraquat exposure affected the lipid content insignificantly,but reduced the protein content and increased the carbohydrate content.Under mixotrophic condition(4g L^-1 glucose),compared with the control,paraquat increased the lipid content,and increased the protein content,but reduced the carbohydrate content.3 days exposure to paraquat under both autotrophic and mixotrophic conditions induced the decrease of chlorophyll content,the increase of superoxide dismutase and peroxidase activities,and the decrease of transcript abundances of three photosynthesis-related genes?chlL,psbA and rbcL?.The inhibition rates of Chl a and Chl b content with exposure to 0.8 to 3.4 ?M paraquat under mixotrophic condition?43.1-52.4%and 54.6-59.7%,respectively?were higher compared with autotrophic condition,revealing the more susceptible reaction of chlorophyll synthesis to paraquat under mixotrophic condition.On the contrary,compared with autotrophic condition,the inhibition rate of rbcL gene transcription under mixotrophic condition?35.7-44.0%?was lower with exposure to 0.8 to 3.4 ?M paraquat.Compared with autotrophic condition,the inhibition rate of chlL gene transcription under mixotrophic condition was lower at 1.8 and 3.4 ?M paraquat,and the inhibition rate of psbA gene transcription under mixotrophic condition was higher at 0.8 and 1.8 ?M paraquat.As for the promotion rates of SOD and POD activities with exposure to 0.8 to 3.4 ?M paraquat,their differences between autotrophic and mixotrophic conditions were not so obvious as those for inhibition of growth rate and inhibition rate of chlorophyll content.This study will provide theoretical reference in industrial production of algal biomass and the rational and efficient treatment of organic wastewater by C.pyrenoidosa.