| Microalgae,as a new,nutrition-rich and renewable resource,has a very broad prospect of development and application.Among them,Haematococcus pluvialis,Arthrospira platensis,and Euglena gracilis are three common economical algal species.They can produce nutritious metabolites,such as astaxanthin,phycocyanin and paramylon,respectively,which have a lot of physiological functions.However,in their natural growth process,the yield of these metabolites is low,and strictly limited by the growth conditions,such as light,pH and temperature.How to effectively promote the growth of these microalgae and increase the content of nutritious metabolites is a hot topic in the field of microalgae research.In this research,H.pluvialis,A.platensis,and E.gracilis were used as experimental materials.Phytic acid and ferulic acid extracted from argo waste rice bran were selected as growth regulators to investigate their effects on the three species of microalgae during the growth process.The effects on cell growth,cell morphology,photosynthetic pigments content,characteristic metabolites such as astaxanthin,phycocyanin and paramylon content,and intracellular antioxidant enzyme activity were assayed,and the differences between the effects of phytic acid and ferulic acid on different strains were also analyzed,which laid the foundation for large-scale use of natural plant additives to increase the biomass of microalgae and the yield of nutrient metabolites.The specific results are as follows:(1)Effect of phytic acid on H.pluvialis:Different concentrations of phytic acid had significant effects on the cell density,dry weight,cell size,intracellular chlorophyll content,astaxanthin content and antioxidant enzyme activity of Hpluvialis.In 0.001%(v/v)phytic acid treatment group,the cell density and dry weight of H.pluvialis was increased by 16.5%and 12.0%,respectively,but the growth was gradually inhibited as the phytic acid concentration increased.At the concentration of 0.05%(v/v),cell density and biomass were decreased by 47.37%and 32.80%,respectively;phytic acid accelerated the process of cell enlargement and resulted in the larger cell size;In 0.001%(v/v)and 0.01%(v/v)phytic acid treatment groups,the content of intracellular chlorophyll and astaxanthin was not significantly changed.In 0.05%(v/v)phytic acid treatment group,the chlorophyll content was decreased by 20.21%compared with the control group,and the astaxanthin content was increased by 33.83%;the activity of superoxide dismutase(SOD)was increased first and then decreased with increasing phytic acid concentrations,reaching a maximum of 165 U/mg·DW at the concentration of 0.01%(v/v)phytic acid,catalase(CAT)was decreased continuously and dropped to 31 U/g·DW at the concentration of 0.05%(v/v).(2)Effect of ferulic acid on Hpluvialis:The co-solvent dimethyl sulfoxide(DMSO)had an inhibitory effect on the growth of H.pluvialis,and the cell density and dry weight in the solvent control group were decreased by 16.67%and 47.48%,respectively;the process of cell enlargement was accelerated by DMSO,and the intracellular chlorophyll content was decreased by 37.89%,but the astaxanthin content was not changed significantly.The addition of ferulic acid relieved the growth inhibitory effect of DMSO.Compared with the solvent control group,the cell density and dry weight in 500 mg/L ferulic acid treatment group were increased by 45.45%and 103.65%,respectively,but the final cell size was decreased by 16.35%.In addition,the intracellular chlorophyll content was increased by 72.11%,but the astaxanthin content was decreased by 32.47%.SOD activity was slightly increased in the solvent control group but the difference was not significant,and then it was decreased to 88 U/mg·DW in the 500 mg/L ferulic acid treatment group.The CAT activity was significantly decreased under DMSO but in 500 mg/L ferulic acid group treatment group it was returned to the level same as the blank control group.(3)Effect of phytic acid on A.platensis:The effect of phytic acid on the growth,cell morphology,chlorophyll and phycocyanin content,and SOD and CAT activity of A.platensis varied with the concentration.When the concentration of phytic acid was less than 0.05%(v/v),the dry weight,the length and the width of the algal filaments,the spiral number of the algae,chlorophyll content,phycocyanin content,and SOD and CAT activity of A.platensis was not significant.When the phytic acid concentration was increased to 0.05%(v/v),dry weight of A.platensis was decreased by 24.35%compared with the control group.The cells were clustered and morphologically changed.The length of the filaments was 3.93 times that of the control group,and the width of the filaments was not significantly different.The spiral number of the cells was increased to 3.63 times that of the control group.The content of phycocyanin in the cells was not significantly changed.The content of chlorophyll was decreased by 14.08%,and SOD and CAT activity were decreased to 8.3 U/mg·DW and 6.3 U/g·DW,respectively.(4)Effect of ferulic acid on A.platensis:In this study,feruic acid at various concentrations had no significant effect on the cell dry weight,the length and the width of the algal filaments,the spiral number of the algae,chlorophyll content,phycocyanin content,and SOD and CAT activity of A.platensis.(5)Effect of phytic acid on E.gracilis:Phytic acid has a significant promoting effect on the growth of E.gracilis.When the concentration of phytic acid is 0.05%(v/v),the cell density and dry weight of E.gracilis are significantly increased by 67.68%and 52.94%,respectively.The median cell length of E.gracilis was also prolonged under light and dark conditions,indicating a more active cell state.Intracellular chlorophyll a content was increased by 76.88%,while chlorophyll b and carotenoids content were not significantly influenced.Paramylon content was also not significantly influenced.However,due to the increase of the cell density,the final paramylon production was increased by 37.83%compared with the control group.When the phytic acid concentration continued to increase to 0.2%(v/v),the growth promotion effect was slightly decreased,but the cell density and dry weight was still 48.17%and 33.33%higher than those of the control group,respectively.The median cell length was decreased relative to that in 0.05%(v/v)phytic acid treatment group,and the carotenoids content was significantly increased by 60.59%compared with the control group.The SOD activity was decreased with phytic acid concentration and dropped to 41 U/mg·DW at 0.05%(v/v),but then it was increased to 52 U/mg·DW at 0.2%(v/v).The variation trend of CAT acivity was similar as that of SOD activity but it was not significant.(6)Effects of Ferulic Acid on E.gracilis:Ferulic acid could not only alleviate the growth inhibitory effect of the co-solvent DMSO,but also significantly promoted the growth of E.gracilis.The cell density and cell dry weight of E.gracilis in the solvent control group were decreased by 30.64%and 12.90%,respectively,and the median cell length was also decreased in the light and dark conditions.The intracellular chlorophyll a content was also decreased by 29.47%,however,the intracellular paramylon content was increased by 77.37%.With the addition of ferulic acid,the inhibitory effect of DMSO wearkened.In 500 mg/L ferulic acid treatment group,compared with the blank control group the cell density and dry weight were increased by 149.65%and 116.13%,respectively.Compared with the solvent control group,they were increased by 259.93%and 148.15%,and the median cell length was also prolonged in both light and dark conditions.The photosynthetic pigments,especially the chlorophyll a content,were also increased significantly.The variation of paramylon content in the cells was not significant,but the final production of paramylon was increased by 69.05%,due to the increase in the number of cells.The SOD and CAT activities were increased significantly in the solvent control group,but both of them were decreased in 500 mg/L ferulic acid treatment group.The effects of phytic acid and ferulic acid derived from rice bran on the growth and highly nutritious metabolites of microalgae varied with the algal species.In this study,we found for the first time that phytic acid and ferulic acid had a certain role in promoting the growth of H.pluvialis and E.gracilis.It is a green and efficient method for enhancing the yield of microalgae,and it provides a theoretical basis for the large-scale use of additives from argo waste to promote the growth of microalgae and the production of high-nutrition metabolites.In addition,in the study of A.platensis we also found that high concentrations of phytic acid could lead to the aggregation of A.platensis cells,which has potential for application in the harvest of the cells. |
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