The Stimulation Of The Growth And Astaxanthin Accumulation Of Haematococcus Pluvialis Induced By Low-temperature Plasma

Plasma is an ionized gas which is generally considered as the fourth state of matter.Plasma can be divided into thermal plasma and low-temperature plasma?LTP?.Low-Temperature Plasma can produce a large amount of active substances such as OH,O₂-,H₂O₂,O₃ and charged particles,and because it maintains at low temperature,thus it has good compatibility with heat-sensitive organisms,it have been widely applied in different fields such as agriculture,animal husbandry,microbiology and biomedicine.Astaxanthin is a keto-carotene with various functions such as coloring and antioxidant,so it is widely used in aquaculture,food,cosmetics,health care,pharmaceutical and other industries.In the nature world,many organisms can accumulate astaxanthin,such as bacteria,fungi,plants and microalgae.Among them,Haematococcus pluvialis is considered to be the best microalgae with the highest accumulation of astaxanthin.Although many techniques have been developed to improve the accumulation of Astaxanthin in H.pluvialis,the slower growth of H.pluvialis is still the main factor limiting the large-scale application of H.pluvialis.Therefore,seeking and developing new techniques and methods to improve the growth rate of H.pluvialis and the production of astaxanthin are still very demanded.In this dissertation,firstly,we compared and analyzed the mechanism of high light stress on astaxanthin production in H.pluvialis from the aspects of photosynthetic ability,pigment and lipid composition and expression of genes,and we used the LTP technique to explore the appropriate conditions that could stimulate the growth and astaxanthin accumulation of H.pluvialis,and adopted transcriptomic methods combined with chlorophyll fluorescence analysis and immunoassay to explore the potential mechanism for stimulation of H.pluvialis induced by LTP.Moreover,we analyzed cell morphology,modes of cell death and changes of redox state in yeast cells to illustrate the LTP interaction with the microorganisms.The main research results are summarized as follows:1?The transcriptome sequencing and metabolic analysis were performed to study a astaxanthin-hyperproducing H.pluvialis mutant M3,which was obtained from LTP,revealing the reason for the differences in astaxanthin accumulation between M3 mutant strain and WT strain under high light stress.Our results indicated that M3 strain had higher utilization efficiency of CO2 to provide the precursors of carotenoid and fatty acid biosynthesis by increasing the expression levels of phosphoenolpyruvate carboxylase?PEPC?,malate dehydrogenase?MDH?,malate dehydrogenase?ME?,ribulose bisphosphate carboxylase/oxygenase?Rubisco?activase?RCA?and glyceraldehyde 3-phosphate dehydrogenase?GAPDH?while decreasing the expression levels of fructose-1,6-bisphosphatase?FBP?.The analysis of pigments,chlorophyll fluorescence and qRT-PCR analysis revealed that M3 strain maintained higher photosynthetic activity by regulating chlororespiration pathway and elevating non-photosynthetic pigment?lutein,?-carotene,and astaxanthin?content to alleviate photooxidative damage.Overall,the combinative analysis of de novo transcriptomic and physiological data provided information necessary for not only a better understanding of the difference in astaxanthin biosynthesis between WT and M3 strains but also the feasibility of genetic engineering of H.pluvialis in the future.2?The stimulation of the growth and astaxanthin accumulation of H.pluvialis by LTP was achieved and the LTP treatment conditions were optimized.Through monitoring by spectrophotometry the changes of biomass and cell number of H.pluvialis cells in the LTP treatment,the results were obtained showing that the biomass and cell numbers of H.pluvialis increased at first and then decreased with the increase of the dose induced by LTP.These results also showed that mild LTP treatment could stimulate the growth of H.pluvialis.In addition,we also found that the LTP treatment could promote the astaxanthin accumulation of H.pluvialis.3?The mechanism of LTP stimulating the growth and astaxanthin accumulation of H.pluvialis was investigated.Through the measurement of chlorophyll fluorescence and biochemical indicators,we found that LTP induced the accumulation of reactive oxygen species?ROS?in H.pluvialis,which triggered the increase in the activity of antioxidant enzymes such as Catalase?CAT?,Glutathione peroxidase?GPx?and Glutathione Reductase?GR?and redox homeostasis in the photosynthetic process,which inhibited the irreversible damage caused by ROS in H.pluvialis.Transcriptomic analysis displayed that LTP regulated the expression of genes related with metabolism,biosynthesis,transport and signal transduction of the phytohormone in H.pluvialis.The results indicated that the LTP could regulate the changes of phytohormone content to stimulate the growth and astaxanthin accumulation of H.pluvialis.The enzyme-linked immunosorbent assay?ELISA?for determining phytohormone revealed that LTP induced an increase in SLs content and decrease in abscisic acid content in H.pluvialis,confirming that phytohormone induced by LTP was the main mechanism for stimulating the growth and astaxanthin accumulation of H.pluvialis.This work not only provided a new technology for improving the growth of H.pluvialis,but also provides a new insight into the role of phytohormone in the growth and astaxanthin accumulation of H.pluvialis.4?A preliminary study on LTP-induced oxidative stress was conducted.The results show that LTP can induce H.pluvialis and Candida utilis cells to undergo different degrees of oxidative stress,thereby inducing different biological effects,such as stimulation effects and mutagenic effects.Taking Candida utilis as a model microbial system,the roles and effects of LTP-induced oxidative stress were explored.The results showed that different LTP treatment times will induce different levels of oxidative stress in the cells,resulting in different degrees of cellular state changes,including changes in the cell's redox state,mitochondrial and other subcellular changes.