The Influence Of Low Intensity Ultrasonic Irradiation Treatment On The Proliferation Of Candida Tropicalis And Its Mechanisms

One of the most important problems in microbial fermentation is big quantity of seed liquid inoculation and high vaccination costs. Some studies found that appropriate preprocess seed liquid by low intensity ultrasound could significantly increase the content of seed solution. However, the mechanism of ultrasound promoting microbial growth is lack of systematic research, which is needed to demonstrate its biological mechanisms. In this study, Candida tropicalis was used as the object to be stimulated by ultrasonic field and the biological changes of proliferation, morphology, cell membrane were studied. Also, the molecular mechanism of the transcriptome sequencing was investigated to explain how the low intensity ultrasonic regulate the cell cycle of Candida tropicalis. The main results are as follows:(1) The ultrasonic treatment on the Candida tropicalis and Saccharomyces cerevisiae were studied. It showed that the proliferation was affected by growth phase,ultrasonic power intensity, ultrasonic frequency and processing time, etc. The optimum ultrasonic treatment conditions for the maximum biomass addition of Candida tropicalis were determined as follows: 28 kHz ultrasound irradiation with power intensity 120 W/L, Candida tropicalis culture at mid logarithmic phase, and treating for 1 hour. Under these conditions, the Candida tropicalis biomass addition reaches to 148.5%. Meanwhile, the optimum conditions of Saccharomyces cerevisiae were 68 kHz ultrasound irradiation with power intensity 120 W/L, culture at early logarithmic phase, and treating for 2 hour. Under these conditions, the Candida tropicalis biomass addition reaches to 44.2%. The results showed that effects of proliferation and stability on Candida tropicalis were better than on Saccharomyces cerevisiae under ultrasonic treatment. So Candida tropicalis was selected for further study.(2) The effects of ultrasonic treatment on the morphology, enzyme activity, cellmembrane permeability and intracellular Ca2+ concentration of Candida tropicalis were investigated. Under the optical microscope, the hyphae were found to be significantly longer than the control and the number of mycelium increased,surrounded by the ball types of Candida tropicalis. Ultrasound has little effect on alkaline protease and neutral protease, but has obvious influence on acid protease in ultrasound after 5 hours(20%). Under conditions of ultrasound promoting Candida tropicalis proliferation, the protease activity did not have many changes. After ultrasonic treatment, the seepage concentration of protein and nucleic acid of the cells gradually increased, 2 hours attached to top(protein increased 5.8% and nucleic increased 12.4%), and then decreased to initial level after 4 hours treatment, after that the concentration values increased again, when treatment time was prolonged to 5 and6 hours.Fluo-4/AM fluorescent probe method was used to explore the transmembrane behavior of intracellular Ca2+ under ultrasonic treatment. The optimal load concentration of Fluo-4/AM fluorescent probes in Candida tropicalis was 7 μmol/L,the incubation time was 60 min and the incubation temperature was 30℃. After a short time of low intensity ultrasound ultrasonic treatment, the number of Candida tropicalis colony increased, while the Ca2+ fluorescence intensity decreased(the maximum value was decreased to 25.8% after 2 hours). But more longer ultrasonic treatment time(about 5 hours) may cause biomass decreased and the Ca2+fluorescence intensity increased. It showed that ultrasonic treatment could change the permeability of the cell membrane and short time ultrasound was good for cell proliferation.(3) Transcriptome sequencing results showed that the influence of low intensity ultrasonic irradiation treatment on the proliferation of Candida tropicalis has a way on gene ontology. The genes involved were CTRG-02711, CTRG-03249,They refer to the cell membrane, Golgi apparatus, signal transduction, cell cycle regulation and DNA replication initiation. So we assumed that the ultrasound effects Candida tropicalis began from cells membrane, gradually extend to the cell interior,and finally affect the DNA replication. Through the way, the corresponding gene and pathway were upregulated, which casued profliferation of Candida tropicalis.It was speculated that CTRG-01717(phosphatidylinositol 3-kinase TOR2) was the key genes of proliferation combined with the differential expression analysis. The expression of TOR2 gene increased 14.4 times. The TOR2 gene related to cell cycle regulation, the express protein product was phosphatidylinositol 3 kinase(PI). Also, it belongs to Protein serine/threonine kinase activity(GO), PI3K-Akt signaling pathway(KEGG) and AMPK signaling pathway(KEGG). Protein serine/threonine kinase activity was an important regulator of apoptosis. The function of PI3K-Akt signaling pathway is inhibited cell apoptosis and promoted cells proliferation. These all reflected the effect of low intensity ultrasound on the proliferation of Candida tropicalis.