Study Of Rhodotorula Glutinis Producing Carotenoid Using Raman Spectroscopy

Raman spectroscopy is a kind of molecular vibration spectrum which can reflect the information about the structure and content of sample, primarily caused by light scatting of molecule and with the advantages of non-destructive, rapid and real-time analysis. In recent years, as a new spectroscopy analysis method, Raman spectroscopy technology has important value of application in the field of physics, chemistry, material and life science. Micro Raman spectroscopy has high spatial resolution. When analyzing samples, the incident laser is focused on the sample by microscope, thus one can obtain precisely the Raman spectrum information of chemical composition, molecular structure, molecular orientation and molecular interactions in a small sample area without interference by the surrounding material. Laser Tweezers Raman Spectroscopy (LTRS) is a kind of optical technology combining Raman micro spectroscopy and laser optical tweezers which fuses optical trap manipulation with the advantages of quick and non-destructice of Raman spectra. It can capture and fix single cells in the solution for a long time so that one can detect and analyze the real-time biochemical kinetic process of single cells, and can also monitor the production of microorganism. Hence, it provides a new detection technology for microbial fermentation industry.Carotenoids are natural pigments, hundreds of millions of tons of natural production are produced annually, which endow variety of life with beautiful color and also participated in some physiological function, such as photic-energy transfer and substance transformation in photosynthesis, quenching singlet oxygen and resisting photosensitive. As an example, ultraviolet radiation threats to human health and ecosystem which caused by ozone hole, carotenoids might have protective effect. On the other hand, carotenoids as an important source of nutrition, can prevent tumors, cancer as well as improve immunity and health care. They have been widely used in many fields, such as medicine, foodstuff, breed and fodder.Carotenoids are C40terpenoid compounds, its chemical properties is not very stable, but one of the main problems in the analysis process is carotenoids is susceptible to light, oxygen and acid, so we must pay attention to it. In traditional analysis methods, the extraction process to a great extent affected the accuracy of test results, in contrast, LTRS can obtain rapidly accurate results without process sample, which is an effective tool for carotenoids analysis. In this thesis, the author designs the experiment based on LTRS as well as MicroRaman spectroscopy technology and some experiment design method. The main work is as follows:1. A method is established based on micro-Raman spectroscopy in situ quantitative detection technology for the screening of carotenoid high-producing mutants from Rhodotorula glutinis colonies growing on solid culture medium.In order to eliminate the interference of medium background signal with the quantitative results,a mathematical routine involving vector correction algebra,is described for the preprocessing of spectra data,which is expected to obtain pure bacterial spectrum regardless of the amount of medium interference signal.Furthermore, the effect of two different spectral collection mode, area-scan excitation and fixed-point detection,on quantification repeatability is investigated. The results show that spectral signal collected with area-scan excitation mode can cover larger areas of microcolonies and possess better repeatability than that with fixed-point detection. Then, confocal micro-Raman spectroscopy is applied to estimate carotenoid content of Rhodotorula glutinis colonies on solid culture medium, which is verified by detecting single cells carotenoid level from the same colony using laser tweezers Raman spectroscopy. A good correlation between carotenoid Raman peak intensity at1512cm-1by above two approaches is observed, and the correlation coefficient is0.9395. The data indicate that confocal micro-Raman spectroscopy is a reliable approach for the quantitative analysis of carotenoids in Rhodotorula glutinis colonies on solid culture medium. Finally, two carotenoid high-producing variants are obtained with the screening procedure established above. This evidence confirms that the strains screening method based on quantitative detection is more objective and more efficient than conventional visual inspection.2. Laser Tweezers Raman Spectroscopy (LTRS) was applied to detect the carotenoid content in Rhodotorula glutinis cells. Based on this, Plackett-Burman design was applied for evaluating the effects of related factors on carotenoid production of Rhodotorula glutinis fermentation. Results showed that the factors including glucose,tryptone, pH and temperature presented the most major affecting on carotenoid yield. Then Box-Benhnken Design combined with response surface methodology was employed to optimize the most important effect factors of carotenoid yield. The optimal fermentation conditions for high carotenoid production consisted of42.86g/L glucose,5g/L tryptone,5g/L yeast extract,1g/L potassium dihydrogen phosphate,0.5g/L Magnesium sulfate, pH7.0with the temperature at28.3℃. This optimization resulted in an increase of carotenoid yield with an improvment of45%when compared with no optimizing.