Study on Measurement Method of Rhodobacter Sphaeroides Based on Plate Counting
LI Long-quan1,2,WANG Zi-quan2,LIU Si-yuan2,ZHOU Guo-ping1,SUI Zhi-wei2,ZHANG Ling2,ZHEN Xiao-xiao3,HUANG Wen-feng2,XU Qian2,4
1. School of Biology and Pharmaceutical Engineering, Wuhan Polytechnic University, Wuhan, Hubei 430023, China
2. Center for Advanced Measurement Science, National Institute of Metrology, Beijing 100029, China
3. Beijing Institute of Metrology, Beijing 100029, China
4. College of Life Sciences,Hunan Normal University, Changsha, Hunan 410006, China
Abstract：Rhodobacter sphaeroides is one of the main microorganisms producing coenzyme Q10. The quantitation detection is of great significance for optimizing the fermentation conditions of Rhodobacter sphaeroides. In this study, the plate counting method of Rhodobacter sphaeroides was optimized by comparing the inoculation method, the inoculation volume, the type of culture medium, and the culture temperature. Additionally, the optimized method was validated by collaborative experiments and uncertainty was analyzed. The final optimization result of the plate counting method is: the inoculation method is coating method, the inoculation amount is 50μL, the culture medium is Nutrient Agar, and the culture temperature is 32℃. The optimized method possessed a good repeatability and reproducibility, of which the RSD of within-laboratory repeatability is 6.0%, and inter-laboratory RSD is 16.4%. With relative expanded uncertainty of 9.2% (k=2), the optimized method could play an important role in the quantitation of Rhodobacter sphaeroides.
Lu W Q, Ye L D, Lü X M, et al. Identification and elimination of metabolic bottlenecks in the quinone modification pathway for enhanced coenzyme Q10 production in Rhodobacter sphaeroides［J］. Metabolic engineering, 2015, 29:208-216
Fu J L, Qian D W, Wu C Q, et al. Screening of high CoQ10 producing Rhodobacter sphaeroides by chemical mutagenesis and its fermentation medium optimization［J］. China Brewing, 2016, 35(6):90-95.
Xu M, Jiang X Z, Huang J Z, et al. Reinforcement of Rhodobacter sphaeroides cofactor NADPH to increase the production of farnesol［J］. Chinese Journal of Biotechnology, 2020, 36(1):90-99.
Xie X, Yang J L, Liu X Y. Optimizing the Determination Conditions of Total Bacterial Colonies in Soil［J］. Chinese Agricultural Science Bulletin, 2020, 36(11):92-95.
Xue L, Lin J, Sui Z W, et al. Preparation of Reference Material for Quantitative Detection of Escherichia coli［J］. Acta Metrologica Sinica, 2015, 36(6):652-656.
［7］
Laverty D J, Kury A L, Kuksin D, et al. Automated quantification of budding Saccharomyces cerevisiae using a novel image cytometry method［J］. Journal of Industrial Microbiology & Biotechnology, 2013, 40(6):581-588.
Parenteral Drug Association. Technical Report No.33: evaluation, validation and implementation of new as microbiological testing methods［R］. PDA, 2000.
Chen M F, Fu B Q, Lin J, et al. Development of Quantitative Reference Materence of Enterobacter Cloacae［J］. Acta Metrologica Sinica, 2017, 38(5):527-531.
Zhang N, Ren D, Li Y J. Study on Detection Technology of Total Bacteria in Milk and Dairy Products［J］. Modern Food, 2018(13):81-82.
Chen F F. Research on Development of Global Coenzyme Q10 Technology Based on Patent Intelligence Analysis［J］. China Invention & Patent, 2019, 16(2):52-58.
［2］
Dhanasekaran M, Ren J. The Emerging Role of Coenzyme Q10 in Aging, Neurodegeneration, Cardiovascular Disease, Cancer and Diabetes Mellitus［J］. Current Neurovascular Research, 2005, 2(5):447-459.
［3］
Littarru G P, Tiano L. Clinical aspects of coenzyme Q10: An update［J］. Current Opinion in Clinical Nutrition and Metabolic Care, 2005, 8(6):641-646.
［5］
Yen H W, Shih T Y. Coenzyme Q10 production by Rhodobacter sphaeroides in stirred tank and in airlift bioreactor［J］. Bioprocess and Biosystems Engineering, 2009, 32(6):711-716.
Han Q L, Zhao Z Y, Zhou Y S, et al. Optimization of Fermentation Conditions for Rhodobacter sphaeroides by Sweet Potato Starch Wastewater［J］. Anhui Agricultural Science Bulletin, 2012, 18(13):35-38.
［13］
Tong X M, Oh Eun K, Lee B H, et al. Production of long-chain free fatty acids from metabolically engineered Rhodobacter sphaeroides heterologously producing periplasmic phospholipase A2 in dodecane-overlaid two-phase culture［J］. Microbial Cell Factories, 2019, 18(1):20.
Mougiakos I, Orsi E, Ghiffary M R, et al. Efficient Cas9-based genome editing of Rhodobacter sphaeroides for metabolic engineering［J］. Microbial Cell Factories, 2019, 18(4):89-561.
［15］
Chen Y G, Li M, Meng F S, et al. Optimal poly (3-hydroxybutyrate/3-hydroxyvalerate) biosynthesis by fermentation liquid from primary and waste activated sludge［J］. Environmental Technology, 2014, 35(13-16):1791-801.
Sui Z W,Liu X X,Wang J, et al. Study on Measurement Method of Serratia Marcescens［J］. Acta Metrologica Sinica, 2018, 39(4):588-592.
2023, Vol. 44 
