High-throughput system for screening of Monascus purpureus high-yield strain in pigment production

Abstract

Background

An economical and integrated high-throughput primary screening strategy was developed for high-aerobic microbe Monascus purpureus cultivation. A novel and effective mixture culture method was proposed and used to realize the whole mutant library being high-throughput screened after mutagenesis.

Results

The good correlation of fermentation results between differing-scale cultivations confirmed the feasibility of utilizing the 48-deep microtiter plates (MTPs) as a scale-down tool for culturing high-aerobic microbes. In addition, the fluid dynamics of 24-, 48-, and 96-deep MTPs and 500-mL shake flask were studied respectively using the computational fluid dynamic (CFD) tool ANSYS CFX 11.0 to get better understanding of their turbulent regimes.

Conclusions

The by-product citrinin production had no significant change while the pigment production had improved. As a result, the high-yield strain T33-6 was successfully screened out and the pigment was more than 50% higher than that of the parental strain in the shake flask.

Keywords

Monascus / High-throughput screening / Mixture cultivation / Computational fluid dynamics

References

[1] Mapari SAS, Nielsen KF, Larsen TO, Frisvad JC, Meyer AS, Thrane U. Exploring fugal biodiversity for production of water-soluble pigments as potential natural food colorants. Curr Opin Biotechnol, 2005, 16: 231-238.

[2] Fabre CE, Santerre AL, Loret MO, Baberian R, Paresllerin A, Goma G, Blanc PJ. Production and food applications of the red pigment of Monascus rubber. J Food Sci, 1993, 58: 1099-1110.

[3] Lee YK, Chen DC, Chauvatcharin S, Seki T, Yoshida T. Production of Monascus pigments by a solid-liquid state culture method. J Ferment Bioeng, 1995, 2: 21-26.

[4] Kim HL, Kim HJ, Oh HJ, Shin CS. Morphology control of Monascus cell and scale up of pigment fermentation. Process Biochem, 2002, 38: 649-655.

[5] Blanc PJ, Loret MO, Goma G. Production of citrinin by various species of Monascus. Biotechnol Lett, 1995, 17: 291-294.

[6] Pisareva E, Savov V, Kujumdzieva A. Pigments and citrinin biosynthesis by fungi belonging to genus Monascus. Z Naturforsch C, 2005, 60: 116-120.

[7] Blanc PJ, Laussac JP, Lebars J, Lebars P, Loret MO, Pareilleux A, Prome D, Prome JC, Santerre AL, Goma G. Characterization of monascidin A from Monascus as citrinin. Int J Food Microbiol, 1995, 27: 201-213.

[8] Li ZQ, Guo F. A further studies on the species of Monascus. Mycosystema, 2004, 23: 1-6.

[9] Kim JY, Kim HJ, Oh JH, Lee IH. Characteristics of Monascus sp. isolated from Monascus fermentation products. Food Sci Biotechnol, 2010, 19: 1151-1157.

[10] Huang L, Wei PL, Zang R, Xu ZN, Cen PL. High-throughput screening of high-yield colonies of Rhizopus oryzae for enhanced production of fumaric acid. Ann Microbiol, 2010, 60: 287-292.

[11] Betts JI, Doig SD, Baganz F. Characterization and application of a miniature 10 mL stirred-tank bioreactor, showing scale-down equivalence with a conventional 7 L reactor. Biotechnol Prog, 2006, 22 3 681-688.

[12] Gao H, Liu M, Zhou XL, Liu JT, Zhuo Y, Gou ZX, Xu B, Zhang WQ, Liu XY, Luo AQ, Zheng CS, Chen XP, Zhang LX. Identification of avermectin-high-producing strains by HTS methods. Appl Microbiol Biotechnol, 2010, 85: 1219-1225.

[13] Harms P, Kostov Y, French JA, Soliman M, Anjanappa M, Ram A, Rao G. Design and performance of a 24-station high throughput microbioreactor. Biotechnol Bioeng, 2006, 93: 6-13.

[14] Chen A, Chitta R, Chang D, Amanullah A. Twenty-four well plate miniature bioreactor system as a scale-down model for cell culture process development. Biotechnol Bioeng, 2008, 102 1 148-160.

[15] Buchs J. Introduction to advantages and problems of shaken cultures. Biochem Eng J, 2001, 7: 91-98.

[16] Du Toit EA, Rautenbach M. A sensitive standardised micro-gel well diffusion assay for the determination of antimicrobial activity. J Microbiol Methods, 2000, 42: 159-165.

[17] Kumar MS, Kumar PM, Sarnaik HM, Sadhukhan AK. A rapid technique for screening of lovastatin-producing strains of Aspergillus terreus by agar plug and Neurospora crassa bioassay. J Microbiol Methods, 2000, 40: 99-104.

[18] Hermann R, Lehmann M, Büchs J. Characterization of gas-liquid mass transfer phenomena in microtiter plates. Biotechnol Bioeng, 2003, 81 2 178-186.

[19] Bujalski JM, Jaworski Z, Bujalski W, Nienow AW. The influence of the addition position of a tracer on CFD simulated mixing times in a vessel agitated by a Rushton turbine. Chem Eng Res Des, 2002, 80: 824-831.

[20] Lane GL, Schwarz MP, Evans GM. Numerical modelling of gas-liquid flow in stirred tanks. Chem Eng Sci, 2005, 60: 2203-2214.

[21] Min J, Gao ZM, Shi LT. CFD simulation of mixing in a stirred tank with multiple hydrofoil impellers. Chin J Chem Eng, 2005, 13: 583-588.

[22] Hristov HV, Mann R, Lossev V, Vlaev SD. A simplified CFD for three dimensional analysis of fluid mixing, mass transfer and bioreaction in a fermenter equipped with triple novel geometry impellers. Food Bioprod Process, 2004, 82: 21-34.

[23] Raimondi MT, Moretti M, Cioffi M, Giordano C, Boschetti F, Lagana K, Pietrabissa R. The effect of hydrodynamic shear on 3D engineered chondrocyte systems subject to direct perfusion. Biorheology, 2006, 43: 215-222.