Screening of Pseudomonas aeruginosa Strains Highly Producing Rhamnolipid

doi:10.13560/j.cnki.biotech.bull.1985.2015.10.028

Abstract

Abstract: This work aims to screen strains highly producing rhamnolipid from multiple sources, analyze the characterization of fermentation and physicochemical characteristics of rhamnolipid. CTAB（cetyltrimethyl ammonium bromide）methylene blue plate was used for primary screening the strains synthesizing the rhamnolipid. Then the strains were identified by analyzing 16S rRNA sequences, and the property of rhamnolipid was analyzed by TLC（thin layer chromatography）and FTIR（Fourier transform infrared spectroscopy）. Total 163 strains with a dark blue halo around the colony were selected for further analysis of producing rhamnolipid. Among them, 10 strains producing 12.2-17.7 g/L rhamnolipid were identified as Pseudomonas aeruginosa. Moreover, strain B12 yielding highest rhamnolipid was selected and used for the optimization of carbon resource, including glycerol, rapeseed oil, peanut cake or sunflower seed cake, and rapeseed oil was recognized as the optimal carbon source for the synthesis of rhamnolipid. Fermentation temperature was also evaluated at 35℃, 37℃ and 40℃, so the production of rhamnolipid was the highest of 26.8 g/L at 37℃. In addition, rhamnolipid produced by strain B12 was purified and analyzed by TLC and FTIR. In conclusion, strain B12 could synthesize a relatively high level of rhamnolipid and could be a candidate strain for industrial production.

Key words: bio-surfactant, Pseudomonas aeruginosa, rhamnolipid, optimization

Zhang Cuikun, Chang Dongmei, Yang Hongjiang. Screening of Pseudomonas aeruginosa Strains Highly Producing Rhamnolipid[J]. Biotechnology Bulletin, 2015, 31(10): 177-183.

References

[1] Thavasi R, Nambaru VR, Jayalakshmi S. Biosurfactant production by Pseudomonas aeruginosa from renewable resources[J]. Indian Journal of Microbiology, 2011, 51（1）：30-36.
[2] Markus MM, Barbara H, Michaela K, et al. Evaluation of rhamnolipid production capacity of Pseudomonas aeruginosa PAO1 in comparison to the rhamnolipid over-producer strains DSM 7108 and DSM 2874[J]. Applied Microbiology Biotechnology, 2011, 89（7）：585-592.
[3] Rahman KS, Rahman TJ, McClean S, et al. Rhamnolipid biosurfactant production by strains of Pseudomonas aeruginosa using low-cost raw materials[J]. Biotechnology Progress, 2002, 18：1277-1281.
[4] Makkar RS, Cameotra SS. Biosurfactant production by microorganisms on unconventional carbon sources-a review[J]. J Surf Det, 1999, 2：237-241.
[5] 包木太, 张金秋, 张娟, 等. 产糖脂类生物表面活性剂菌株鉴定及发酵条件优化[J]. 环境工程学报, 2013, 7（1）：365-370.
[6] Nur AM, Amirul AA, Mohamad NM. Rhamnolipid produced by Pseudomonas aeruginosa USM-AR2 facilitates crude oil distillation[J]. The Journal of General and Applied Microbiology, 2012, 58：153-161.
[7] Siegmund I, Wagner F. New method for detecting rhamnolipids excreted by Pseudomonas species during growth on mineral agar[J]. Biotechnology Technology, 1991, 5：265-268.
[8] Pinzon N, Ju LK. Improved detection of rhamnolipid production using agar plates containing methylene blue and cetyl trimethylammonium bromide[J]. Biotechnology Letter, 2009, 31：1583-1588.
[9] Benincasa M, Accorsini FR. Pseudomonas aeruginosa LBI production as an integrated process using the wastes from sunflower-oil refining as a substrate[J]. Bioresource Technology, 2008, 99：3843-3849.
[10] Sambrook J, Fritsch EF, Maniatis T. Molecular cloning：A laboratory manual[M]. 2nd ed. New York：Cold Spring Harbor Laboratory Press, 1989：20-25.
[11] Kutter E. Bacteriophages：biology and applications[J]. Boca Raton, FL：CRC Press, 2004：528.
[12] Zhang JH, Madden TL. Power BLAST：A new network BLAST application for interactive or automated sequence analysis and annotation[J]. Genome Research, 1997, 7（6）：649-656.
[13] Tamura K, Peterson D, Peterson N, et al. MEGA5：Molecular evolutionary genetics analysis using maximum likelihood, evolutionary distance, and maximum parsimony methods[J]. Molecular Biology and Evolution, 2011, 28：2731-2739.
[14] Zhao JF, Wu YJ, Akateh TA, et al. Chemical structures and biological activities of rhamnolipid biosurfactants produced by Pseudomonas aeruginosa M14808[J]. Journal of Chemical and Pharmaceutical Research, 2013, 5（12）：177-182.
[15] Dwivedi S, Singh BR, Al KA, et al. Biodegradation of isoproturon using a novel Pseudomonas aeruginosa strain JS-11 as a multi-functional bioinoculant of environmental significance[J]. Journal of Hazardous Materials, 2011, 185（2-3）：938-944.
[16] Abalos A, Pinazo A, Infante MR, et al. Physicochemical and antimicrobial properties of new rhamnolipids produced by Pseudomonas aeruginosa AT10 from soybean oil refinery wastes[J]. Langmuir, 2001, 17（5）：1367-1371.
[17] Thavasi R, Jayalakshmi S, Balasubramanian T, et al. Biosurfactant production by Corynebacterium kutscheri from waste motor lubricant oil and peanut oil cake[J]. Letters in Applied Microbiology, 2007, 45（1）：686-691.
[18] George S, Jayachandran K. Production and characterization of rhamnolipid biosurfactant from waste frying coconut oil using a novel Pseudomonas aeruginosa D[J]. Journal of Applied Microbiology, 2013, 114（2）：373-383.
[19] Li AH, Xu MY, Sun W, et al. Rhamnolipid production by Pseudomonas aeruginosa GIM 32 using different substrates including molasses distillery wastewater[J]. Applied Biochemistry Biotechnology, 2011, 163（5）：600-611.
[20] Milena GR, Ahmad MA, Eric DZ. Comparative analysis of rhamno-lipids from novel environmental isolates of Pseudomonas aeruginosa[J]. Journal of Surfactants and Detergents, 2013, 16（3）：673-682.
[21] Wadekar SD, Kale SB, Lali AM, et al. Microbial synthesis of rhamnolipids by Pseudomonas aeruginosa（ATCC 10145）on waste frying oil as low cost carbon source[J]. Preparative Biochemistry and Biotechnology, 2012, 42（3）：249-266.