Aims: Rhamnolipids are seeking utmost attention as a new class of biosurfactants having promising potential in diverse fields as they offer a wide range of advantages over chemically synthesised surfactants. However, the high extraction costs make large scale production face difficulty. In present study, hydrocarbon degrading bacteria Pseudomonas aeruginosa UKMP14T was exploited for its biosurfactant producing ability including a comparative study between different extraction procedures for its recovery. In addition to this, the recovered biosurfactant was explored for its potential application as an antimicrobial agent. Methodology and results: The production of rhamnolipid biosurfactant was confirmed through various detection methods which are drop-collapse test, oil spreading assay, emulsification index, cetyltrimethylammonium bromide (CTAB) assay and hemolytic assay. The test strain P. aeruginosa UKMP14T showed positive results for all the detection assays. Following this, shake flask cultivation was carried out for several time intervals (1, 3, 5, 7 and 9 days) to discover the optimum time for rhamnolipid biosurfactant production. The results were evaluated by quantifying the rhamnolipid yield using Anthrone method and maximum yield was obtained on day 7. Then, three commonly employed rhamnolipid biosurfactant extraction methods (acid precipitation, solvent extraction and zinc sulphate precipitation) were incorporated for the extraction of rhamnolipid biosurfactant. Among these methods, organic solvent extraction (using methanol, chloroform and acetone in 1:1:1 ratio) gave the highest yield (7.37 ± 0.81 g/L) of biosurfactant, followed by zinc sulphate precipitation (5.83 ± 0.02 g/L), whereas acid precipitation gave the lowest yield (2.8 ± 0.12 g/L) and required longer time (30 days). Finally, the antimicrobial activity of several concentrations of rhamnolipid was tested using modified microdilution method and highest antibacterial activity (in the form of percent reduction in growth) of 95.05% and 91.89% was recorded for Escherichia coli ATCC 10536 and Staphylococcus aureus ATCC 11632, respectively, at 100 µg/mL concentration of rhamnolipid biosurfactant. Conclusion, significance and impact of study The ability of P. aeruginosa UKMP14T in producing rhamnolipid biosurfactant was confirmed. Despite the higher yield obtained by organic solvent extraction method, the recovery technique (involving the separation of solvent system) caused some loss in product. In addition, the transfer and storage of rhamnolipid was challenging using solvent extraction in comparison to acid precipitation and zinc sulphate precipitation. On the other hand, recovery using acid precipitation suffered from lowest yield of rhamnolipid. Therefore, zinc sulphate precipitation is prioritised over the other two methods. Furthermore, the antimicrobial potential of rhamnolipid biosurfactant was tested successfully for as low as 10 µg/mL concentration against E. coli ATCC 10536 and S. aureus ATCC 11632. Therefore, the recovery cost of a high value product like rhamnolipid can be reduced by incorporating the results of this study in the downstream processing and promote rhamnolipid biosurfactant as a potential antimicrobial agent.
Glycolipids--biosynthesis ; Surface-Active Agents ; Pseudomonas aeruginosa

Mannosylerythritol lipids (MELs), mainly produced by Ustilago and Pseudozyma, are surface active compounds that belong to the glycolipid class of biosurfactants. MELs have potential application in food, pharmaceutical and cosmetics industries due to their excellent surface activities and other peculiar bioactivities. In recent years, the research field of MELs has regained much attention abroad. However, MELs are rarely studied in China. In this review, the producing microorganisms and production conditions, diverse structures, biochemical properties, structure-function relationship and biosynthetic pathways of MELs are described. Some research problems and prospects are summarized and discussed as well.
Glycolipids ; biosynthesis ; genetics ; Metabolic Networks and Pathways ; genetics ; Surface-Active Agents ; metabolism ; Ustilaginales ; classification ; genetics ; metabolism ; Ustilago ; genetics ; metabolism

Biosurfactants (BSs) are highlighted owing to their multiple advantages in diverse applications. To screen a superior strain that producing a blend-biosurfactant of lipopeptide and glycolipid, the hemolytic activity assay on blood agar plates, the modified oil-red spreading test and MALDI-TOF Mass Spectrometry identification of the purified products was carried out. Bacillus subtilis THY-7 was selected and its principal products were surfactin and dirhamnolipid. The medium component and culture conditions of THY-7 were optimized by both single factor and orthogonal experiments. After 48 h optimal batch culture in flask, the cell density (OD600) was 37.0 and the product titer was 2.4 g/L, which was 3.4 folds and 3.1 folds of that under original condition, respectively. A fed-batch culture in a 5 L fermentor was further performed coupling with in situ recovery of foam, in which the titer of blend-BS increased to 4.5 g/L at 25 h. Quantification by HPLC and anthrone colorimetry revealed that surfactin and dirhamnolipid accounted for 74% and 22% of the blend-BS, respectively.
Bacillus subtilis ; growth & development ; isolation & purification ; metabolism ; Chromatography, High Pressure Liquid ; Culture Media ; Glycolipids ; biosynthesis ; Industrial Microbiology ; Lipopeptides ; biosynthesis ; Surface-Active Agents ; metabolism

Differences in ability to produce the specific phenolic glycolipid-Tb (PGL-Tb) antigen among Mycobacterium tuberculosis strains have been reported. One of the explanations would be the genotypic variation between the strains. In this study, we compared the DNA fragment patterns after digestion of DNA with various restriction enzymes between the PGL-Tb producing and non-producing strains of M. tuberculosis. Three clinical isolates of M. tuberculosis producing the PGL-Tb antigen detectable by thin-layer chromatography, and M. tuberculosis H37Rv and M. bovis BCG not producing the antigen were grown in Sauton medium. The chromosomal DNA was digested with the restriction endonucleases, Eco RI, Sau3A I, BamH I, Xho I, Sma I, Pst I, Hinc II, and Bst EII. Most of the restriction enzymes used gave no clear DNA bands or no DNA fragment common just to the PGL-Tb producing strains. When DNAs were digested with Bst EII, however, there was a 13 kb DNA fragment common to the PGL-Tb producing isolates of M. tuberculosis and not present in the H37Rv strain and M. bovis BCG. This study thus suggests that there might be differences in DNA fragment patterns between the PLG-Tb producing and non-producing strains of M. tuberculosis.
Base Sequence ; Comparative Study ; DNA Restriction Enzymes ; DNA, Bacterial/*metabolism ; Glycolipids/*biosynthesis ; Molecular Sequence Data ; Mycobacterium tuberculosis/*genetics/metabolism ; Support, Non-U.S. Gov't ; Tuberculosis/microbiology

The cause responsible for the lack of an efficient cell-mediated immunity or a delayed type hypersensitivity to M. leprae in lepromatous patients is poorly understood. But the resistance to M. leprae infection in humans is likely mediated by the activated macrophages to present M. leprae antigen to T cells for cell-mediated immunity. Phenolic glycolipid-I (PGL-I) is a M. leprae-specific antigen and is supposed to play a significant role in the long lasting unresponsiveness in lepromatous leprosy. In this study, IL-1 activities were tested among leprosy patients to evaluate monocyte function and the role of IL-1 in the immunosuppression in leprosy. We found that peripheral blood mononuclear cells (PBMCs) from tuberculoid patients were strongly reactive to M. leprae (mean cpm; 28,853 +/- 28,916), but the proliferative responses of PBMCs from lepromatous patients (mean cpm; 6,051 +/- 803) were significantly lower. IL-1 concentration in culture supernatant of monocytes from lepromatous patients was similar to that from tuberculoid patients with stimulation of M. leprae (lepromatous: 1,014 +/- 637 pg/ml, tuberculoid: 1,012 +/- 167 pg/ml) or lipopolysaccharides (IPS) (lepromatous: 3,479 +/- 2,188 pg/ml, tuberculoid: 4,246 +/- 2,432 pg/ml). The IL-1 concentration is sera from lepromatous patients (42 +/- 30 pg/ml) tended to be higher than those from tuberculoid patients (28 +/- 69 pg/ml). And there was no significant difference in IL-1 production between peritoneal macrophages from mice sensitized with PGL-1 and those from nonsensitized mice. In conclusion, this study suggests that the immunosuppression in lepromatous patients may not be due to the decreased production of IL-1. And the increased IL-1 activity in sera may affect the inflammatory response of lepromatous patients.
Glycolipids/pharmacology ; Human ; Immunity, Cellular ; Interleukin-1/*biosynthesis ; Leprosy, Lepromatous/blood/*metabolism ; Lymphocyte Activation ; Monocytes/*metabolism ; Mycobacterium leprae/metabolism ; Support, Non-U.S. Gov't

Rhamnolipid biosurfactant is mainly produced by Pseudomonas aeruginosa that is the opportunistic pathogenic strain and not suitable for future industrial development. In order to develop a relatively safe microbial strain for the production of rhamnolipid biosurfactant, we constructed engineered Escherichia coli strains for rhamnolipid production by expressing different copy numbers of rhamnosyltransferase (rhlAB) gene with the constitutive synthetic promoters of different strengths in E. coli ATCC 8739. We further studied the combinatorial regulation of rhlAB gene and rhaBDAC gene cluster for dTDP-1-rhamnose biosynthesis with different synthetic promoters, and obtained the best engineered strain-E. coli TIB-RAB226. Through the optimization of culture temperature, the titer of rhamnolipd reached 124.3 mg/L, 1.17 fold higher than that under the original condition. Fed-batch fermentation further improved the production of rhamnolipid and the titer reached the highest 209.2 mg/L within 12 h. High performance liquid chromatography-mass spectrometry (LC-MS) analysis showed that there are total 5 mono-rhamnolipid congeners with different nuclear mass ratio and relative abundance. This study laid foundation for heterologous biosynthesis of rhanomilipd.
Bacterial Proteins ; genetics ; Batch Cell Culture Techniques ; Decanoates ; Escherichia coli ; metabolism ; Fermentation ; Glycolipids ; biosynthesis ; Hexosyltransferases ; genetics ; Industrial Microbiology ; methods ; Multigene Family ; Promoter Regions, Genetic ; Pseudomonas aeruginosa ; Rhamnose ; analogs & derivatives ; biosynthesis ; Surface-Active Agents ; metabolism