Two protocols for the detection of oleaginous bacteria using Oil Red O.

The selection of oleaginous bacteria, potentially applicable to biotechnological approaches, is usually carried out by different expensive and time-consuming techniques. In this study, we used Oil Red O (ORO) as an useful dye for staining of neutral lipids (triacylglycerols and wax esters) on thin-layer chromatography plates. ORO could detect minimal quantities of both compounds (detection limit, 0.0025 mg of tripalmitin or 0.005 mg of cetylpalmitate). In addition, we developed a specific, rapid, and inexpensive screening methodology to detect triacylglycerol-accumulating microorganisms grown on the agar plate. This staining methodology detected 9/13 strains with a triacylglycerol content higher than 20% by cellular dry weight. ORO did not stain polyhydroxyalkanoates-producing bacteria. The four oleaginous strains not detected by this screening methodology exhibited a mucoid morphology of their colonies. Apparently, an extracellular polymeric substance produced by these strains hampered the entry of the lipophilic dye into cells. The utilization of the developed screening methodology would allow selecting of oleaginous bacteria in a simpler and faster way than techniques usually used nowadays, based on unspecific staining protocols and spectrophotometric or chromatographic methods. Furthermore, the use of ORO as a staining reagent would easily characterize the neutral lipids accumulated by microorganisms as reserve compounds. KEY POINTS: • Oil Red O staining is specific for triacylglycerols • Oil Red O staining is useful to detect oleaginous bacteria • Fast and inexpensive staining to isolate oleaginous bacteria from the environment.

Isolation, characterization and optimization of oleaginous Providencia vermicola as a feedstock for biodiesel production using Response Surface Methodology.

Oleaginous organisms accrue more than twenty percent of their biomass as lipids and hence are promising feedstocks for biodiesel production. In this study, lipid accumulating bacteria were isolated from diesel-contaminated soils and screened with Sudan black B stain. The most oleaginous was done using 16s rRNA gene sequencing. Lipid production was initially optimized based on media, nitrogen source, pH and temperature. Response surface methodology (RSM) was then employed for the enhancement of lipid weight and content. Obtained lipid was converted to biodiesel using direct transesterification, and both lipid and biodiesel were characterized using FTIR. A total of thirteen bacteria were isolated and the most prominent lipid producer was identified as Providencia vermicola with lab number BA6. Preliminary optimization studies revealed optimum lipid production when nutrient broth and acetic acid served as carbon source; KNO3 as nitrogen source, pH 7.0 and 30 °C. Optimization using RSM resulted in a 5.1% and 74.1% increase in the biomass and lipid content of BA6 respectively. FTIR analyses confirmed the presence of functional groups characteristic of lipids and biodiesel. P. vermicola is a novel oleaginous organism that represents a promising feedstock for biodiesel production.HIGHLIGHTSThe bacterium designated as BA6 identified as Providencia vermicola has the highest lipid contents of the oleaginous bacteria isolated.It accumulates lipids up to 47.73 % of its biomassThe percentage lipids accumulation increased to about 74 % when RSM was used.Providencia vermicola is being reported as an oleaginous organism for the first time.