Characterization of bacterial strains of rhamnolipid surfactants from palm kernel cake and its applications

Abstract

Rhamnolipid is a glycolipid surfactant used in various sectors due to its versatile action. The major problem of rhamnolipid production is an expensive substrate and high production microbial strains. With this in mind, a novel substrate from palm waste, palm kernel cake (PKC) was explored for rhamnolipid production using co-culture to maximize the return. A mixture of seven bacterial population was isolated from PKC and labelled as VS1 to VS7. All the isolates were identified as biosurfactant producers through haemolytic assay, drop collapse, surface tension, oil spreading and emulsification index. However, only VS2, VS3, VS5 and VS7 were rhamnolipid producers. Biochemical analysis and 16S rRNA sequence analysis disclosed that they were Enterococcus faceium (VS2), Pantoea ananatis LMG 5342 (VS3), Enterococcus hirae (VS5) and Stenotrophomonas maltophilia K279 (VS7). The selection of co-culture in this investigation was based on the compatibility test with Pseudomonas aeruginosa ATCC 9027, a commercial strain. Isolated bacteria Stenotrophomonas maltophilia K279 was the most compatible bacteria in this study. Out of the eleven screened factors, four factors, namely sucrose, glucose, NaNO3 and KH2PO4, were the most significant components for rhamnolipid production in Plackett Burman experimental design. As PKC functioned as the primary substrate, sucrose was chosen as the co-substrate. One factor at a time (OFAT) experiment showed that PKC (8%), sucrose (4 g/L), NaNO3 (1.4 g/L), KH2PO4 (1.3 g/L), temperature (35C), pH (7) and inoculum size (6%) were the optimum concentrations and conditions required for best rhamnolipid production. Media optimization using Face centered central composite design (FCCCD) showed that sucrose (4.1 g/L), NaNO3 (1.9 g/L) and KH2PO4 (1.29 g/L) produced the highest E24 value indicating maximum rhamnolipid production. Process optimization for aeration and agitation in a bioreactor using 2k factorial design indicated that aeration of 1 vvm and agitation above 250 rpm was suitable for maximum production of rhamnolipid. An increase of 25% in rhamnolipid recovery was recorded with mixed culture compared to using a single strain in a production comparison study. The brown viscous extract showed the presence of mono-rhamnolipid with a Rf value of 0.70 in TLC analysis. The presence of hexadecanoic acid, methyl ester, was the fatty acid detected in GS-MS analysis for our rhamnolipid. Both 1H NMR and 13C NMR detected the presence of rhamnose ring in the chromatogram. In vitro antibacterial experiment showed that rhamnolipid was more potent towards Gram negative bacteria compared to Gram positive bacteria. Likewise, rhamnolipid recovered in this study successfully removed 91.3% (Zn), 91% (Cu) and 90.7% (Fe) at 10 ppm that is common in agriculture soil.