Production and characterization of rhamnolipid biosurfactant from thermophilic Geobacillus stearothermophilus bacterium isolated from Uhud mountain

Abstract

Introduction: Biosurfactants have been given considerable attention as they are potential candidates for several biotechnological applications.

Materials and methods: In this study, a promising thermophilic biosurfactant-producing HA-2 was isolated from the volcanic and arid region of Uhud mountain, Madinah, Saudi Arabia. It was identified using 16S rRNA gene sequence analysis. The biosurfactant production ability was screened using different methods such as the drop collapse test, oil spreading test, hemolytic activity test, CTAB test, and emulsification index. The ability of rhamnolipid production by the tested strain was confirmed by the polymerase chain reaction (PCR) of rhlAB. The affinity of thermophilic HA-2 to hydrophobic substrates was also investigated. Optimization of biosurfactant production was conducted. The biological activities of produced surfactant were investigated.

Results and discussion: The isolated HA-1 was identified as Geobacillus stearothermophilus strain OR911984. It could utilize waste sunflower frying oil (WSFF) oil as a low-cost carbon source. It showed high emulsification activity (52 ± 0.0%) and positive results toward other biosurfactant screening tests. The strain showed high cell adhesion to hexane with 41.2% cell surface hydrophobicity. Fourier-transform infrared (FTIR) spectra indicated the presence of hydrophobic chains that comprise lipids, sugars, and hydrophilic glycolipid components. The optimization results showed the optimal factors included potato peel as a carbon source with 68.8% emulsification activity, yeast extract as a nitrogen source with 60% emulsification activity, a pH of 9 (56.6%), and a temperature of 50° (72%). The kinetics showed that optimum biosurfactant production (572.4 mg/L) was recorded at 5 days of incubation. The produced rhamnolipid biosurfactant showed high antimicrobial activity against some human and plant pathogenic bacterial and fungal isolates and high antioxidant activity (90.4%). In addition, it enhanced wheat (Triticum aestivum) growth, with the greatest enhancement obtained with the 5% concentration. Therefore, thermophilic G. stearothermophilus is a promising rhamnolipid biosurfactant producer that utilizes many organic wastes. The produced biosurfactant could be applied as a promising emulsifier, antimicrobial, antioxidant, and plant growth promoter.

Keywords: Geobacillus; antimicrobial activity; biosurfactants; rhamnolipid; rhl gene; thermophilic bacteria.

Figure 1

(A) Agarose gel stained with ethidium bromide resolved the 1.5-Kb PCR-amplified fragment of the 16S rRNA gene sequence from the AH-2 isolate. (B) The maximum likelihood phylogenetic tree constructed based on 16S rRNA gene sequences showed the association between strain HA-2 and the closely related strains within the genus Geobacillus. Bootstrap values (expressed as percentages of 1,000 replications) of more than 50% are shown at the branch points. The scale bar indicates 0.01. Substitutions per nucleotide position. (C) WSFF oil emulsification and biosurfactant production in the MBM at 50°C.

Figure 2

The emulsification index (%) of the produced biosurfactant from the cells of thermophilic AH-2 at different environmental and nutritional conditions; (A) impact of pH, (B) impact of temperature, (C) impact of different nitrogen sources (1%), and (D) impact of different organic carbon sources (1%). Plotted values are the means of triplicate treatments ± standard deviation of the mean. The impact of the revelators corresponding to each treatment and not sharing the same letters are significantly different according to the Fisher LSD method and 95% confidence intervals.

Figure 3

(A) Kinetics of the production of biosurfactants by AH-2 in MBM with 10 mL of the initial volume of the bacterial culture at 50°C. (B) Critical micelle concentration of the biosurfactant. The plotted values are the means of triplicate treatments ± standard error of the mean.

Figure 4

Agarose gel electrophoresis analysis after rhlAB amplification from the bacterial isolates. PCR-amplified products were run on 1% agarose gel. Lane M indicates 1-kb DNA ladder. Lane 1 indicates the PCR products (770 bp) of the respective bacterial isolate HA-2.

Figure 5

(A) Thin-layer chromatography (TLC): TLC plate run in a solvent system of chloroform: methanol: acetic acid (6.5:1.5:0.2), developed with Molisch reagent (I, standard and II, AH-2 surfactant) showing typical brown color spots of rhamnolipids and with iodine vapor (III, standard and IV, AH-2 surfactant) developed yellow color. (B) FTIR spectrum of the biosurfactants produced by AH-2 on waste sunflower frying oil.

Figure 6

Antibacterial (A) and Antifungal (B) activity of biosurfactants. The inhibition zone diameter was measured against MBM as a control.