PARTIAL CHARACTERIZATION OF BIOSURFACTANTANTS PRODUCED BY HYDROCARBON-DEGRADING Pseudomonas spp

CARMIN ZANGARI; RUBY TERRANOVA; LAURA J. RAIGER IUSTMAN

Congreso; SAIB-SAMIGE 2021; 2021

SAIB- SAMIGE

Availability of hydrophobic compounds is a key factor for their biodegradation. Because of that, the use of surfactants was proposed for Surfactant Enhanced Remediation (SER) or Surfactant Enhanced Oil Recovery (SEOR) and the use of biosurfactants became interesting because of their chemical properties and biodegradability. Previous studies from our group showed that Pseudomonas extremaustralis and Pseudomonas sp. KA-08 were able to produce biosurfactants of different chemical nature using diesel or kerosene as their sole carbon source. In this work we continued the analysis of those compounds, using different growth conditions, carbon sources and extraction methods to improve their production. P. extremaustralis was able to degrade long chain alkanes only when cultures were carried in microaerobiosis, but a recombinant strain carrying a plasmid pGEc47, that contains the alk genes from P. putida GPo1, allowed the use of medium chain alkanes and to develop in aerobic growth conditions. On the other hand, Pseudomonas sp. KA-08 showed to be an excellent xylene and toluene degrader but was unable to use alkanes as carbon source. For P. extremaustralis and P. extremaustralis/ pGEc47 two growth conditions were assayed. Microaerobiosis cultures were carried out in 50 ml E2 minimum medium supplemented with 2% diesel and KNO3 as electron acceptor, in 100 ml capped bottles without agitation. Aerobic cultures (only for P. extremaustralis/ pGEc47) were carried out in the same media but using 50 ml of culture in 500 ml bottles and 280 rpm. To analyze if an extra carbon source could enhance surfactant production, 0.05% glucose addition was also tested. For Pseudomonas sp. KA-08, cultures were grown in aerobiosis with three different carbon sources: 10% kerosene, 0.1% toluene and 1% xylene. After 7 days, cultures were centrifuged, and the supernatants were separated into two halves. One half was filtered with a 0,22μm pore cellulose ester filter and the second half remained without filtration. All the samples were then acidified up to pH 2, left overnight at 4°C and centrifuged at 12000 rpm, 4ºC for 20 minutes. The pellets were resuspended in 1 ml 0.1mM TrisHCl (pH 8), extracted with ethyl acetate and concentrated by Rotavap. Finally, these crude extracts wereresuspended in 0.5mL ethyl acetate and analyzed by TLC. P. extremaustralis and P. extremaustralis/ pGEc47 showed similar glycosidic compounds (Molisch staining), but only P. extremaustralis/ pGEc47 presented also a putative aminoacidic surfactant in the unfiltered samples (Ninhydrin staining). On the other hand, Pseudomonas sp. KA-08 showed glycosidic compounds when it was grown with kerosene or toluene as carbon source. In this case, also unfiltered samples showed spots with different Rf than the filtered ones. Glucose addition seems to have no effect on the produced biosurfactants. This work allows us to continue the study of these compounds and to evaluate their potential as biosurfactants.