Diversity of efficient biosurfactant-producing yeasts and biosurfactant production by Cyberlindnera samutprakarnensis sp. nov. JP52T

Abstract

In the course of a survey on the biosurfactants-producing yeasts, 368 strains were isolated from soil, agricultural wastes and samples contaminated with vegetable or palm oils in Thailand. The efficient biosurfactant producing strains were selected by their activities of lowering surface tension to below 40 mN m-1. As a result, 94 strains were selected and further identified by molecular DNA analysis using the D1/D2 sequencing method. Among them, 93.6% were known species, 2.1% were undescribed species and 4.3% were novel yeast strains. Based on the phylogenetic sequence analysis of the D1/D2 region of the large subunit (LSU) rRNA gene, the internal transcribed spacer region (ITS1-5.8S rRNA gene-ITS2; ITS1-2) and their physiological characteristics, the three strains (JP52, JP59 and JP60) were found to represent two novel species of the anamorphic ascomycetous yeast. Strain JP52 represent a novel species which was named Cyberlindnera samutprakarnensis sp. nov. (Type strain JP52T; = BCC46825T = JCM17816T = CBS12528T, MycoBank no. MB800879), it is classified as a members of the Cyberlindnera clade which was different from the closely related species, Cyberlindnera mengyuniae CBS10845T by 2.9% and 4.4% sequence divergence in the D1/D2 region and ITS1-2, respectively. Strain JP59 and JP60 were identical in the D1/D2 region and ITS1-2 which were closely related to Scheffersomyces spartinae CBS6059T by 0.9 and 1.3% sequence divergence respectively. Gene analysis of 6.5 and 4.8% of sequence divergence in the actin and translational elongation factor gene and their differences from the closely related species in some biochemical and physiological characteristics were also supported a distinct species. These two strains were assigned as a single novel species which was named Candida thasaenensis sp. nov. (Type strain JP59T = BCC46828T = JCM17817T = CBS12529T, MycoBank no. MB800880). In this work, Cyberlindnera samutprakarnensis JP52T was found to be an efficient biosurfactant-producing yeast that showed strong activity of surface tension reduction and oil displacement test when cultured in glucose and palm oil medium (medium broth consisted of 0.02% KH₂PO₄, 0.02% MgSO₄.7H₂O, 0.4% NaNO₃, 0.1% yeast extract, 2% (w/v) glucose and 2% (v/v) palm oil), initial pH 5.5. The optimum cultivation was performed at 30 °C in shake flask at 200 rpm. After 7 days of cultivation the biosurfactant produced possessed minimum surface tension of 30.9 mN m-1, the critical micelle concentration (CMC) of 457.1 mg L-1 and yield of 1.89 g L-1. The physicochemical studies of the product obtained revealed that this biosurfactant has optimum pH as well as pH stability at pH 8.0. It was stable at high temperature up to 121 °C and could tolerate to NaCl at 1.0-10.0%. In addition, it could form emulsion with various oils including sesame oil, palm oil, olive oil, safflower oil, cotton seed oil and salad oil by showing E24 value over 80%. For purification, thin layer chromatography revealed that strain JP52T produced three major biosurfactants in GP medium, namely F1GP, F2GP and F3GP with Rf values of 0.89, 0.77 and 0.69, respectively. F3GP gave the highest oil displacement activity and was glycolipid positive by the Molish test. Structural analyses by MALDI-TOF MS and NMR suggested that F3GP contained sophorolipid-type biosurfactants with molecular weights of 574 and 662 that are likely to be non acetylated lactonic sophorolipids (SL-C18) and either a monoacetylated acidic sophorolipid (SL-Ac-C19:1) or diacetylated lactonic sophorolipids (SL-Ac-Ac-C16), respectively. We propose Cyberlindnera samutprakarnensis JP52T as new sophorolipid biosurfactant-producing yeast that produce attractive biosurfactants for diverse applications.