Gupta, R. (2012) Biosynthesis of novel sophorolipids using candida bombicola ATCC 22214: characterization and applications. PhD thesis, CSIR-National Chemical Laboratory, Pune, India..

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Abstract

Preparation of new sophorolipid (SL) analogues with different functionalities has widespread use in pharmaceutical and industrial applications. SL composition can be modified by using both in vivo and in vitro methods. Different lipophilic substrates have been used by researchers for SL production such as oleic acid, stearic acid, palmitic acid and different vegetable oils. To our knowledge, there is no such report on the analysis of individual SL molecule produced using pure α-linolenic acid (ALA) as a lipophilic substrate. SL production using α-linolenic acid as the lipophilic substrate may become a valuable product of interest. In order to biosynthesize novel SLs using Candida bombicola, Linolenic acid was used as the lipophilic and glucose as a hydrophilic source in the fermentation medium. The present study is expected to provide information on the production under optimized fermentation conditions, purification , characterization and application of the SL derived using α-linolenic acid (ALA) as the lipophilic source. The thesis will be divided in to the following chapters: Chapter 1: General Introduction The first chapter is the general introduction of the thesis and it gives detailed literature survey, significance of the sophorolipids (SLs) and objectives of the study. It also describes the biosynthesis and various industrial applications of different sophorolipids derived using various lipophilic substrates. Chapter specific introduction will be discussed in respective chapters. Chapter 2: Optimization of Fermentation parameters for production of linolenic acid derived Sophorolipids from Candida bombicola ATCC 22214 Sophorolipid production is regulated by various parameters of culture condition and media components. This chapter describes the fermentation parameters of Candida bombicola (ATCC 22214) for the production of linolenic acid derived Sophorolipids. These parameters included testing of different medium, medium pH, temperature, inoculum size and age, glucose, fatty acid and yeast extract concentration etc. as well as time kinetics of SL and biomass production. Chapter 3: Production and Purification of Linolenic acid derived Sophorolipids The sophorolipid was produced using optimum fermentation condition in shake flasks. The SL from Candida bombicola is secreted in the culture broth. After fermentation, culture broth was centrifuged and the supernatant was extracted twice with equal volumes of ethyl acetate, the organic layer was dried over anhydrous sodium sulphate and the solvent was removed by rotary-evaporation. The brownish semi-crystalline product (SL mixture) was washed twice with n-hexane to remove un-reacted fatty-acid and was stored at 4°C. Sophorolipid mixture (LNNSL) was analyzed by reverse phase high performance liquid chromatography (HPLC) with a Waters 2487 separation module (Waters Co. Milford, Massachusetts) using 250 × 4.6 mm2 analytical symmetry C18, 5 µm column. The gradient solvent elution profile used was as follows: water/ acetonitrile (95:5, v/v) holding for 10 min; to a final composition of water/acetonitrile (5:95, v/v) with a linear gradient over 50 min and holding for 10 min. The flow rate was 0.5 ml min-1. The peaks were detected at 220 nm wavelength by absorbance detector. Fractions from different peaks were collected and pooled separately in many runs. Further, for the ease of purification, chemical esterification reaction from the literature was followed in order to get single homogenous product (Bisht et al., 1999). Chapter 4: Structural Determination and physical properties of Linolenic acid derived Sophorolipid and its methyl ester form Positive and negative ESI and CID (collision induced dissociation) mass spectra were obtained with an API QSTAR PULSAR hybrid MS/MS quadrupole TOF system (Applied biosystems). Samples from different fractions (collected from HPLC) were injected separately into mass spectrometer for analysis. ESI and CID mass spectral analysis confirmed that the Candida bombicola when grown on glucose and α-linolenic acid produces a mixture of glycolipids consisting of free acid, lactone and diacetylated lactone forms of C18:3 (linolenic acid) SLs as well as diacetylated lactone form of C18:1 SL. The composition of the fermentation product (SL mixture) was 7.5 % free acid, 80 % lactone and 4.5 % diacetylated lactone of C18:3 molecules and 8 % of diacetylated lactone of C18:1 SL molecules. This composition was determined from the initial crude SL loaded on the column. Further, for the ease of purification, chemical esterification reaction from the literature was followed in order to get single homogenous product (Bisht et al. 1999). SL mixture was converted into the sophorolipid methyl ester and ESI-MS analysis confirmed the presence of C18:3 moieties in the fatty acid chain of this product and further the structure of this SL methyl ester (LNNSLME) was confirmed by 1H and 13C nuclear magnetic resonance spectroscopy which showed the presence of ester group in LNNSLME. Physical properties such as surface tension and critical micelle concentration were determined for SL mixture containing 80 % Lactone and its purified sophorolipid methyl ester form and it was found that both the compounds are good surface active agents. Chapter 5: Antibacterial properties of Linolenic acid derived Sophorolipid and its methyl ester form Antibacterial properties of Sophorolipid mixture containing 80% Lactone of C18:3 fatty acid (linolenic acid) and its chemically derived Sophorolipid methyl ester form were checked against Gram-positive (B. subtilis) and Gram-negative (E. coli and P. aeruginosa) bacteria. Antibacterial tests of SL mixture (LNNSL) and sophorolipid methyl ester were performed using standard dilution micromethod. SL mixture and its methyl ester form were diluted to 5, 10, 20 µg ml-1 with sterile millipore water. Bacterial suspensions at a concentration of 106 CFU ml-1 were added into each of these dilutions of SL mixture and SL methyl ester separately and incubated for 6 h at their respective temperatures. 100 l aliquots were taken out from the respective suspensions at 2 h intervals and plated on LB agar plates followed by incubation at their respective temperatures. Colonies were visualized after 24 h and digital images of the plates were captured. The effectiveness of compounds was analyzed by plotting percentage cell survival versus incubation time. It was observed that bacterial colonies of both the Gram-positive and Gram-negative bacteria decreased with increasing amount of compounds as well as increasing incubation time. SL mixture (LNNSL) containing 80 % Lactone of C18:3 molecules were found more effective as compared to Sophorolipid methyl ester against both Gram-positive and Gram-negative bacteria. The antibacterial action of LNNSL containing 80 % Lactone of C18:3 fatty acid (linolenic acid) and its methyl ester derivative on Gram-positive and Gram-negative bacteria is investigated with the help of atomic force microscopy (AFM). Mode of action of SL mixture containing 80 % Lactone and Sophorolipid methyl ester was in agreement with other biosurfactants that act on the integrity of cell membrane (Baek et al. 2003) which involves bactericidal action as confirmed from AFM study. Further, The peptidoglycan cell wall of bacteria are mainly open networks of macromolecules and generally do not offer significant permeability barriers to compounds of molecular mass less than 50 kDa. So it was hypothesized that sophorolipids due to their amphiphilic nature and molecular mass in the range of 600-800 Da can make entry into the bacterial cell through both the lipid bilayer as well as through porin channels. At some concentrations, sophorolipid becomes toxic to the bacterial cell and show bactericidal action. Sophorolipid mixture (LNNSL) containing 80 % Lactone and Sophorolipid methyl ester (LNNSLME) both showed the similar mode of bactericidal action. Summary and Conclusions This study successfully demonstrated the analysis of chemically distinct forms in the SL mixture produced by Candida bombicola when grown on glucose and α-linolenic acid as well as its conversion into the single homogenous product by chemical esterification reaction. These novel Sophorolipids were good surface active and antibacterial agents. They have potential for various applications and offer the advantages of further modifications and can produce functionalized SLs according to their applications. Future aspects of these novel SLs will be discussed in this section of the thesis.

Item Type: Thesis (PhD)
Subjects: Biological Sciences
Divisions: UNSPECIFIED
Depositing User: Ms Reetika Gupta
Date Deposited: 13 Jul 2012 09:38
Last Modified: 07 Jul 2014 07:23
URI: http://ncl.csircentral.net/id/eprint/1032

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