Candida Bombicola ATCC Research Articles

Efficient production of acidic sophorolipid from rapeseed oil by Candida bombicola

AbstractOwing to the high surface activity and good biological activity, sophorolipid (SL) has been considered as one of the most promising biosurfactants in recent years. Among all of the structures of SL, acid‐type SL has gained increasing attention and application in the areas of bioremediation and daily chemical industry owing to its excellent foamability and water solubility. However, few studies focused on acid‐type SL biological production have been reported. In this study, acid‐type SL production by the yeast Candida bombicola ATCC 22214 was comprehensively evaluated and optimized. Under the optimal medium composition and fermentation conditions, the yield of SL in the shake flask reached 68.8 g L−1, in which acid‐type SL accounted for 70%. With subsequent scale‐up to a 5 L bioreactor, the yield of SL using fed‐batch fermentation strategy reached 131.2 g L−1, and the acid‐type SL accounted for 84.7%. To the best of our knowledge, the yield of acid‐type SL in this study is the highest reported currently. In addition, the SL mixture (containing 84% acid‐type SL) produced by C. bombicola ATCC 22214 had better surface activity, emulsification properties and environmental stability than current commercial SL products. This study provided a promising microbial chassis and fermentation strategy for commercial production of acid‐type SL. © 2022 Society of Chemical Industry and John Wiley & Sons, Ltd

Efficient Preparation of Sophorolipids and Functionalization with Amino Acids to Furnish Potent Preservatives.

The development of new preservatives is an ongoing investigation in the food industry, especially those which are safe and environmentally friendly. In this study, biosurfactant sophorolipids (SLs) functionalized with amino acids were developed as efficient preservative agents. SLs were first isolated from fermentation broth by Candida bombicola ATCC 22214, hydrolyzed, and purified by extraction. The typical recovery is around 70%, while the extracted material consists of over 90% deacetylated acidic SLs (SL-COOH). Four types of SL derivatives were then synthesized via dicyclohexylcarbodiimide amidation reactions from prepared SL-COOH. Among the derivatives produced, the arginine SL conjugates (SL-d-Arg) displayed the highest activity against Gram-positive bacteria and fungi and even inhibited the cell growth of Gram-negative bacteria and mildew. Furthermore, the arginine conjugates performed the broadest antimicrobial activity among the derivatives evaluated. The sterilization dosage of the arginine conjugates against the food-spoilage pathogen Bacillus spp. was 63-125 mg/L, in contrast to 250 mg/L for the enterotoxin producer Staphylococcus aureus and 500 mg/L for fungi. More importantly, SL-d-Arg displayed excellent biocompatibility, with a therapeutic index of over 7.94. SL-d-Arg has excellent potential as an alternative to traditional chemical preservatives.

Advances in sophorolipid-producing strain performance improvement and fermentation optimization technology.

Sophorolipids (SLs), currently one of the most promising biosurfactants, are secondary metabolites produced by many non-pathogenic yeasts, among which Candida bombicola ATCC 22214 is the main sophorolipid-producing strain. SLs have gained much attention since they exhibit anti-tumor, anti-bacterial, anti-inflammatory, and other beneficial biological activities. In addition, as biosurfactants, SLs have a low toxicity level and are easily degradable without polluting the environment. However, the production cost of SLs remains high, which hinders the industrialization process of SL production. This paper describes SL structure and the metabolic pathway of SL synthesis firstly. Furthermore, we analyze factors that contribute to the higher production cost of SLs and summarize current research status on the advancement of SL production based on two aspects: (1) the improvement of strain performance and (2) the optimization of fermentation process. Further prospects of lowering the cost of SL production are also discussed in order to achieve larger-scale SL production with a high yield at a low cost. KEY POINTS: • Review of advances in strain performance improvement and fermentation optimization. • High-throughput screening and metabolic engineering for high-performance strains. • Low-cost substrates and semi-continuous strategies for efficient SL production.

Sophorolipid Biosurfactants Activate Taste Receptor Type 1 Member 3‐Mediated Taste Responses and Block Responses to Bitter Taste In Vitro and In Vivo

AbstractSophorolipids (SL) are typically produced and secreted by select nonpathogenic yeast species (i.e., Candida) from renewable substrates. They are currently being used by industry on a limited basis in formulations for cleaning solutions as well as laundry and dishwashing detergents. Due to the nature of their chemical structure, it was hypothesized that SL would demonstrate taste‐sensory properties. In this study, SL were produced via fermentation on a mixed substrate platform with glucose and either palmitic acid, stearic acid, or oleic acid using Candida (currently reclassified as Starmerella) bombicola ATCC 22214. The taste properties of SL were determined using a single‐cell manual calcium imaging technique on cultured human fungiform taste papillae (HBO) cells. The results of those studies demonstrated that sweetener‐responsive HBO cells also respond to SL, and these responses are mediated by the type 1 taste receptors 3 (T1R3), because they were blocked by lactisole (a T1R3 receptor‐specific blocker). The involvement of the T1R3 receptor in SL recognition was confirmed via the chorda tympani nerve recording (CTNR) study in a (−/−) T1R3 knockout (KO) mouse model. We further demonstrated that SL are capable of blocking the bitter stimuli‐elicited responses both in HBO cells and in the CTNR study. This is the first report demonstrating that SL have taste‐sensory properties, which opens up numerous possibilities for practical applications of SL to ameliorate bitter tastes in foods and drugs and understand the potential source of dysgeusia in some patients.

Metabolic profiling and flux distributions reveal a key role of acetyl-CoA in sophorolipid synthesis by Candida bombicola

Sophorolipids are important biosurfactants produced by the wild-type strain of Candida bombicola ATCC 22214, with antimicrobial, antiviral, and anticancer activities. Our previous study showed that a mutant strain lacking multifunctional enzyme type 2 displayed extremely low sophorolipid production capacity compared to the wild-type strain. In the present study, we aimed to reveal the possible reasons for this deficiency through a series of analytical methods. First, comparative metabolic profiling analysis between the wild-type and mutant strains was performed. Multivariate statistical analysis, including principal component analysis and orthogonal partial least squares discriminant analysis, showed that nine potential metabolites were closely associated with the metabolic differences between strains. Second, metabolic flux analysis revealed that an inadequate supply of intracellular acetyl-CoA in the mutant strain could significantly affect the biosynthesis of sophorolipids. In addition, supplementation of citric acid recovered the sophorolipid production in the mutant strain by approximately 83%, suggesting that citrate metabolism plays an important role in sophorolipid biosynthesis. These findings provide novel theoretical insights for further improving the production of sophorolipids in C. bombicola.

Lubricant Properties of ω − 1 Hydroxy Branched Fatty Acid-Containing Natural and Synthetic Lipids

Hydroxy fatty acids, such as those derived from castor and lesquerella seed oils, make ideal substrates for the synthesis of bio-lubricants, cosmetics, coatings, plastics, and lubricant additives. However, feedstocks of such fatty acids suffer from major drawbacks, such as a lack of a cropping system to produce those seeds or toxic by-products in generating the seed oil, all of which limit availability and thus add to costs. In this study, we explore lubrication properties of microbially derived hydroxy fatty acids and demonstrate that such microbial ω − 1 hydroxy fatty acids, and their derivatives, exhibit lubrication traits (e.g., anti-friction and anti-wear properties) comparable to those of seed-derived hydroxy fatty acids. These ω − 1 hydroxy fatty acids can be recovered from sophorolipids produced by the yeast Candida bombicola ATCC 22214, or by bioengineering bacterial systems to produce them from sugar (Garg et al. in Metab Eng 35:9–20, 2016). Optimization of this latter system can pave the way for a less costly and sustainable alternative to plant-derived bio-lubricants.

Sophorolipids Production by Candida bombicola ATCC 22214 and Its Potential Application in Soil Bioremediation

The sophorolipids are biosurfactants composed by a disaccharide sophorose linked by a hydroxyl group of a fatty acid. Due to their surface-active properties, sophorolipids have a great potential for using in different industrial sectors. They are synthesized by various strains of the genus Candida in the presence of hydrophilic and hydrophobic sources. In this study, it was evaluated the production of sophorolipids by Candida bombicola ATCC 22214 using sugar cane molasses, sugar cane juice, sucrose or glucose as hydrophilic source and chicken fat or sunflower oil as hydrophobic source. The sophorolipids were characterized and applied in bioremediation of soils contaminated with lubricating oil. The production of sophorolipids was 39.81 g L−1 in the optimized condition of 75 g L−1 of chicken fat, 77.5 g L−1 of glucose, 2.5 g L−1 of yeast extract and without urea. The sophorolipids produced present the diacetylated lactonic form, exhibiting the surface tension properties of 35 mN/m and of CMC 65 mg L−1. The sophorolipids showed great emulsification index in lubricant oil, toluene and n-heptane, suggesting the potential application in bioremediation of lubricating-oil-contaminated soils.

Sophorolipids Production by Candida bombicola ATCC 22214 and its Potential Application in Microbial Enhanced Oil Recovery.

Biosurfactant production using Candida bombicola ATCC 22214, its characterization and potential applications in enhancing oil recovery were studied at laboratory scale. The seed media and the production media were standardized for optimal growth and biosurfactant production. The production media were tested with different carbon sources: glucose (2%w/v) and corn oil (10%v/v) added separately or concurrently. The samples were collected at 24 h interval up to 120 h and checked for growth (OD660), and biosurfactant production [surface tension (ST) and interfacial tension (IFT)]. The medium with both glucose and corn oil gave better biosurfactant production and reduced both ST and IFT to 28.56 + 0.42mN/m and 2.13 + 0.09mN/m, respectively within 72 h. The produced biosurfactant was quite stable at 13–15% salinity, pH range of 2–12, and at temperature up to 100°C. It also produced stable emulsions (%E24) with different hydrocarbons (pentane, hexane, heptane, tridecane, tetradecane, hexadecane, 1-methylnaphthalene, 2,2,4,4,6,8-heptamethylnonane, light and heavy crude oil). The produced biosurfactant was extracted using ethyl acetate and characterized as a mixture of sophorolipids (SPLs). The potential of SPLs in enhancing oil recovery was tested using core-flooding experiments under reservoir conditions, where additional 27.27% of residual oil (Sor) was recovered. This confirmed the potential of SPLs for applications in microbial enhanced oil recovery.

A new class of bacterial quorum sensing antagonists: glycomonoterpenols synthesized using linalool and alpha terpineol.

With increasing burden of antibiotic resistant microorganism search for newer drug targets and potent drug molecules is a never ending scenario. Quorum sensing (QS), the phenomenon of bacterial cross-talk, is one such target that has captured the attention of many and has been touted as the future of new age antimicrobials. Quorum sensing has the potential to regulate a plethora of bacterial virulence phenotypes and search of molecules with powerful quorum sensing inhibitory (QSI) capacity are underway. Monoterpene alcohols like linalool and alpha terpineol have been shown to possess antimicrobial and anti-biofilm activity. However in this article we attempt to bring forth a new class of compounds, glycomonoterpenols, derived from monoterpenoids alcohols. These glycomonoterpenols have been synthesized using Candida bombicola ATCC 22214 by feeding the cells with linalool and alpha terpineol respectively as substrates in 10% glucose, production medium. The advantage of these molecules over their parent compound is their additional surfactant like property, increased solubility and enhanced QSI potential. A variety of gram-negative bacteria capable of elaborating quorum sensing mediated phenotypes have been selected and both these glycoterpenoid derivatives have been shown to possess strong anti-QS activity.

Antimicrobial properties of sophorolipids produced by Candida Bombicola ATCC 22214 against gram positive and Gram-negative bacteria

Antimicrobial properties of sophorolipids produced by Candida Bombicola ATCC 22214 against gram positive and Gram-negative bacteria

Structure Elucidation and Characterization of Microbial 2-Tridecyl Sophorosides (Biosurfactants)

To produce novel types of sophorose lipids containing an odd number of carbon atoms in the lipophilic moiety, Candida bombicola ATCC 22214 was grown in 500-ml flask cultures with glucose as main carbon source, and additionally, 2-tridecanone as co-substrate. After solvent extraction, the crude product mixture was separated into pure fractions, and each fraction was analysed via NMR and mass spectroscopy. This effective strategy generated five new glycolipids, 2-tridecyl sophorosides, which differed in the number of glucose units, and acetyl and hydroxy groups, respectively. Based on these compounds, a proposal for the possible biosynthetic pathway was deduced. Two compounds of the mixture, mono- and diacetylated 2-tridecyl sophorosides, respectively, were able to lower the surface tension of water from 72 mN m(-1) to 32 mN m(-1) and the interfacial tension between water and n-hexadecane from 43 mN m(-1) down to 4 and 3 mN m(-1). Thus, both compounds possess a very good surfactant behaviour. Moreover, it was observed that the new products inhibit the growth of particular Gram-positive bacteria, and they indicate potential for antitumour-promoting activity.

Antimicrobial and SEM Studies of Sophorolipids Synthesized Using Lauryl Alcohol

AbstractIn recent years, researchers have developed biosurfactants for industrial, pharmaceutical and medical applications revealing the promising biological activities of these biomolecules. One of the best studied microbial surfactants are glycolipids, especially sophorolipids (SLs) produced by selected non‐pathogenic yeast species of Candida. They are biodegradable, non‐toxic and are environmentally friendly. Sophorolipid production was carried out using glucose as the hydrophilic source and lauryl alcohol C12–14, as the hydrophobic source using Candida bombicola ATCC 22214. Primary characterization of the SL obtained using lauryl alcohol (SLLA) was done by FTIR which depicted the presence of alkyl sophorosides/SLs. Antimicrobial activity testing revealed that SLLA showed complete inhibition against gram negative bacteria, Escherichia coli (ATCC 8739) Pseudomonas aeruginosa (ATCC 9027) at 30 and 1 μg/ml at a contact time of 2 and 4 h respectively. Whereas for gram positive bacteria Staphylococcus aureus (ATCC 6358), Bacillus subtilis (ATCC 6633), complete inhibition was observed at 6 and 1 μg/ml respectively at a contact time of 4 h. The formed SLLA showed noteworthy inhibition against the pathogenic yeast Candida albicans (ATCC 2091) at 50 μg/ml with a contact time of 4 h. These values are remarkably low compared to reported values of oleic acid SLs and linolenic acid SLs which were studied for antimicrobial properties. Scanning electron microscopy analysis of the treated cells revealed the changes in morphology and topography of the microorganisms.

Cloning and functional characterization of the UDP‐glucosyltransferase UgtB1 involved in sophorolipid production by Candida bombicola and creation of a glucolipid‐producing yeast strain

Sophorolipids produced by the non-pathogenic yeast Candida bombicola ATCC 22214 are glycolipid biosurfactants applied commercially as biodegradable and eco-friendly detergents. Their low cell toxicity, excellent wetting capability and antimicrobial activity attract the attention of high-value markets, such as the cosmetic and pharmaceutical industries. Although sophorolipid production yields have been increased by the optimization of fermentation parameters and feed sources, the biosynthetic pathway and genetic mechanism behind sophorolipid production still remains unclear. Here we identify a UDP-glucosyltransferase gene, UGTB1, with a key function in this economically important pathway. The protein shows sequence and structural homology to several bacterial glycosyltransferases involved in macrolide antibiotic synthesis. Deletion of UGTB1 in C. bombicola did not affect cell growth and resulted in a yeast producing glucolipids, thereby opening the route for in vivo production of these glycolipid intermediates. Activity assays on cell lysates confirmed that the identified gene is responsible for the second glucosylation step during sophorolipid production and illustrated that sophorolipid production in C. bombicola involves the stepwise action of two independent glucosyltransferases. The complete UGTB1 sequence data have been submitted to the GenBank database (http://www.ncbi.nlm.nih.gov) under Accession No. HM440974.

Identification of the UDP-glucosyltransferase gene UGTA1, responsible for the first glucosylation step in the sophorolipid biosynthetic pathway of Candida bombicola ATCC 22214

Candida bombicola ATCC 22214 is applied commercially for the production of sophorolipids from renewable resources such as vegetable oils or waste streams. Although much research has been performed on optimization of fermentation conditions and on the influence of feed source and process parameters on sophorolipid structures and yields, the metabolic pathway of these important bioproducts remains unclear. Here, we identify a glucosyltransferase gene UGTA1 and show that the gene product is responsible for the first glucosylation step in the biosynthetic pathway of sophorolipids. Moreover, we provide evidence that the second glucosylation step is catalysed by a different glucosyltransferase that acts independently from the first. Therefore, the biosynthesis of sophorolipids by C. bombicola involves two glucosyltransferases that act in a stepwise manner. The UGTA1 gene described here is the first identified gene with a clear function in sophorolipid production by this economically important yeast.

On the production and identification of medium‐chained sophorolipids

AbstractSophorolipids are biologically produced surfactants that consist of a hydrophilic sophorose moiety bound to a hydroxylated lipophilic substrate. The highest levels of production are currently achieved using Candida bombicola ATCC 22214, an ascomycotous yeast, as the production organism. The lipophilic moiety is typically found to have a length between C16 and C18 and thus the applications of readily produced sophorolipids with regard to cleaning purposes are somewhat limited. Experiments were performed to evaluate the conditions under which a β‐oxidation deficient strain of C. bombicola is able to convert 1‐dodecanol into sophorolipids. Both shakeflask and bioreactor cultures yielded C12, or medium‐chained, sophorolipids and the effect of aeration on the production level was investigated and found to favour limited aeration. Analysis of the conversion products was performed through RP‐HPLC and the formation of new‐to‐nature sophorolipids was confirmed by MS. The use of the β‐oxidation deficient strain under limited aeration yielded at least 15 g/L of medium‐chained sophorolipids, which is more than in the wild‐type strain or when employing full aeration.

Sophorolipids from Candida bombicola: Cell separation by ultrasonic particle manipulation

AbstractThe yeast Candida bombicola ATCC 22214 is well‐known to produce mixtures of glycolipids containing the sugar sophorose, the so‐called sophorolipids, especially when cultivated on hydrophobic carbon sources as co‐substrates. To improve cultivation efficiency, an integrated process was developed using ultrasound separation technology. Since this technology is new for use with C. bombicola, it was first characterized in batch experiments and afterwards implemented in an integrated production process. In this process, separation efficiencies of about 99% C. bombicola cells could be achieved, leading to 8 g/L of nearly cell‐free sophorolipid product and a total amount of 73.8 g/L sophorolipids. Furthermore, a technical mixture of unusual branched fatty alcohols containing mainly 2‐hexyl‐1‐decanol was used as co‐substrate with glucose in a shake flask study. This resulted in the production of a new product, 1‐O‐β‐glucopyranosyl‐2‐hexyldecanol, a molecule containing glucose as the sugar moiety and 2‐hexyl‐1‐decanol as a branched hydrophobic side chain.

Production and characterization of methyl ester sophorolipids with 22-carbon-fatty acids

In this study, various fatty acids of 18 and 22 carbons were used as substrates to produce sophorolipids in Candida bombicola ATCC 22214. Methyl esterification of erucic acid and C22-enriched rapeseed oil generated mainly acidic sophorolipids with different degrees of saturation in the fatty acid chain, which was demonstrated by structural analysis using HPLC, LC–MS and NMR spectroscopy. Regardless of methyl esterification, the rapeseed oil served as the best substrate for high production of sophorolipids among the tested. Methyl esterification also had no noticeable effect on the interfacial properties of sophorolipids. However, 22 carbons introduced in the fatty acid chain increased the hydrophobicity of sophorolipids, and therefore improved surface-active properties and biodegradability.

Enhanced sophorolipid production by feeding-rate-controlled fed-batch culture

To develop the easier control method for fed-batch culture of sophorolipid production, we chose rapeseed oil as the most productive oil and compared their productivities in relation to different concentrations of glucose. The optimal concentration of glucose was 30 g/L for sophorolipid production. A fed-batch method was conducted using Candida bombicola ATCC 22214 with rapeseed oil as a secondary substrate. The feeding rate of rapeseed oil was dependent on pH and was calculated by the consumption rate of NaOH and rapeseed oil. The glucose concentration was constantly maintained between 30 and 40 g/L. As a result, we have produced a crude sophorolipid up to 365 g/L for 8 days through a feeding-rate-controlled fed-batch process.

Amino Acid Conjugated Sophorolipids: A New Family of Biologically Active Functionalized Glycolipids

Sophorolipids (SLs) are extra cellular glycolipids produced by Candida bombicola ATCC 22214 when grown in the presence of glucose and fatty acids. These compounds have a disaccharide head group connected to a long-chain hydroxyl-fatty acid by a glycosidic bond. To explore structure-activity of modified SLs, a new family of amino acid-SL derivatives was prepared. Synthesized analogs consist of amino acids linked by amide bonds formed between their alpha-amino moiety and the carboxyl group of ring-opened SL fatty acids. Their preparation involved the following: (i) hydrolysis of a natural SL mixture with aqueous alkali to give SL free acids, (ii) coupling of free acids to protected amino acids using dicarbodiimide, and (iii) removing amino acid carboxyl protecting groups. These conjugates were evaluated for their antibacterial, anti-HIV, and spermicidal activity. All tested analogs showed antibacterial activity against both gram +ve and gram -ve organisms. Leucine-conjugated SL was most efficient. For example, the minimum inhibitory concentrations (MIC) for Moraxella sp. and E. coli were 0.83 and 1.67 mg/mL, respectively. Among the alkyl esters of amino acid conjugated SLs, the ethyl ester of leucine-SLs was most active. Against Moraxella sp., S. sanguinis, and M. imperiale, MIC values are 7.62 x 10(-4), 2.28 x 10-(3) and 1.67 mg/mL, respectively. All compounds displayed virus-inactivating activity with 50% effective concentrations (EC50) below 200 microg/mL. The EC50 of leucine-SL ethyl ester was 24.1 microg/mL, showing that it is more potent than commercial spermicide nonoxynol-9 (EC50 approximately 65 microg/mL).

Production of Sophorolipids fromCandida bombicola ATCC 22214 Using Turkish Corn Oil and Honey

The ability of Candida bombicola ATCC 22214 to produce sophorolipids using Turkish corn oil and honey was investigated. Shake flask experiments were carried out both with and without the addition of glucose as the second carbon source. The organism could produce sophorolipids under both conditions but higher production was obtained when corn oil was combined with glucose. The 3 L bioreactor was first operated in batch mode, using both corn oil and glucose. When all the glucose was consumed, 1/4th of the broth was pumped out and was replaced by freshly prepared medium containing 10 % [w/v] of cheap market honey as the sole carbon source. Feed was comprised of corn oil. High concentrations of sophorolipids (&n62; 400 g/L) were produced. The crude products obtained from the batch cultivation could be solidified as very light brown solids when unused oil was removed by hexane, while the products of the two-stage cultivation remained as viscous, honey-like liquids after identical treatments.