Sophorolipids: improvement of the selective production by Starmerella bombicola through the design of nutritional requirements

Microtiter plates were used as minireactors to study Starmerella bombicola growth and sophorolipid (SL) production. Compositional analysis of SL mixtures by liquid chromatography with electrospray ionization tandem mass spectrometry showed similar results on SLs produced using the laboratory scale (shake flask) and the microscale (24-well microtiter plates (MTP)) approach. MTP suitability on SL production was proven, being this approach, especially advantageous on SL screening. Several hydrophilic carbon sources, hydrophobic co-substrates and nitrogen sources were supplied to culture media, and their influence on SL production was evaluated. The selection of specific hydrophobic co-substrate and nitrogen sources influenced the ratio acidic/lactonic SLs. In fact, it was observed that the production of acidic C18:1 diacetylated hydroxy fatty acid SLs was favoured when culture media was supplied with avocado, argan, sweet almond and jojoba oil or when NaNO₃ was supplied instead of urea. This last case was observed after 144 h of cultivation. A new SL, lactonic C18:3 hydroxy fatty acid diacetylated SL, was detected when borage and onagra oils were used individually as co-substrates. Overall results indicated the potential of the selective production of different and new sophorolipids by Starmerella bombicola based on the selection of carbon and nitrogen sources to culture media.     

Enzymatic conversion of a sophorolipid into a glucose lipid

The diacetylated lactonic sophorolipid from Candida bombolica was converted into the deacetylated acidic form by alkaline hydrolysis and subsequently treated with several glycosidases. One of these enzymes, a hesperidinase (E.C. 3.2.1.40), was most active in the specific release of one glucose molecule from the disaccharide lipid. The novel glucolipid was isolated and characterized. The surface and interfacial tension of aqueous solutions were measured and compared with the lactonic and acidic sophorolipid.     

Production of sophorolipids with eicosapentaenoic acid and docosahexaenoic acid from Wickerhamiella domercqiae var. sophorolipid using fish oil as a hydrophobic carbon source

Sophorolipids (SLs) were synthesized by Wickerhamiella domercqiae var. sophorolipid CGMCC 1576 grown on fish oil and glucose. They were purified using preparative HPLC and their structures were identified by MS/MS. The yields of total and lactonic SLs were 47 and 19 g l^?1 in shake-flasks when fish oil 4 % (v/v) was used with glucose in the medium. The composition of SL mixture contained more than 20 SL molecules. Several unconventional SL molecules with eicosapentaenoic acid (EPA) or docosahexaenoic acid (DHA) including zero-, mono- and di-acetylated acidic SLs with EPA and a di-acetylated acidic SL with DHA were obtained. Two unconventional lactonic SL molecules, non-acetylated lactonic SL with 22:3 and non-acetylated lactonic SL with 20:0, were also obtained.     

Sophorolipid production from delignined corncob residue by Wickerhamiella domercqiae var. sophorolipid CGMCC 1576 and Cryptococcus curvatus ATCC 96219

Delignined corncob residue hydrolysate (DCCRH) and detoxified DCCRH were used for single cell oil (SCO) and single cell protein (SCP) production of Cryptococcus curvatus ATCC 96219 and for sophorolipid (SL) production of Wickerhamiella domercqiae var. sophorolipid CGMCC 1576. Both C. curvatus and W. domercqiae could utilize glucose in DCCRH to grow and accumulate lipids or particle-shaped SLs. DCCRH detoxification by activated carbon adsorption not only improved cell growth and lipid accumulation of C. curvatus but also increased SL production and proportion of lactonic SL in total SL. A total biomass of 17.36 g/l with a lipid content of 44.36 % could be achieved after cultivation of C. curvatus on the detoxified DCCRH. The predominant fatty acids of the produced SCO were oleic, stearic, and palmitic acids (27.2, 20.5, and 15.7 %, respectively). When W. domercqiae cells were cultivated on DCCRH and SCO, total SL production of 39.08 g/l (DCCRH?+?SCO) and 42.06 g/l (detoxified DCCRH?+?SCO) were obtained. Furthermore, when cell lysate of C. curvatus, oleic acid, and DCCRH/detoxified DCCRH was used as nitrogen and carbon sources, total SL production reached 37.19 g/l and 48.97 g/l, respectively. These results demonstrated that renewable DCCRH can be utilized for the production of high-value SCO and SLs.     

High‐titer production and strong antimicrobial activity of sophorolipids from Rhodotorula bogoriensis

Rhodotorula bogoriensis produces sophorolipids (SLs) that contain 13‐hydroxydocosanoic acid (OH‐C₂₂) as the lipid moiety. A systematic study was conducted to further understand the fermentative production of SLs containing OH‐C₂₂ (C₂₂‐SL) by R. bogoriensis. Shake‐flask studies showed that R. bogoriensis consumed glucose at a slow pace. HPLC analysis of the C₂₂‐SL products from shake‐flask fermentations at different glucose concentrations showed a correlation between glucose depletion and the extent of C₂₂‐SL deacetylation. A large‐scale bioreactor fermentation resulted in the isolation of C₂₂‐SL at a volumetric product yield of 51 g/L. HPLC analysis of C₂₂‐SL product from the bioreactor fermentation corroborated the finding that glucose depletion correlated with extensive deacetylation of C₂₂‐SL. The antimicrobial activity of C₂₂‐SL was established for the first time to be stronger than the C₁₈‐SL from Candida bombicola against Propionibacterium acnes in a plate assay. © 2015 American Institute of Chemical Engineers Biotechnol. Prog., 31:867–874, 2015     

Purification of lactonic sophorolipids by crystallization

Various experimental methods for purifying lactonic sophorolipids (SLs) via crystallization were studied. The commonly used solvent, ethanol, was found to have much higher solubility of the lactonic SLs than the acid SLs. Consequently, for purification of lactonic SLs, ethanol not only lacked the selectivity in removing acidic SLs but also resulted in significant loss of desired products. Aqueous buffers were subsequently studied as the solvent, based on the rationale that acidic SLs, having the free carboxylic acid group(s), are more hydrophilic than the lactonic SLs, especially at higher pH. Both phthalate and phosphate buffers were found more suitable for purifying lactonic SLs than ethanol. A practical and effective method for purifying lactonic SLs to about 99% purity using phosphate buffers was developed. The FTIR spectra also showed significantly less SL components with free acid groups in the purified SLs than in the crude SLs, confirming the removal of acidic SLs in the purification.     

Effects of structured lipids containing eicosapentaenoic or docosahexaenoic acid and caprylic acid on serum and liver lipid profiles in rats

The physiological activity and effect on lipid metabolism of four types of structured lipids (SLs), that contain caprylic acid (C8) and either eicosapentaenoic (EPA) or docosahesaenoic acid (DHA), were evaluated in male Wistar rats fed experimental diets containing 7% (wt %) of each SL and 3% (wt %) soybean oil for 28 days. Control rats were fed a diet containing 10% (wt %) soybean oil. The relative perirenal adipose tissue weights of rats fed D-8-8 and 8-D-8 diets were significantly lower than those of other groups. We observed significantly lower serum cholesterol concentrations in rats fed SLs than those of control group over experimental period. The serum lipids concentrations in rats fed diets containing SLs were significantly lower P < 0.05) than those of soybean oil group. The fatty acid compositions of WAT did not reflect the structural differences in the triglyceride. These results suggest that the physiological effects of the SLs used in this study were due to the fatty acids rather than the structural specificity. Therefore, further study will be needed to ascertain the most desirable structural configuration.     

Effects of nitrogen sources on production and composition of sophorolipids by <Emphasis Type="Italic">Wickerhamiella domercqiae</Emphasis> var. <Emphasis Type="Italic">sophorolipid</Emphasis> CGMCC 1576

The effects of nitrogen sources on growth of sophorolipid-producing yeast, Wickerhamiella domercqiae var. sophorolipid CGMCC 1576 and on production and composition of sophorolipids were studied. Organic nitrogen sources are more favorable for accumulation of biomass than inorganic ones. Presence of ammonium ion from different inorganic nitrogen sources (except NH₄HCO₃) greatly inhibited the production of lactonic sophorolipids. However, when organic nitrogen sources were used, lactonic sophorolipid production was strongly increased. Production of crystalline lactonic sophorolipids from organic/inorganic nitrogen sources was enhanced with the increase of pH value adjusted by sodium hydroxide or sodium citrate solution. Fourier-transform infrared (FT-IR), gas chromatography mass spectrometry (GC-MS), high-performance liquid chromatography (HPLC), and mass spectra (MS) were employed to compare the composition of sophorolipid mixture obtained from different nitrogen sources. More than 15 acidic sophorolipid molecules and only 4 lactonic sophorolipid molecules were produced by using 1.27 g/l ammonium sulfate as nitrogen source; they were separated by preparative HPLC and their structures were elucidated by MS. These results suggest extraordinary regulatory effects of nitrogen source on growth and sophorolipid synthesis of W. domercqiae var. sophorolipid.     

Identification and quantification of sophorolipid analogs using ultra-fast liquid chromatography–mass spectrometry

An ultra-fast liquid chromatographic method combined with atmospheric pressure chemical ionization mass detection (UHPLC/APCI-MS) has been developed for the separation and quantification of sophorolipid analogs produced by the yeast Candida bombicola. The sophorolipid mixture was produced by growing the yeast in the presence of glucose and oleic acid under higher aeration. It was found that more than 95% of the analogs are lactonic sophorolipids and all the produced sophorolipids produced were either mono- or di-acetylated. Also observed was a sophorolipid analog with a tri-unsaturated fatty acid, which has not been reported previously.     

Semicontinuous sophorolipid fermentation using a novel bioreactor with dual ventilation pipes and dual sieve‐plates coupled with a novel separation system

Sophorolipids (SLs) are biosurfactants with widespread applications. The yield and purity of SLs are two important factors to be considered during their commercial large‐scale production. Notably, SL accumulation causes an increase in viscosity, decrease in dissolved oxygen and product inhibition in the fermentation medium. This inhibits the further production and purification of SLs. This describes the development of a novel integrated system for SL production using Candida albicans O‐13‐1. Semicontinuous fermentation was performed using a novel bioreactor with dual ventilation pipes and dual sieve‐plates (DVDSB). SLs were separated and recovered using a newly designed two‐stage separation system. After SL recovery, the fermentation broth containing residual glucose and oleic acid was recycled back into the bioreactor. This novel approach considerably alleviated the problem of product inhibition and accelerated the rate of substrate utilization. Production of SLs achieved was 477 g l^?1, while their productivity was 1.59 g l^?1 h^?1. Purity of SLs improved by 23.3%, from 60% to 74%, using DVDSB with the separation system. The conversion rate of carbon source increased from 0.5 g g^?1 (in the batch fermentation) to 0.6 g g^?1. These results indicated that the integrated system could improve the efficiency of production and purity of SLs.     

Production of sophorolipid biosurfactant by Pichia anomala

Biosurfactant production by Pichia anomala PY1, a thermotorelant strain isolated from fermented food, was examined as grown in media containing various carbon and nitrogen sources. The optimal conditions for biosurfactant production included 4% soybean oil as carbon source at pH 5.5 at 30 degrees C for 7 d. Under these conditions, the surface tension of the medium decreased to 28 mN/m with oil displacement measured at 69.43 cm(2). Comparative studies of biosurfactant production in media containing glucose or soybean oil were performed. The biosurfactants obtained were isolated and purified by chromatographic methods. The molecular weights of samples were further investigated by mass spectrometry. In medium containing glucose, biosurfactants of molecular weights of 675, 691, and 707 were obtained, while those isolated from medium containing soybean oil were of molecular weights of 658, 675, and 691. These results reveal that sophorolipid compounds containing fatty acids of C20 and C18:1 were produced from both media.     

Selective recovery of acidic and lactonic sophorolipids from culture broths towards the improvement of their therapeutic potential

Sophorolipids (SLs) were produced by Starmerella bombicola. The separation and purification of SLs are a complex process, since they are produced as a mixture of compounds with few structural differences. Solvent extraction is commonly used in downstream processing. In this work, an environmental friendly approach was developed for SLs recovery and purification, based on neutral polymeric sorbents, Amberlite XAD16N^TM, XAD18^TM, and XAD1600N^TM. In batch microassays, key parameters of sorption/desorption process (e.g., contact time, temperature, sorbents, and SLs concentrations) were optimized for separation of acidic and lactonic SLs. Sorption equilibrium was reached after 2–3 h, for all the sorbents tested. Among them XAD1600N^TM showed a higher sorption capacity (q _max 230 mg g^?1), a higher removal (≈100 %) of acidic and lactonic SLs [1 and 2.5 % (w/v)], and the best selectivity. Methanol, ethanol, and acetone were suitable for SLs elution. A selective desorption of SLs was attained with acetonitrile aqueous solutions (v/v): (1) 25 % led to 88.3 % of acidic SLs and (2) 55 % followed by methanol solution (100 %) led to 93.2 % of purified lactonic SLs. This achievement was particularly important regarding SLs potential therapeutic applications, since acidic and lactonic SLs show different biologic activities. In fact, acid SLs show higher virucidal and pro-inflammatory cytokine activity, while lactonic SLs show stronger spermicidal and anti-cancer activity.     

Sophorolipid production by <Emphasis Type="Italic">Candida bombicola</Emphasis> on oils with a special fatty acid composition and their consequences on cell viability

Sophorolipids production by the yeast Candia bombicola is most favourable when glucose is used as a carbon source in combination with a hydrophobic carbon source such as a common vegetable oil. Most vegetable oils are comprised of C16–C18 fatty acids, an ideal range for sophorolipid production. The use of oils with either shorter or longer fatty acids, such has coconut oil or meadowfoam oil, respectively, was evaluated. Such oils did not contribute to enhanced sophorolipid production when compared to cultures run on glucose as the sole carbon source. Moreover, a toxic effect of medium-chain fatty acids towards stationary C. bombicola cells was demonstrated.     

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.     

代谢改造熊蜂生假丝酵母提高酸型槐糖脂产量

【目的】槐糖脂是一类生物表面活性剂,不仅具有常规表面活性剂所具有的增溶、乳化、润湿、发泡、分散、降低表面张力等通用性能,且对环境的耐受性极强。熊蜂生假丝酵母(Starmerella bombicola)能够发酵生产槐糖脂,但槐糖脂具有酸型、内酯型和乙酰化型等不同类型,结构多样,难以分离。本文拟通过代谢工程改造,构建高产酸型槐糖脂的熊蜂生假丝酵母工程菌株。【方法】利用潮霉素抗性基因构建了标记基因重复利用系统Rec-six基因编辑系统,在此基础上将合成内酯型槐糖脂的关键基因——内酯酶基因SBLE敲除获得一株只产酸型槐糖脂的工程菌株Δsble,进一步同源过量表达葡萄糖基转移酶基因UGTB并敲除过氧化物酶体膜转运蛋白编码基因PXA1,构建了高产酸型槐糖脂的酵母工程菌。【结果】与出发菌株相比,重组熊蜂生假丝酵母发酵油酸能够合成单一的酸型槐糖脂,而不再合成内酯型槐糖脂,同时酸型槐糖脂的产量由20 g/L提高到44 g/L,提高了2.1倍。【结论】通过敲除PXA1、SBLE和过表达UGTB来改造熊蜂生假丝酵母,能够有效提高重组菌的酸型槐糖脂产量,为发酵法生产酸型槐糖脂奠定了基础。     

Integrated sophorolipid production and gravity separation

A novel method for the integrated gravity separation of sophorolipid from a fermentation broth has been developed, enabling removal of a sophorolipid phase of either higher or lower density than the bulk fermentation broth, while cells and other media components are recirculated and returned to the bioreactor. The capability of the separation system to recover an enriched sophorolipid product phase was demonstrated on three sophorolipid producing fed batch fermentations using Candida bombicola, giving an 11% reduction in fermenter volume required whilst maintaining sophorolipid production. Sophorolipid recoveries of up to 86% (280 g) of the total produced over a whole fermentation were achieved at an enrichment of up to 9. Furthermore, the broth viscosity reduction achieved by removal of the sophorolipid phase enabled a 34% reduction in mixing power to maintain the same dissolved oxygen level by the end of the fermentation, with a 9% average reduction over the course of the fermentation. Fermentation duration could be extended to 1023 h, allowing production of 623 g sophorolipid from 1 l initial batch volume. These benefits could lead to a substantial decrease in the cost of sophorolipid production, making high volume applications such as enhanced oil recovery economically feasible.     

The Use of Fatty Acid Esters to Enhance Free Acid Sophorolipid Synthesis

Fatty acid esters were prepared by transesterification of soy oil with methanol (methyl-soyate, Me-Soy), ethanol (ethyl-soyate, Et-Soy) and propanol (propyl-soyate, Pro-Soy) and used with glycerol as fermentation substrates to enhance production of free-acid sophorolipids (SLs). Fed-batch fermentations of Candida bombicola resulted in SL yields of 46 ± 4 g/l, 42 ± 7 g/l and 18 ± 6 g/l from Me-Soy, Et-Soy, and Pro-Soy, respectively. Liquid chromatography with atmospheric pressure ionization mass spectrometry (LC/API-MS) showed that Me-Soy resulted in 71% open-chain SLs with 59% of those molecules remaining esterified at the carboxyl end of the fatty acids. Et-Soy and Pro-Soy resulted in 43% and 80% open-chain free-acid SLs, respectively (containing linoleic acid and oleic acid as the principal fatty acid species linked to the sophorose sugar at the omega-1 position), with no evidence of residual esterification. Mention of trade names or commercial products in this publication is solely for the purpose of providing specific information and does not imply recommendation or endorsement by the U.S. Department of Agriculture.     

Conversion of oil waste to valuable fatty acids using Oleaginous yeast

Oil waste poses a highly dangerous threat to the environment, mainly because it is considered a high energy demanding degradation process. Oleaginous yeast utilizing oil waste to produce microbial fatty acids is considered an innovative method for oil waste elimination. In the present study, fifteen yeast isolates were screened for their lipid content, three of which were chosen for their high lipid content as compared to the standard strain Phaffia rhodozyma NRRL-Y-10921. The three selected isolates were further screened for their fatty acid profile. Yeast isolate (NC-I), identified as Yarrowia lipolytica, was chosen because it exceeded the lipid production of the standard strain by 21%, it also produced the highest C 14:0 (myristic acid), C 18:1 (oleic acid) and C18:2 (linoleic acid), compared to the other two isolates. Growth on different oil wastes resulted in an increase in total lipid content which reached its maximum when oil waste of frying vegetables was added to the media (57.89%). A variation in the fatty acid profile was detected when different types of oil waste were used before and after fermentation. The addition of different glucose concentrations to the vegetable oil waste media resulted in the appearance of C 22:0 (behenic acid) which was not present when the basal medium was used. Scanning Electron Microscopy indicated morphologic changes when the yeast was grown in high glucose concentration as compared to those grown in oil waste media. The activity of malate dehydrogenase (MDH) enzyme exhibited a correlative relationship with the lipid content under various glucose concentrations. The obtained results indicate that vegetable oil waste is suitable for microbial fatty acid production and that the fatty acid profile could be maneuvered through the manipulation of the fermentation media.     

Utilization of restaurant waste oil as a precursor for sophorolipid production

Approximately 100 billion liters of oil is generated per week as waste from restaurants around the country. Because of health, environmental, and economic factors, current methods of disposal are ineffective for disposal of the restaurant oil wastes. In this study we have investigated the ability of Candida bombicola to fermentatively transform the restaurant oil waste into glycolipids called sophorolipids. Batch and fed-batch studies were carried out using oil waste as the lipid feedstock in Erlenmeyer flasks and in a fermentor. Batch fermentation in a fermentor gave the highest yield of sophorolipids of 34 g L-1. Fermentation using oleic acid as control feedstock were also carried out. Batch fermentation in the fermentor using this pure fatty acid gave a highest yield of 42 g L-1. The difference in the sophorolipid yield was attributed to the fatty acid composition of restaurant oil waste.     

Extracellular production of a glycolipid biosurfactant, mannosylerythritol lipid, by Candida sp. SY16 using fed-batch fermentation

Candida sp. strain SY16 produces a glycolipid-type biosurfactant, mannosylerythritol lipid (MEL-SY16), which can reduce the surface tension of a culture broth from 72 to 30 dyne cm⁻¹ and highly emulsify hydrocarbons when cultured in soybean-oil-containing media. As such, laboratory-scale fermentation for MEL-SY16 production was performed using optimized conditions. In batch fermentation, MEL-SY16 was mainly produced during the stationary phase of growth, and the concentration of MEL-SY16 reached 37 g l⁻¹ after 200 h. The effect of pH control on the production of MEL-SY16 was also examined in batch fermentation. The highest production yield of MEL-SY16 was when the pH was controlled at 4.0, and the production was significantly improved compared to batch fermentation without pH control. In fed-batch fermentation, glucose and soybean oil (1:1, w/w) were used in combination as the initial carbon sources for cell growth, and soybean oil was used as the feeding carbon source during the MEL production phase. The feeding of soybean oil resulted in the disappearance of any foam and a sharp increase in the MEL production until 200 h, at which point the concentration of MEL-SY16 was 95 g l⁻¹. Among the investigated culture systems, the highest MEL-SY16 production and volumetric production rate were achieved with fed-batch fermentation.