Microbial production and application of sophorolipids

Sophorolipids are surface-active compounds synthesized by a selected number of yeast species. They have been known for over 40 years, but because of growing environmental awareness, they recently regained attention as biosurfactants due to their biodegradability, low ecotoxicity, and production based on renewable resources. In this paper, an overview is given of the producing yeast strains and various aspects of fermentative sophorolipid production. Also, the biochemical pathways and regulatory mechanisms involved in sophorolipid biosynthesis are outlined. To conclude, a summary is given on possible applications of sophorolipids, either as native or modified molecules.     

Simplified soy molasses-based medium for reduced-cost production of sophorolipids by <Emphasis Type="Italic">Candida bombicola</Emphasis>

A simplified medium containing only soy molasses and oleic acid as ingredients was developed for the production of sophorolipids (SLs) from Candida bombicola. We achieved a product yield of 53 ± 3 g of purified sophorolipids per liter of starting culture volume, which is 71 ± 4% of the yield obtained with growth medium that also additionally contains the costly yeast extract and urea as nitrogen source. The large majority of the SL components existed in the lactone form (87%), and the predominant component is SL containing (ω-1)-hydroxyoleic acid as the lipid moiety. The study demonstrated for the first time the usefulness of the low-value soy molasses as a combined nitrogen- and carbon-source for SL production at a reduced cost. Mention of trade names or commercial products in this article is solely for the purpose of providing specific information and does not imply recommendation or endorsement by the U.S. Department of Agriculture.     

Structural characterization and surface-active properties of a new glycolipid biosurfactant,mono-acylated mannosylerythritol lipid,produced from glucose by <Emphasis Type="Italic">Pseudozyma antarctica</Emphasis>

Mannosylerythritol lipids (MELs), which are glycolipid biosurfactants produced by Pseudozyma yeasts, show not only excellent interfacial properties but also versatile biochemical actions. In the course of MEL production from glucose as the sole carbon source, P. antarctica was found to produce unknown glycolipids more hydrophilic than conventional “di-acylated MELs,” which have two fatty acyl esters on the mannose moiety. Based on a detailed characterization, the most hydrophilic one was identified as 4-O-(3′-O-alka(e)noyl-β-d-mannopyranosyl)-d-erythritol namely, “mono-acylated MEL.” The mono-acylated MEL reduced the surface tension of water to 33.8 mN/m at a critical micelle concentration (CMC) of 3.6 × 10⁻⁴ M, and its hydrophilic–lipophilic balance was tentatively calculated to be 12.15. The observed CMC was 100-fold higher than that of the MELs hitherto reported. Interestingly, of the yeast strains of the genus Pseudozyma, only P. antarctica and P. parantarctica gave the mono-acylated MEL from glucose, despite a great diversity of di-acylated MEL producers in the genus. These strains produced MELs including the mono-acylated one at a rate of 20–25%. From these results, the new MEL is likely to have great potential for use in oil-in-water-type emulsifiers and washing detergents because of its higher water solubility compared to conventional MELs and will thus contribute to facilitating a broad range of applications for the environmentally advanced surfactants.     

Production of a novel glycolipid biosurfactant,mannosylmannitol lipid,by <Emphasis Type="Italic">Pseudozyma parantarctica</Emphasis> and its interfacial properties

The development of a novel glycolipid biosurfactant was undertaken using the high-level producers of mannosylerythritol lipids (MELs) such as Pseudozyma parantarctica, Pseudozyma antarctica, and Pseudozyma rugulosa. Besides the conventional MELs (MEL-A, MEL-B, and MEL-C), these yeasts produced an unknown glycolipid when they were cultivated in a medium containing 4% (w/v) olive oil and 4% (w/w) mannitol as the carbon source. The unknown glycolipid extracted from the culture medium of P. parantarctica JCM 11752^T displayed the spot with lower mobility than that of known MELs on TLC and provided mainly two peaks identical to mannose and mannitol on high-performance liquid chromatography after acid hydrolysis. Based on structural analysis by ¹H and ¹³C nuclear magnetic resonance, the novel glycolipid was composed of mannose and mannitol as the hydrophilic sugar moiety and was identified as mannosylmannitol lipid (MML). Of the strains tested, P. parantarctica JCM 11752^T gave the best yield of MML (18.2 g/L), which comprised approximately 35% of all glycolipids produced. We further investigated the interfacial properties of the MML, considering the unique hydrophilic structure. The observed critical micelle concentration (CMC) and the surface tension at CMC of the MML were 2.6 × 10⁻⁶ M and 24.2 mN/m, respectively. In addition, on a water-penetration scan, the MML efficiently formed not only the lamella phase (Lα) but also the myelins at a wide range of concentrations, indicating its excellent self-assembling properties and high hydrophilicity. The present glycolipid should thus facilitate the application of biosurfactants as new functional materials.     

Surface and physico-chemical properties of a glycolipid biosurfactant, mannosylerythritol lipid, from Candida antarctica

Candida sp. SY16 produces a glycolipid-type biosurfactant, mannosylerythritol lipid (MEL-SY16) which, at low concentrations, emulsifies hydrocarbons, vegetable oil, and crude oil. The molecular weight of MEL-SY16 was 634 Da and its hydrophilic lipophilic balance (HLB) was 8.8. It decreased the surface tension to 30 dyne cm^–1 at a critical micelle concentration (CMC) of 15.8 M, and the minimum interfacial tension against kerosene (a mixture of petroleum hydrocarbons) was 0.1 dyne cm^–1. MEL-SY16 was stable from pH 4 to pH 10, up to 90 °C for 1 h, and against NaCl up to 100 mM. It was readily degraded by microorganisms in activated sludge within a short time, and exhibited very low toxicity to mouse fibroblast cells. These properties confirm that MEL-SY16 is suitable for a variety of environmental applications.     

Extracellular production of a glycolipid biosurfactant, mannosylerythritol lipid, from Candida antarctica

Candida antarctica (sp. SY16) required avegetable oil as the carbon source to produce a biosurfactant, mannosylerythritol lipid (MEL-SY16). Biosurfactant production was 31 g l^–1 after 7 days in a batch culture and was not growth associated. In a two-stage culture, glycerol and oleic acid were used as an initial and a feeding carbon source, respectively, and 41 g l^–1 biosurfactant was produced after 8 days.     

Isolation of lipid activated pseudomurein precursors from Methanobacterium thermoautotrophicum

From cell extracts of the pseudomurein possessing methanogen Methanobacterium thermoautotrophicum two putative pseudomurein precursors were isolated and characterized: (1) an undecaprenyl pyrophosphate activated disaccharide pentapeptide composed of N-acetylglucosamine, N-acetyltalosaminuronic acid, alanine, glutamic acid and lysine in a molar ratio of 1:1:2:2:1 and (2) the corresponding undecaprenyl pyrophosphate activated tetrapeptide lacking one alanine residue. The isolation of these precursors show that the biosynthesis of the eubacterial murein and the methano-bacterial pseudomurein differs not only in the cytoplasmic step, as recently described, but also in the lipid stage.Abbreviations GlcNitol glucosaminitol - NAcTalNUA N-acetyltalosaminuronic acid - Udp undecaprenol - TLC thin layer chromatography     

Preliminary evaluation of the ethanol production from enzymatically hydrolyzed sugarcane bagasse

Ethanol production from sugarcane bagasse using immobilizedSaccharomyces cerevisiae was increased when coupled to a saccharification process involving the cellulase fromTrichoderma viride. However, the fixed costs of production, estimated to be about $US 0.4/tonne 95% ethanol, were high. Some key areas that influence these costs were identified.     

Enhancing of erythromycin production by <Emphasis Type="Italic">Saccharopolyspora erythraea</Emphasis> with common and uncommon oils

The enhancing effect of various concentrations of 18 oils and a silicon antifoam agent on erythromycin production by Saccharopolyspora erythraea was evaluated in a complex medium containing soybean flour and dextrin as the main substrates. The oils used consisted of sunflower, pistachio, cottonseed, melon seed, water melon seed, lard, corn, olive, soybean, hazelnut, rapeseed, sesame, shark, safflower, coconut, walnut, black cherry kernel and grape seed oils. The biomass, erythromycin, dextrin and oil concentrations and the pH value were measured. Also, the kinds and frequencies of fatty acids in the oils were determined. The productivity of erythromycin in the oil-containing media was higher than that of the control medium. However, oil was not suitable as a main carbon source for erythromycin production by S. erythraea. The highest titer of erythromycin was produced in medium containing 55 g/l black cherry kernel oil (4.5 g/l). The titers of erythromycin in the other media were also recorded, with this result: black cherry kernel > water melon seed > melon seed > walnut > rapeseed > soybean > (corn = sesame) > (olive = pistachio = lard = sunflower) > (hazelnut = cotton seed) > grape seed > (shark = safflower = coconut). In media containing various oils, the hyphae of S. erythraea were longer and remained in a vegetative form after 8 days, while in the control medium, spores were formed and hyphae were lysed.     

Optimization of fermentation conditions for thermostable cellulase production byThielavia terrestris

Summary Of the eighteen different carbon sources, solka floc was optimal for the induction of cellulases by the thermophilic fungusThielavia terrestris. The temperature optimum for growth was between 44–52°C. The effect of initial and controlled pH on fungal growth and cellulase production was investigated and the results obtained showed that the maximum volumetric productivity (6.07 I.U./1 per h) of filter paper activity was achieved when the pH was controlled at 4.5–5.0.     

Fatty acid composition in lipid fractions lengthwise the mycelium of Mortierella isabellina and lipid production by solid state fermentation

This paper investigates the correlation between mycelial age and fatty acid biosynthesis. The correlation was investigated by analyzing the lipid composition lengthwise the mycelium of the oleaginous fungus Mortierella isabellina, a potential producer of γ-linolenic acid (GLA). Young mycelia were rich in polar lipids (glycolipids plus sphingolipids and phospholipids), while neutral lipid content increased in aged mycelia. In young mycelia, each polar lipid fraction contained almost 40% (w/w) polyunsaturated fatty acids (PUFAs), but this content decreased to less than 30% (w/w) in aged mycelia. On the other hand, PUFA content in neutral lipids fluctuated slightly with age. These results indicate that PUFA biosynthesis is favored in young, fast growing mycelia, while it decreases significantly in aged mycelia. This trend was also observed when we grew M. isabellina on pear pomace, an agro-industrial waste. Pear pomace cultures yielded significant amounts of lipid, which reached 12% (w/w) in dry fermented mass. The produced lipid was rich in GLA and the maximum GLA content in dry fermented mass was 2.9 mg/g.     

Optimization of BLyS production and purification from Escherichia coli

B lymphocyte stimulator (BLyS) is a member of the tumor necrosis factor superfamily of cytokines. When the 152 amino acids of the C-terminus are associated into a homotrimer, this protein exhibits the ability to stimulate B cell proliferation and differentiation. Since numerous potential therapeutic indications have been identified for BLyS and other BLyS-derived products, large quantities of the protein are needed to further basic research and clinical trials. In this work, we have developed a high yield recombinant expression system that utilizes Escherichia coli as the host organism. Recombinant soluble BLyS (rsBLyS) production was achieved through the use of the phoA promoter system. This expression system, coupled to a semi-defined fermentation process, resulted in final purified yields of 435 mg/L of properly folded, trimeric, biologically active rsBLyS. This level of production is an 11-fold increase in volumetric yields compared to the process currently being used for clinical production. Furthermore, the increased rsBLyS production obtained from this process enabled the development of a conventional purification scheme that eliminated the use of a BLyS-affinity resin.     

The effect of organic nitrogen and glucose on the production of recombinant human insulin-like growth factor in high cell densityEscherichia coli fermentations

Summary Two kinds of fed batch fermentation processes were compared at a 10-liter scale to examine their effect on recombinant human insulin-like growth factor (IGF-1) gene expression inEscherichia coli. The difference between the two processes was the feed medium composition and whether the process used a single or dual feed during the course of the fermentation. In the dual feed system, organic nitrogen was delivered at a higher rate (50 g/h) than in the single feed system (5 g/h). The dual feed process resulted in a significant increase in IGF-1 yield. 30 mg IGF-1/g dry cell weight was synthesized in the dual feed system compared to 3 mg IGF-1/g dry cell weight obtained in the single feed system. The presence of high levels of organic nitrogen during the induction period may enhance IGF-1 synthesis by protecting the IGF-1 from proteolytic degradation. The IGF-1 yield decreased to 17 mg/g dry cell weight when the glucose supply was decreased from 17 g/h to 8 g/h during the induction period; however, an increase in glucose supply from 17 g/h to 50 g/h during the induction period did not enhance the IGF-1 synthesis. Thus, the enhancement of IGF-1 gene expression in the dual feed process was mainly dependent on a high level of organic nitrogen and an appropriate level of glucose in the medium during the induction period.     

Oil biodegradation by Bacillus strains isolated from the rock of an oil reservoir located in a deep-water production basin in Brazil

Sixteen spore forming Gram-positive bacteria were isolated from the rock of an oil reservoir located in a deep-water production basin in Brazil. These strains were identified as belonging to the genus Bacillus using classical biochemical techniques and API 50CH kits, and their identity was confirmed by sequencing of part of the 16S rRNA gene. All strains were tested for oil degradation ability in microplates using Arabian Light and Marlin oils and only seven strains showed positive results in both kinds of oils. They were also able to grow in the presence of carbazole, n-hexadecane and polyalphaolefin (PAO), but not in toluene, as the only carbon sources. The production of key enzymes involved with aromatic hydrocarbons biodegradation process by Bacillus strains (catechol 1,2-dioxygenase and catechol 2,3-dioxygenase) was verified spectrophotometrically by detection of cis,cis-muconic acid and 2-hydroxymuconic semialdehyde, and results indicated that the ortho ring cleavage pathway is preferential. Furthermore, polymerase chain reaction (PCR) products were obtained when the DNA of seven Bacillus strains were screened for the presence of catabolic genes encoding alkane monooxygenase, catechol 1,2-dioxygenase, and/or catechol 2,3-dioxygenase. This is the first study on Bacillus strains isolated from an oil reservoir in Brazil.     

The structure of lipid A from the lipopolysaccharide of Rhodopseudomonas gelatinosa 29/1

The structure of the lipid A component of Rhodopseudomonas gelatinosa 29/1 lipopolysaccharide was established. It constitutes a -1,6-glucosamine disaccharide substituted on either side by ester-and glycosidically-bound phosphate residues. Both phosphate groups are in turn nonstoichiometrically substituted by ethanolamine. The amino groups of the disaccharide are N-acylated by 3-acyloxyacyl residues: that at the reducing glucosamine by 3-O-(14:0) 10:0, and that at the non-reducing one by 3-O-(12:0)10:0. Hydroxyl groups at C-3 and C-3 are esterified by hydroxycapric acid. Hydroxyl groups at C-4 and C-6 in free hydroxycapric acid. Hydroxyl groups at C-4 and C-6 in free lipid A were shown to be unoccupied by methylation with diazomethane. A similar methylation of the intact lipopolysaccharide revealed a free hydroxyl group only at C-4, indicating that C-6 is the attachment site of 3-deoxy-d-anno-octulosonic acid.By preparative thin-layer chromatography free lipid A could be resolved into at least two major and one minor fractions. Lipid A of R. gelatinosa 29/1 shows high lethal toxicity, comparable to that of Salmonella lipid A.Abbreviations GlcN d-Glucosamine - dOclA 2-keto-3-deoxy-d-manno-octonate - GC-MS combined gas liquid chromatographymass spectrometry - LPS lipopolysaccharide Dedicated to Prof. Dr. Gerhart Drews on the occasion of his 60th birthday     

Phenotypic character of the lipid hydroperoxide-resistant mutant: Positive relationship between flocculation and resistance against lipid hydroperoxide inSaccharomyces cerevisiae

A lipid hydroperoxide-resistant mutant was isolated from a strain ofSaccharomyces cerevisiae. The mutant was resistant to 1.5mm tert-butylhydroperoxide and 1.0mm linoleic acid hydroperoxide. It flocculated in a Ca²⁺-dependent manner and the resistance against lipid hydroperoxide was suppressed by mannose, which also inhibited flocculation. A positive relationship between the acquirement of, the flocculent phenotype and resistance against lipid hydroperoxide is suggested. A protein with a molecular weight of 33 kDa was found on the surface of the mutant cell.     

Modeling of the pyruvate production with<Emphasis Type="Italic"> Escherichia coli</Emphasis> in a fed-batch bioreactor

A family of 10 competing, unstructured models has been developed to model cell growth, substrate consumption, and product formation of the pyruvate producing strain Escherichia coli YYC202 ldhA::Kan strain used in fed-batch processes. The strain is completely blocked in its ability to convert pyruvate into acetyl-CoA or acetate (using glucose as the carbon source) resulting in an acetate auxotrophy during growth in glucose minimal medium. Parameter estimation was carried out using data from fed-batch fermentation performed at constant glucose feed rates of q_VG=10 mL h^–1. Acetate was fed according to the previously developed feeding strategy. While the model identification was realized by least-square fit, the model discrimination was based on the model selection criterion (MSC). The validation of model parameters was performed applying data from two different fed-batch experiments with glucose feed rate q_VG=20 and 30 mL h^–1, respectively. Consequently, the most suitable model was identified that reflected the pyruvate and biomass curves adequately by considering a pyruvate inhibited growth (Jerusalimsky approach) and pyruvate inhibited product formation (described by modified Luedeking–Piret/Levenspiel term).List of symbols c_A acetate concentration (g L^–1) - c_A,0 acetate concentration in the feed (g L^–1) - c_G glucose concentration (g L^–1) - c_G,0 glucose concentration in the feed (g L^–1) - c_P pyruvate concentration (g L^–1) - c_P,max critical pyruvate concentration above which reaction cannot proceed (g L^–1) - c_X biomass concentration (g L^–1) - K_I inhibition constant for pyruvate production (g L^–1) - K_I^A inhibition constant for biomass growth on acetate (g L^–1) - K_P saturation constant for pyruvate production (g L^–1) - K_P inhibition constant of Jerusalimsky (g L^–1) - K_S^A Monod growth constant for acetate (g L^–1) - K_S^G Monod growth constant for glucose (g L^–1) - m_A maintenance coefficient for growth on acetate (g g^–1 h^–1) - m_G maintenance coefficient for growth on glucose (g g^–1 h^–1) - n constant of extended Monod kinetics (Levenspiel) (–) - q_V volumetric flow rate (L h^–1) - q_VA volumetric flow rate of acetate (L h^–1) - q_VG volumetric flow rate of glucose (L h^–1) - r_A specific rate of acetate consumption (g g^–1 h^–1) - r_G specific rate of glucose consumption (g g^–1 h^–1) - r_P specific rate of pyruvate production (g g^–1 h^–1) - r_P,max maximum specific rate of pyruvate production (g g^–1 h^–1) - t time (h) - V reaction (broth) volume (L) - Y_P/G yield coefficient pyruvate from glucose (g g^–1) - Y_X/A yield coefficient biomass from acetate (g g^–1) - Y_X/A,max maximum yield coefficient biomass from acetate (g g^–1) - Y_X/G yield coefficient biomass from glucose (g g^–1) - Y_X/G,max maximum yield coefficient biomass from glucose (g g^–1) - growth associated product formation coefficient (g g^–1) - non-growth associated product formation coefficient (g g^–1 h^–1) - specific growth rate (h^–1) - _max maximum specific growth rate (h^–1)