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.     

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.     

Production of sophorolipids by Candida bombicola grown on soy molasses as substrate*,**

Sophorolipids (SLs) were produced from Candida bombicola using soy molasses and oleic acid as co-substrates. The purified SLs were obtained at 21 g l(-1) and were 97% in lactone form. The major SL constituent (81% relative abundance) of the product mixture contains an oleoyl chain. The surface properties of the SLs obtained from the soy molasses/oleic acid fermentation had minimum surface-tension values of 37 mN m(-1) (pH 6) and 38 mN m(-1) (pH 9), and critical micelle concentration values of 6 mg l(-1) (pH 6) and 13 mg l(-1) (pH 9).     

Cerulenin inhibits de novo sophorolipid synthesis of Candida bombicola

Synthesis of medium-chain sophorolipids by Candida bombicola is a challenging objective. One of the difficulties is that the obtained sophorolipids always represent a mixture of medium-chain and native de novo formed or long-chain sophorolipids. The fatty acid moiety of de novo sophorolipids is derived from the de novo synthesis of fatty acids. Fatty acid synthesis can be blocked by the antifungal agent cerulenin, an inhibitor if the fatty acid synthase (FAS) complex acting on the β-ketoacyl thioester synthetase reaction. The toxic effect of cerulenin on C. bombicola was evaluated and 20 mg/ml was added in the stationary growth phase. No de novo formed sophorolipids were observed when the cells were cultured on merely glucose. Also when the hydrophilic substrate, 1,12-dodedanediol, was added, no de novo formed sophorolipids were detected, leading to a reduced complexity of the sophorolipid mixture.     

Cloning and characterisation of the glyceraldehyde 3-phosphate dehydrogenase gene of Candida bombicola and use of its promoter

The glyceraldehyde-3-phosphate dehydrogenase gene (GPD) of the sophorolipid producing yeast Candida bombicola was isolated using degenerated PCR and genome walking. The obtained 3,740 bp contain the 1,008 bases of the coding sequence and 1,613 and 783 bp of the upstream and downstream regions, respectively. The corresponding protein shows high homology to the other known GPD genes and is 74% identical to the gyceraldehyde-3-phosphate dehydrogenase of Yarrowia lipolytica. The particular interest in the C. bombicola GPD gene sequence originates from the potential use of its promoter for high and constitutive expression of homologous and heterologous genes. Southern blot analysis did not give any indication for the presence of multiple GPD genes and it can therefore be expected that the promoter can be used for efficient and high expression. This hypothesis was further confirmed by the biased codon usage in the GPD gene. GDP promoter fragments of different lengths were used to construct hygromycin resistance cassettes. The constructs were used for the transformation of C. bombicola and all of them, even the ones with only 190 bp of the GPD promoter, were able to render the cells resistant to hygromycin. The efficacy of a short GPD promoter can be a convenient characteristic for the construction of compact expression cassettes or vectors for C. bombicola. The GenBank accession number of the sequence described in this article is EU315245.     

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.     

Microbial oxidases of acidic -amino acids

Two microbial oxidases of acidic -amino acids have been purified to homogeneity. One is a -aspartate oxidase of the yeast Cryptococcus humicolus UJ1 that was induced markedly with -aspartate and is far more active toward -aspartate than -glutamate. The other is a -glutamate oxidase of Candida boidinii 2201 that preferred -glutamate to -aspartate as a substrate in terms of k_cat/K_m, but was not induced very effectively by -glutamate. The most potent competitive inhibitor of the C. humicolus -aspartate oxidase was malonate, and that of the C. boidinii -glutamate oxidase was -malate. The former enzyme was a homotetramer of 160 kDa consisting of subunits of 40 kDa, each of which contained 1 mol of FAD, while the latter was a monomer of 45 kDa. The N-terminal sequences of both enzymes were similar to those of other FAD enzymes and contained a consensus sequence common to most enzymes binding ADP-containing nucleotides. Peroxisomal localization of the C. humicolus -aspartate oxidase was shown by subcellular fractionation and morphological analysis via electron microscopy of C. humicolus cells, where induction of the enzyme was accompanied by induction of catalase and development of peroxisomes. The apo-form of C. humicolus -aspartate oxidase, prepared by removal of FAD was a monomeric protein of 40 kDa, and its binding with FAD proceeded in two stages. The K_d for the apoprotein-FAD complex was very low (8.2×10⁻¹² M) consistent with the observed tight binding. The C. humicolus -aspartate oxidase was essentially similar to other flavoprotein oxidases of acidic and neutral -amino acids with respect to its spectral properties and sensitivity to specific modifying reagents for arginyl and histidyl residues.     

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.     

Transformation of arachidonic acid to 19-hydroxy- and 20-hydroxy-eicosatetraenoic acids using Candida bombicola

Candida bombicola (ATCC 22214) and C. apicola (ATCC 96134), grown on glucose (100 g l^–1) and arachidonic acid (5Z, 8Z, 11Z, 14Z-eicosatetraenoic acid; AA), 1.25 g l^–1, synthesized sophorolipid up to 0.93 g l^–1. Acid hydrolysis of sophorolipid yielded 19-hydroxy-5Z, 8Z, 11Z, 14Z-eicosatetraenoic acid (19-HETE) and 20-hydroxy-5Z, 8Z, 11Z, 14Z-eicosatetraenoic acid (20-HETE) which were identified by TLC and GC-MS; the ratio of synthesis was 73:27, respectively. Conversion of AA by immobilized Candida bombicola, suspended in beads of 2% (w/v) calcium alginate for 96 h, gave an 83% conversion of 1 g AA l^–1 to 19- and 20-HETE. There was no significant loss in the efficiency of the immobilized cells after ten uses.     

Experimental design for the production of tensio-active agent by <Emphasis Type="Italic">Candida lipolytica</Emphasis>

The strategy of optimization using sequential factorial design was employed to enhance the tensio-active emulsifying agent produced by Candida lipolytica using soybean oil refinery residue as substrate. A full factorial design was used to evaluate the impact of three fermentation factors—amounts of refinery residue, glutamic acid and yeast extract. This allowed exclusion of the yeast extract. Full factorials designs were then sequentially used to optimize the levels of the residue and glutamic acid. The surface tension value was finally reduced to 25.29 mN/m. The maximum emulsifier activity using different substrates was within 40 h of cultivation. The surface tension of the cell-free broth containing the biosurfactant remained very stable during exposure to a wide range of pH (2–12), temperatures (0–120°C) and salinity (2–10% NaCl). The combination of an industrial waste and a cheap substrate therefore seems to be very promising for the low-cost production of potent biosurfactant.     

On-line biomass estimation in biosurfactant production process by <Emphasis Type="Italic">Candida lipolytica</Emphasis> UCP 988

Biomass is an important variable in biosurfactant production process. However, such bioprocess variable, usually, is collected by sampling and determined by off-line analysis, with significant time delay. Therefore, simple and reliable on-line biomass estimation procedures are highly desirable. An artificial neural network model (ANN) is presented for the on-line estimation of biomass concentration, in biosurfactant production by Candida lipolytica UCP 988, as a nonlinear function of pH and dissolved oxygen. Several configurations were evaluated while developing the optimal ANN model. The optimal ANN model consists of one hidden layer with four neurons. The performance of the ANN was checked using experimental data. The results obtained indicate a very good predictive capacity for the ANN-based software sensor with values of R2 of 0.969 and RMSE of 0.021 for biomass concentration. Estimated biomass using the ANN was proved to be a simple, robust and accurate method.     

Sophorose lipid production from lipidic precursors: Predictive evaluation of industrial substrates

Summary Sophorose lipids stand out as biosurfactants with a wide potential for industrial application and which can be produced in good yield from glucose and a lipidic cosubstrate.Candida bombicola CBS 6009 (ATCC 22214) was used in the present study. The influence of the lipidic cosubstrate on various aspects of production performance of these glycolipids (final concentration, yield) and on product composition (in particular, the structure of the hydroxy fatty acid vegetable and animal oils, markedly influenced product composition. In terms of production performance, the best substrates were oils or esters rich in C18:0 and C18:1 fatty acids. Optimal overall performance was obtained with esters (340 g L^–1 sophorose lipids with rapeseed esters). Conclusions drawn from the results allow predictive evaluation of lipidic industrial substrates.     

Enhancement of stability of biosurfactant produced by <Emphasis Type="Italic">Candida lipolytica</Emphasis> using industrial residue as substrate

This work describes experimental results carried out on the fermentation of Candida lipolytica, which produced a new biosurfactant when grown on a vegetable oil refinery residue as substrate. The cell-free culture broth containing the biosurfactant formed stable emulsions with hydrophobic natural compounds. Emulsification properties of the biosurfactant were not affected by salinity; however, treatment at a higher temperature decreased the emulsification activity, indicating applications in oil recovery. The isolated biosurfactant corresponds to a yield of 4.5 g/l, and the surface tension of water was reduced from 71 to 32 mN/m. Preliminary chemical characterizations showed that the biosurfactant consisted of protein (50%), lipid (20%), and carbohydrate (8%).     

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。     

Microbial challenges of poultry meat production

Food safety and shelf-life are both important microbial concerns in relation to broiler meat production. Focus is mainly placed on the absence or control of potentially pathogenic microbes such as Salmonella spp. and Campylobacter spp. but, from the commercial point of view, other spoilage bacteria also play a role as potential threats. Regarding food safety, the primary target should be the production of pathogen-free live animals, thus allowing slaughter plants to keep the processing line free of those microorganisms.Consumers believe that quality of foods from organic production is superior to foods from conventional production. The aim of the present study was to evaluate and compare the bacterial quality of chicken meat from organic and conventional production on the basis of traditional meat quality criteria. Fresh free grazing broiler carcasses were purchased directly from rural households (n = 80) and fresh retail chicken parts from conventional broiler carcasses from the local supermarkets in the region of Epirus (Poultry Producers Association. Arta) (n = 200).The samples were microbiologically tested for the presence of bacteria such as: Salmonella spp., Listeria monocytogenes, Staphylococcus aureus, Enterobacteriaceae, Escherichia coli, Campylobacter spp., and C. perfringens. Total count of aerobic mesophilic bacteria was also determined. Bacteriological tests were performed by means of standard methods of isolation and identification of individual species of bacteria according to ISO requirements. API-tests (bioMerieux) and Vitek 2 Identification System (bioMerieux) were used for biochemical determination. High levels of microbial contamination and occurrence of pathogenic bacteria at then fresh free grazing broiler carcasses reflect the poor hygienic quality of the slaughter conditions in the rural households.     

Kinetic studies on surfactant production byPseudomonas aeruginosa 44T1

Summary Biosurfactant accumulation occurred in the exponential and stationary phases. Production started when the nitrogen level was very low. Surfactant was produced with a diauxic pattern. Rhamnolipid concentration increased as nitrogen levels increased. Maximum product yield (Y _p/x) 2.9 was detected when C/N ratio was 6.6 and specific rate of product formation (p _q) was calculated. The examination of these kinetics parameters such as product yield and specific rate of product formation should be taken into account to develop a high efficient production process.     

Analysis of Candida utilis genomic DNA,homologous to cDNA of chicken A1 (1) collagen gene

Genomic fragments, homologous to chicken A1(1) collagen cDNA encoding triple-helical domain, were revealed by Southern analysis in various fungi. Such a genomic fragment from Candida utilis was cloned and sequenced. Analysis of the obtained DNA sequence revealed the 119 bp segment, which has possibly originated from the 54bp module common for the fibrillar collagen genes of higher eukaryotes.     

One-step production of unacetylated sophorolipids by an acetyltransferase negative Candida bombicola

Sophorolipids from the non-pathogenic yeast Candida bombicola are applied commercially as biodegradable, eco-friendly surface active agents. These sophorolipids are produced by cultivation in presence of a hydrophobic carbon source and are always constituted of a mixture of structurally related molecules. For some applications however, certain structural variants perform better than others. Acetylation of the sophorolipid molecule is such a parameter that gains interest because of its influence on water solubility, foaming properties, and biological activity. Fully unacetylated sophorolipids therefore are interesting metabolites but cannot be produced in a pure way by conventional cultivation. Here we report the identification of the acetyltransferase gene AT, responsible for acetylation of de novo synthesized sophorolipids in Candida bombicola. By the creation of a Δat deletion mutant, we could create a yeast strain producing purely unacetylated sophorolipids with a yield of 5 ± 0.7 g/L using rapeseed oil as hydrophobic carbon source. In contrast to the chemical production of unacetylated sophorolipids used nowadays, the microbial production leads to mainly lactonic sophorolipids, in addition to minor amounts of acidic sophorolipids.     

Microbial and bioconversion production of D-xylitol and its detection and application

D-Xylitol is found in low content as a natural constituent of many fruits and vegetables. It is a five-carbon sugar polyol and has been used as a food additive and sweetening agent to replace sucrose, especially for non-insulin dependent diabetics. It has multiple beneficial health effects, such as the prevention of dental caries, and acute otitis media. In industry, it has been produced by chemical reduction of D-xylose mainly from photosynthetic biomass hydrolysates. As an alternative method of chemical reduction, biosynthesis of D-xylitol has been focused on the metabolically engineered Saccharomyces cerevisiae and Candida strains. In order to detect D-xylitol in the production processes, several detection methods have been established, such as gas chromatography (GC)-based methods, high performance liquid chromatography (HPLC)-based methods, LC-MS methods, and capillary electrophoresis methods (CE). The advantages and disadvantages of these methods are compared in this review.     

Biosurfactant production and hydrocarbon-degradation by halotolerant and thermotolerant Pseudomonas sp.

A hydrocarbon degrading and biosurfactant producing, strain DHT2, was isolated from oil-contaminated soil. The organism grew and produced biosurfactant when cultured in variety of substrates at salinities up to 6 g l⁻¹ and temperatures up to 45°C. It was capable of utilizing crude oil, fuels, alkanes and PAHs as carbon source across the wide range of temperature (30–45°C) and salinity (0–6%). Over the range evaluated, the salinity and temperature did not influence the degradation of hydrocarbon and biosurfactant productions. Isolate DHT2 was identified as Pseudomonas aeruginosa by analysis of 16S rRNA sequences (100% homology) and biochemical analysis. PCR and DNA hybridization studies revealed that enzymes involved in PAH metabolism were related to the naphthalene dioxygenase pathway. Observation of both tensio-active and emulsifying activities indicated that biosurfactants were produced by DHT2 during growth on both, water miscible and immiscible substrates, including PAH. The biosurfactants lowered the surface tension of medium from 54.9 to 30.2 dN/cm and formed a stable emulsion. The biosurfactant produced by the organism emulsified a range of hydrocarbons with hexadecane as best substrate and toluene was the poorest. These findings further indicate that the isolate could be useful for bioremediation and bio-refining application in petroleum industry.