Optimizing the medium components in bioemulsifiers production by Candida lipolytica with response surface method

A response surface methodology was used to study bioemulsifier production by Candida lipolytica. A 2(4) full experimental design was previously carried out to investigate the effects and interactions of the concentrations of corn oil, urea, ammonium sulfate, and potassium dihydrogen orthophosphate on the emulsification activity (EA) of the bioemulsifier produced by C. lipolytica. The best EA value (3.727 units of emulsification activity (UEA)) was obtained with a medium composed of 0.4 g of urea, 1.1 g of ammonium sulfate, 2.04 g of potassium dihydrogen orthophosphate, 5 mL of corn oil, 50 mL of distilled water, and 50 mL of seawater. A curvature check was performed and revealed a lack of fit of the linear approximation. The proximity of the optimum point was evident, as was the need for quadratic model and second-order designs that incorporate the effect of the curvature. Medium constituents were then optimized for the EA using a three-factor central composite design and response surface methodology. The second-order model showed statistical significance and predictive ability. It was found that the maximum EA produced was 4.415 UEA, and the optimum levels of urea, ammonium sulfate, and potassium dihydrogen orthophosphate were, respectively, 0.544% (m/v), 2.131% (m/v), and 2.628% (m/v).     

Comparative study of Candida lipolytica yeasts with various abilities to produce citrate

The growth and the activity of some enzymes were studied in a Candida lipolytica strain 12a which did not synthesize acids in a medium with glucose under the conditions of nitrogen deficiency. The substrate was not assimilated and cyanide-resistant respiration did not develop in the strain under the conditions of profound nitrogen deficiency. The inability of cells to assimilate glucose at the stationary phase of growth resulted, apparently, from an abrupt decrease of phosphofructokinase and pyruvate dehydrogenase activities in the cells. The activities of pyruvate carboxylase and citrate synthase fell down abruptly at the same time.     

An integrated approach to produce biodiesel and monoglycerides by enzymatic interestification of babassu oil (Orbinya sp)

Two screenings of commercial lipases were performed to find a lipase with superior performance for the integrated production of biodiesel and monoglycerides. The first screening was carried out under alcoholysis conditions using ethanol as acyl acceptor to convert triglycerides to their corresponding ethyl esters (biodiesel). The second screening was performed under glycerolysis conditions to yield monoglycerides (MG). All lipases were immobilized on silica–PVA composite by covalent immobilization. The assays were performed using babassu oil and alcohols (ethanol or glycerol) in solvent free systems. For both substrates, lipase from Burkholderia cepacia (lipase PS) was found to be the most suitable enzyme to attain satisfactory yields. To further improve the process, the Response Surface Methodology (RSM) was used to determine the optima operating conditions for each biotransformation. For biodiesel production, the highest transesterification yield (>98%) was achieved within 48 h reaction at 39 °C using an oil-to-ethanol molar ratio of 1:7. For MG production, optima conditions corresponded to oil-to-glycerol molar ratio of 1:15 at 55 °C, yielding 25 wt.% MG in 6 h reaction. These results show the potential of B. cepacia lipase to catalyze both reactions and the feasibility to consider an integrated approach for biodiesel and MG production.     

Fermentation of 1-hexadecene by Candida lipolytica

Candida lipolytica, strain Phaff, was grown on 1.0% 1-hexadecene as sole source of carbon. Several oxidative intermediates were isolated and identified. Based on these intermediates two pathways are proposed for the degradation of the 1-alkene via the methyl group and the double bond. Subterminal oxidation of the 1-alkene was also indicated. Cell yield, lipid content, fatty acid profile and 1, 2-diol concentration are given for various rates of aeration during growth in a fermentor.     

解脂假丝酵母(Candida lipolytica)对铜的吸附

研究了解脂假丝酵母的表面特性及培养时间、pH值、铜浓度、菌体投加量、吸附时间等因素对铜吸附的影响,并探讨了吸附动力学特征。结果表明,菌体表面可能有-OH和-PO₄^3-,培养96 h的菌体吸附性能最佳,适宜pH为4.0-6.0,适宜菌体投加量为25.0g·L^-1(湿重)。在初始浓度为20mg·L^-1的铜溶液中投加25.0g·L^-1(湿重)的菌体,吸附2h,铜的去除率最高达86.5%。铜浓度为5,10mg·L^-1时,铜的去除率高达95%以上。动力学分析表明,在实验设定的浓度范围内解脂假丝酵母对铜的吸附基本符合Freundlich吸附模型。红外光谱分析表明吸附后-OH吸收峰蓝移18cm^-1,其它吸收峰没有明显的变化。     

Production of uricase by Candida tropicalis using n-alkane as a substrate.

Production of uricase (urate oxidase, EC 1.7.3.3) by n-alkane-utilizing Candida tropicalis pK233 was studied. Although the yeast showed very low enzyme productivity under growing conditions on glucose or an n-alkane mixture (C10 to C13) (less than 2 U/g of dry cells), enzyme formation was enhanced markedly in an induction medium consisting of potassium phosphate buffer, MgSO4, uric acid, and an n-alkane mixture (47 U/g of dry cells) or glucose (21 U/g of dry cells). Of the carbon sources tested, the n-alkane mixture was the most suitable for enzyme production. Appropriate aeration also stimulated uricase formation. In addition to uric acid, xanthine, guanine, adenine, and hypoxanthine were also effective for inducing uricase. Under optimum conditions, the maximum yield of the enzyme was 91 U/g of dry cells. Uricase thus induced was localized in the microbodies of the yeast.     

Production of uricase by Candida tropicalis using n-alkane as a substrate.

Production of uricase (urate oxidase, EC 1.7.3.3) by n-alkane-utilizing Candida tropicalis pK233 was studied. Although the yeast showed very low enzyme productivity under growing conditions on glucose or an n-alkane mixture (C10 to C13) (less than 2 U/g of dry cells), enzyme formation was enhanced markedly in an induction medium consisting of potassium phosphate buffer, MgSO4, uric acid, and an n-alkane mixture (47 U/g of dry cells) or glucose (21 U/g of dry cells). Of the carbon sources tested, the n-alkane mixture was the most suitable for enzyme production. Appropriate aeration also stimulated uricase formation. In addition to uric acid, xanthine, guanine, adenine, and hypoxanthine were also effective for inducing uricase. Under optimum conditions, the maximum yield of the enzyme was 91 U/g of dry cells. Uricase thus induced was localized in the microbodies of the yeast.     

Lipolytic fermentations of stickwater by Geotrichum candidum and Candida lipolytica.

Stickwater, a by-product of the fish meal and oil industry, is an aqueous suspension of fish proteins, lipids, and other materials, and also contains soluble nonprotein nitrogen but not carbohydrate. It is usually partially evaporated by heat to a marketable form called "fish solubles," which is sold with an acid preservative as an animal feed supplement. However, fish solubles are only used to a limited extent in feeds, because the lipids of solubles (averages 11%) are relatively prone to oxidative rancidity development. An investigation was undertaken to digest and/or stabilize lipids in stickwater by lipolytic fermentations and, at the same time, to attempt to increase the protein content as single cell protein. Strains of the yeast Candida lipolytica and the yeast like mold Geotrichum candidum were employed for these investigations. Stickwater fermentations were performed in a laboratory bench top fermentor. Respirometric studies of lipid metabolic activity and microbial observations were periodically performed during these fermentations. Rapid microbial growth and metabolic activity were observed in well aerated cultures. Fermented products were evaluated for chemical composition. Lipid residues were characterized by thin-layer chromatographic procedures. There was evidence of abundant microbial growth, increased lipolytic activity, and decreased lipid content. However, evidence was lacking to show that the protein content of stickwater was actually increased.     

Yarrowia lipolytica as a biotechnological chassis to produce usual and unusual fatty acids

One of the most promising alternatives to petroleum for the production of fuels and chemicals is bio-oil based chemistry. Microbial oils are gaining importance because they can be engineered to accumulate lipids enriched in desired fatty acids. These specific lipids are closer to the commercialized product, therefore reducing pollutants and costly chemical steps. Yarrowia lipolytica is the most widely studied and engineered oleaginous yeast. Different molecular and bioinformatics tools permit systems metabolic engineering strategies in this yeast, which can produce usual and unusual fatty acids. Usual fatty acids, those usually found in triacylglycerol, accumulate through the action of several pathways, such as fatty acid/triacylglycerol synthesis, transport and degradation. Unusual fatty acids are enzymatic modifications of usual fatty acids to produce compounds that are not naturally synthetized in the host. Recently, the metabolic engineering of microorganisms has produced different unusual fatty acids, such as building block ricinoleic acid and nutraceuticals such as conjugated linoleic acid or polyunsaturated fatty acids. Additionally, microbial sources are preferred hosts for the production of fatty acid-derived compounds such as γ-decalactone, hexanal and dicarboxylic acids. The variety of lipids produced by oleaginous microorganisms is expected to rise in the coming years to cope with the increasing demand.     

Isolation of a bioemulsifier from Candida lipolytica.

The yeast Candida lipolytica produced an inducible extracellular emulsification activity when it was grown with a number of water-immiscible carbon substrates. Negligible emulsification activity was produced by this yeast when it was grown with glucose as the carbon substrate. In hexadecane-supplemented cultures, emulsification activity was first detected after 36 h of growth, with maximum production after 130 h. A water-soluble emulsification activity was partially purified by repeated solvent extractions of the culture filtrate. This emulsifier, which we named liposan, was primarily composed of carbohydrate. Maximum emulsification activity was obtained when the ratio of hexadecane to liposan was 50:1. Maximum emulsification activity was obtained from pH 2 to 5. Liposan was heat stable to temperatures up to 70 degrees C, with a 60% loss in activity after heating for 1 h at 100 degrees C. Liposan effected stable oil-in-water emulsions with a variety of hydrocarbons.     

Cultivation of the yeast Candida lipolytica on hydrocarbons. II. One-stage continuous cultivation on gas oil

Continuous cultivation of the yeast Candida lipolytica on gas oil was studied from the viewpoint of biomass production and oil deparaffination. Optimum conditions wore found at the dilution rate D = 0.16–0.19 when biomass productivity 1.7 g/l/hr and yield coefficient. y = 0.92 were achieved. At deparaffination to the same freezing point, more than double the production of biomass and deparaffined oil during a given time unit was achieved in a continuous process than in batch cultivation. Consumption of substrate was followed in both cultivation processes and it was confirmed that individual n-alkanes of gas oil were degraded at various rates and yields. Results proved optimum cultivation conditions to depend on concentration and composition of the paraffinic fraction of gas oil used. To achieve these conditions the continuous process may be controlled by choice; of suitable dilution rate and concentration of gas oil.     

Properties of Candida lipolytica mutants with the modified glyoxylate cycle and their ability to produce citric and isocitric acid

Summary Enzyme activities of the tricarboxylic acid (TCA) cycle and the anaplerotic pathways, as well as the cell cytology of two C. lipolytica mutants with the modified glyoxylate cycle and their parent strain were studied during the exponential growth phase on glucose or hexadecane.Among the TCA cycle enzymes, the key enzyme citrate synthase had the highest activity in all three strains grown on both substrates. NAD-dependent isocitrate dehydrogenase had the minimum activity. All strains had well-developed mitochondria.Pyruvate carboxylation was active in the wild strain and mutant 2 grown on glucose, where this reaction is the basic anaplerotic pathway for oxal-acetate synthesis; mutant 1 had actively functioning enzymes for both anaplerotic pathways — pyruvate carboxylase, isocitrate lyase and malate synthase.During hexadecane assimilation, the number of peroxisomes in all strains increased sharply, accompanied by a simultaneous increase in isocitrate lyase activity.The low activities of both isocitrate lyase and pyruvate carboxylase in mutant 2 give reason to believe that this strain has an additional pathway for oxalacetic acid synthesis during the assimilation of n-alkane.     

解脂假丝酵母对重金属工业废水的吸附特性

利用解脂假丝酵母对Cr(Ⅵ)、Ni(Ⅱ)和Cu(Ⅱ)共存的模拟重金属废水及3种实际重金属废水进行了微生物吸附,结果表明,pH、吸附时间和菌浓度等均是显著的影响因素.Cr(Ⅵ)、Ni(Ⅱ)和Cu(Ⅱ)的去除均符合准一级和准二级动力学模型,其中准二级模型的拟合效果最理想,证明该菌种对重金属的吸附包括了多个步骤,其中化学吸附是限速步骤.解脂假丝酵母对共存重金属的生物吸附效果理想,1g·L^-1菌体在120min时,对18.7～37.86mg·L^-1Cr、2.39～9.21mg·L^-1Cu、2.27～9.87mg·L^-1Ni和0.43～1.32mg·L^-1Zn的去除率分别为81.6%～84.6%、84.0%～100%、84.1%～100%和93.9%～100%.菌体的蛋白质、脂质和多糖均参与了重金属吸附,起作用的主要功能团是-OH、-NH₂、-CH₂、-CH₃、-COOH、-CHO、C=C、-PO₄^3-和-SO₃H.     

Degradation of Hydrocarbons by Members of the Genus Candida II. Oxidation of n-Alkanes and 1-Alkenes by Candida lipolytica

Candida lipolytica ATCC 8661 was grown in a mineral-salts hydrocarbon medium. n-Alkanes and 1-alkenes with 14 through 18 carbon atoms were used as substrates. Ether extracts of culture fluids and cells obtained from cultures grown on the various substrates were analyzed by thin-layer and gas-liquid chromatography. Analyses of fluids from cultures grown on n-alkanes indicated a predominance of fatty acids and alcohols of the same chain length as the substrate. In addition, numerous other fatty acids and alcohols were present. Analyses of saponifiable and nonsaponifiable material obtained from the cells revealed essentially the same products. The presence of primary and secondary alcohols, as well as fatty acids, of the same chain length as the n-alkane substrate suggested that attack on both the methyl and α-methylene group was occurring. The significance of these two mechanisms in the degradation of n-alkanes by this organism was not evident from the data presented. Analyses of fluids from cultures grown on 1-alkenes indicated the presence of 1,2-diols, as well as ω-unsaturated fatty acids, of the same chain length as the substrate. Alcohols present were all unsaturated. Saponifiable and nonsaponifiable material obtained from cells contained essentially the same products. The presence of 1,2-diols and ω-unsaturated fatty acids of the same chain length as the substrate from cultures grown on 1-alkenes indicated that both the terminal methyl group and the terminal double bond were being attacked.     

Role and control of isocitrate lyase in Candida lipolytica.

Mutants of Candida lipolytica that were unable to grow on acetate but able to utilize succinate or glycerol as a sole carbon source were isolated. Amongst the mutants isolated, one strain (Icl-) was specifically deficient in isocitrate lyase activity, whereas another strain (Acos-) was deficient in acetyl coenzyme A synthetase activity. Since the Icl- mutant could not grow either on n-alkane or its derivatives, such as fatty acid and long-chain dicarboxylic acid, any anaplerotic route other than the glyoxylate pathway was inconceivable as far as growth on these carbon sources was concerned. Acetyl coenzyme A is most likely a metabolic inducer of isocitrate lyase and malate synthase, because the Acos- mutant was characterized by the least susceptibility to induction of these enzymes by acetate. The structural gene for isocitrate lyase was most probably impaired in the Icl- mutant, since revertants (Icl-) produced thermolabile isocitrate lyase. The production of isocitrate from n-alkane by the revertants was enhanced in comparison with the parental strain.