Efficient utilization of inulin and glycerol as fermentation substrates in erythritol and citric acid production using <Emphasis Type="Italic">Yarrowia lipolytica</Emphasis> expressing inulinase

Inulin and glycerol were used as substrates for efficient erythritol and citric acid production by newly engineered Yarrowia lipolytica strains. Hydrolysis of inulin by the Y. lipolytica Wratislavia K1 strain was established by expressing the Kluyveromyces marxianus INU1 gene. Erythritol was produced in two stages: inulin was used for biomass formation, followed by erythritol biosynthesis initiated by glycerol addition. The highest titer of erythritol obtained, 120.9 g L^?1 with the yield of 0.6 g g^?1, was produced by the K1 INU 6 strain. Moreover, the K1 INU 6 strain in fed-batch culture produced a high amount of citric acid: 105.2 g L^?1 after 235 h from 200 g L^?1 of inulin. Maximum activity of inulinase during this culture was 14000 U g^?1 of cell dry mass. The presented study proves the potential of new Y. lipolytica transformants for efficient erythritol and citric acid production from inexpensive raw materials such as inulin and glycerol.     

Mineral Supplementation Increases Erythrose Reductase Activity in Erythritol Biosynthesis from Glycerol by Yarrowia lipolytica

The aim of this study was to examine the impact of divalent copper, iron, manganese, and zinc ions on the production of erythritol from glycerol by Yarrowia lipolytica and their effect on the activity of erythrose reductase. No inhibitory effect of the examined minerals on yeast growth was observed in the study. Supplementation with MnSO₄·7H₂O (25 mg l^?1) increased erythritol production by Y. lipolytica by 14.5 %. In the bioreactor culture with manganese ion addition, 47.1 g l^?1 of erythritol was produced from 100.0 g l^?1 of glycerol, which corresponded to volumetric productivity of 0.87 g l^?1 h^?1. The addition of Mn²⁺ enhanced the intracellular activity of erythrose reductase up to 24.9 U g^?1 of dry weight of biomass (DW), hence, about 1.3 times more than in the control.     

<Emphasis Type="Italic">Yarrowia lipolytica</Emphasis> application as a prospective approach for biosynthesis of pyruvic acid from glycerol

With the problems related to chemical methods of pyruvic acid (PA) synthesis, a fast-growing interest has been observed in research aiming at reducing the production cost of PA by applying biotechnological methods. This study aimed to investigate the potential applicability of Yarrowia lipolytica Wratislavia 1.31 yeast strain for valorisation of pure and crude glycerol through the production of industrially desired PA. Conditions required for the effective PA biosynthesis, i.e., pH value, thiamine concentration, agitation, and substrate concentration, were examined in batch and fed-batch cultivation modes. The efficient production of PA occurred under the limitation of thiamine (1 µg L^?1) and was stimulated by moderate pH (4.5) and agitation (800 rev min^?1) of the culture. Under optimal conditions, Y. lipolytica Wratislavia 1.31 was able to produce 85.2 g L^?1 of PA with volumetric productivity of 0.90 g L^?1 h^?1. The yield of PA biosynthesis reached a high level of 1.03 g g^?1. Obtained results confirmed the aptitude of Y. lipolytica yeast to produce high amounts of PA from simple glycerol-containing media. Presented process was very promising and might be considered as an attractive alternative for currently used chemical methods of PA synthesis.     

Simultaneous production of citric acid and erythritol from crude glycerol by Yarrowia lipolytica Wratislavia K1

This study shows a possible microbial process for utilization of crude glycerol generated by the biodiesel industry for citric acid and erythritol production. Simultaneous production of citric acid and erythritol under nitrogen-limited conditions with glycerol as the carbon source was achieved with an acetate negative mutant of Y. lipolytica Wratislavia K1 in fed-batch cultivations. The effect of the initial glycerol concentration (from 30–180 g dm⁻³) on the citrate and erythritol production was investigated. As a result of the experiments, maximum citric acid production (110 g dm⁻³) and a very high amount of erythritol (81 g dm⁻³) were determined after 168 h of fed-batch cultivation with the initial glycerol concentration of 150 g dm⁻³ and the total glycerol concentration of 250 g dm⁻³. In addition, the citric acid to isocitric acid ratio of the products from this strain was 35.5:1. Presented at the 34th International Conference of the Slovak Society of Chemical Engineering, Tatranské Matliare, 21–25 May 2007.     

Oxygen transfer rate and pH as major operating parameters of citric acid production from glycerol by <Emphasis Type="Italic">Yarrowia lipolytica</Emphasis> W29 and CBS 2073

The amount of citric acid (CA) produced by Yarrowia lipolytica is dependent on the yeast strain and growth conditions such as pH, oxygen availability and medium composition. In this work, an experimental design based on the Taguchi method was applied to evaluate the effect of parameters: pH, carbon/nitrogen (C/N) ratio in the medium, oxygen mass transfer rate (OTR) and salts concentration, on the CA production by two Y. lipolytica strains, W29 (ATCC 20460) and CBS 2073. OTR and pH showed higher influence on the CA production for both strains. The increase of OTR from air to the culture medium led to a two- and three-fold improvement of the CA production by Y. lipolytica CBS 2073 and W29, respectively. Besides the individual effects of the parameters, a significant influence of the interaction between these parameters was observed, mainly between OTR and salts. Different values of the parameters were found at the optimum conditions for each strain, but the theoretically predicted and experimentally obtained citric acid concentrations (c_CA) were approximately 10 g L^?1 for both strains. The optimal conditions were also validated employing crude glycerol from biodiesel industry as a substrate, and similar behavior of the strains was observed.     

The Overexpression of YALI0B07117g Results in Enhanced Erythritol Synthesis from Glycerol by the Yeast Yarrowia lipolytica

The unconventional yeast Yarrowia lipolytica is used to produce erythritol from glycerol. In this study, the role of the erythrose reductase (ER) homolog YALI0B07117g in erythritol synthesis was analyzed. The deletion of the gene resulted in an increased production of mannitol (308%) and arabitol (204%) before the utilization of these polyols began. The strain overexpressing the YALI0B07117g gene was used to increase the erythritol yield from glycerol as a sole carbon source in batch cultures, resulting in a yield of 0.4 g/g. The specific consumption rate (qs) increased from 5.83 g/g/L for the WT strain to 8.49 g/g/L for the modified strain and the productivity of erythritol increased from 0.28 g/(L h) for the A101 strain to 0.41 g/(L h) for the modified strain. The application of the research may prove positive for shortening the cultivation time due to the increased rate of consumption of the substrate combined with the increased parameters of erythritol synthesis.     

Elucidating the Effect of Glycerol Concentration and C/N Ratio on Lipid Production Using <Emphasis Type="Italic">Yarrowia lipolytica</Emphasis> SKY7

The high demand for renewable energy and increased biodiesel production lead to the surplus availability of crude glycerol. Due to the above reason, the bio-based value addition of crude glycerol into various bioproducts is investigated; among them, microbial lipids are attractive. The present study was dedicated to find the optimal glycerol concentration and carbon/nitrogen (C/N) ratio to produce maximum lipid using Yarrowia lipolytica SKY7. The glycerol concentration (34.4 to168.2 g/L) and C/N ratio (25 to 150) were selected to investigate to maximize the lipid production. Initial glycerol concentration 112.5 g/L, C/N molar ratio of 100, and with 5 % v/v inoculum supplementation were found to be optimum for biomass and lipid production. Based on the above optimal parameters, lipid concentration of 43.8 % w/w with a biomass concentration of 14.8 g/L was achieved. In the case of glycerol concentration, the maximum Yp/s (0.192 g/g); Yx/s (0.43 g/g) was noted when the initial glycerol concentration was 112.5 g/L with C/N molar ratio 100 and inoculum volume 5 % v/v. The glycerol uptake was also noted to increase with the increase in glycerol concentration. At low C/N ratio, the glycerol consumption was found to be high (79.43 g/L on C/N 25) whereas the glycerol consumption was observed to decrease when the C/N ratio was raised to 150 (40.8 g/L).     

Simultaneous Production of Amyloglucosidase and Exo-Polygalacturonase by <Emphasis Type="Italic">Aspergillus niger</Emphasis> in a Rotating Drum Reactor

Simultaneous production of amyloglucosidase (AMG) and exo-polygalacturonase (exo-PG) was carried out by Aspergillus niger in substrate of defatted rice bran in a rotating drum bioreactor (RDB) and studied by a 3¹ × 2² factorial experimental design. Variables under study were A. niger strains (A. niger NRRL 3122 and A. niger t0005/007-2), types of inoculum (spore suspension and fermented bran), and types of inducer (starch, pectin, and a mix of both). Solid-state fermentation process (SSF) was conducted at 30 °C under 60-vvm aeration for 96 h in a pilot scale. Production of AMG and exo-PG was significantly affected by the fungal strain and the type of inoculum, but inducers did not trigger any significant effect, an evidence of the fact that these enzymes are constitutive. The maximum activity of exo-PG was 84 U g_dm ^?1 whereas the maximum yield of AMG was 886.25 U g_dm ^?1.     

Synthesis and biotests of 2-aryl-5-arylmethylidene-substituted 1,3-oxazol-5(4<Emphasis Type="Italic">H</Emphasis>)-ones and <Emphasis Type="Italic">N</Emphasis>-methyl-3,5-dihydro-4<Emphasis Type="Italic">H</Emphasis>-imidazol-4-ones as combretastatin A-4 analogs

A series of 2-aryl-5-arylmethylidene-1,3-oxazol-5(4H)-ones and 2-aryl-5-arylmethylidene-N-methyl-3,5-dihydro-4H-imidazol-4-ones was synthesized as structural analogs of combret- astatin A-4 (a compound possessing antitumor activity). (5Z)-5-[(4-Methoxyphenyl)methyl-idene]-3-methyl-2-(4-methylphenyl)-3,5-dihydro-4H-imidazol-4-one was found to exhibit the highest cytotoxicity against cells of human A549 lung carcinoma line (EC₅₀ = 6±0.8 μmol L^?1).     

Production and characterization of surfactant-stable fungal keratinase from <Emphasis Type="Italic">Gibberella intermedia</Emphasis> CA3-1 with application potential in detergent industry

Surfactant-stable keratinases with good properties are promising candidates for extensive applications in detergent industries. A novel fungal keratinase-producing strain, Gibberella intermedia CA3-1, is described in this study. The keratinase production medium was optimized and composed of 10 g L^?1 of wool powder, 5 g L^?1 of tryptone, 10 g L^?1 of maltodextrin and 0.5 g L^?1 of NaCl. Keratinase activity was increased up to 109 U mL^?1 from 15 U mL^?1 by culture optimization. The optimal reaction pH and temperature of the enzyme were 9.0 and 60°C, respectively. The keratinase activity could be improved by sodium dodecyl sulphate (SDS), and it remained stable in the presence of several surfactants and commercial detergents. G. intermedia keratinase was proved to completely remove blood stains from cotton cloth when combined with detergents. These findings indicate that this fungal keratinase is a promising catalyst for the application in detergent industry. To our knowledge, this is the first report on keratinase production by Gibberella genus.     

Thermodynamic Study on the Protonation and Na<Superscript>+</Superscript>, Ca<Superscript>2+</Superscript>, Mg<Superscript>2+</Superscript>-Complexation of a Biodegradable Chelant (HEIDA) at Different Ionic Strengths and Temperatures

A potentiometric method has been used for the determination of the protonation constants of N-(2-hydroxyethyl)iminodiacetic acid (HEIDA or L) at various temperatures 283.15?≤?T/K?≤?383.15 and different ionic strengths of NaCl_(aq), 0.12?≤?I/mol·kg^?1?≤?4.84. Ionic strength dependence parameters were calculated using a Debye–Hückel type equation, Specific Ion Interaction Theory and Pitzer equations. Protonation constants at infinite dilution calculated by the SIT model are \( \log_{10} \left( {{}^{T}K_{1}^{\text{H}} } \right) = 8.998 \pm 0.008 \) (amino group), \( \log_{10} \left( {{}^{T}K_{2}^{\text{H}} } \right) = 2.515 \pm 0.009 \) and \( \log_{10} \left( {{}^{T}K_{3}^{\text{H}} } \right) = 1.06 \pm 0.002 \) (carboxylic groups). The formation constants of HEIDA complexes with sodium, calcium and magnesium were determined. In the first case, the formation of a weak complex species, NaL, was found and the stability constant value at infinite dilution is log₁₀K_NaL?=?0.78?±?0.23. For Ca²⁺ and Mg²⁺, the CaL, CaHL, CaL₂ and MgL species were found, respectively. The calculated stability constants for the calcium complexes at T?=?298.15 K and I?=?0.150 mol·dm^?3 are: log₁₀β_CaL?=?4.92?±?0.01, log₁₀β_CaHL?=?11.11?±?0.02 and \( \log_{10} \beta_{\text{Ca{L}}_{2}} \)?=?7.84?±?0.03, while for the magnesium complex (at I?=?0.176 mol·dm^?3): log₁₀β_MgL?=?2.928?±?0.006. Protonation thermodynamic functions have also been calculated and interpreted.     

Ethanol Production from Starch Hydrolyzates using <Emphasis Type="Italic">Zymomonas mobilis</Emphasis> and Glucoamylase Entrapped in Polyvinylalcohol Hydrogel

The glucoamylase from Aspergillus niger, immobilized into poly(vinylalcohol) hydrogel lens-shaped capsules LentiKats®, was used for simultaneous saccharification and fermentation (SSF) with Zymomonas mobilis in free form. This system was stable in both the repeated batch and continuous mode of SSF. The microorganism was found to adsorb on the capsules with immobilized enzyme. This increased the ethanol productivity of the repeated batch system with 5% w/v of immobilized glucoamylase almost 2.1 times (7.2 g l^?1 h^?1) compared to free enzyme–free microorganism system (3.5 g l^?1 h^?1). The continuous SSF with the immobilized glucoamylase (11.5% w/v) tested for 15 days had productivity 10 g l^?1 h^?1, which is comparable to continuous experiments on semi-defined glucose medium (10 g l^?1 h^?1). These two systems were stable in both glucoamylase activity and microorganism productivity.     

Mechanism of the oxidation of hydrazoic acid by tetrachloroaurate(III) ion

The oxidation of hydrazoic acid in perchloric acid in the absence of added chloride under pseudo first-order conditions ([HN₃] » [AuCl ₄ ^? ]) is first order in [Au(III)]. Michaelis–Menten type of dependence (linear plots of k _obs ^?1 vs [HN₃]^?1) is observed with respect to [HN₃]. The k _obs is independent of ionic strength and the plot between k _obs ^?1 and [H⁺] is linear. The inner-sphere mechanism is consistent with the formation of an axial complex (K = 25 dm³ mol^?1) between AuCl₃(HO)^? ion and HN₃ prior to its rate determining decomposition (k = 0.0182 s^?1). It is inferred that the free radicals N ₃ ^? do not oxidise Au(II). The reaction becomes outer-sphere in the presence of added Cl^? ions which are inferred to form a cage around the hydronium ion surrounding the AuCl ₄ ^? ions. The penetration of N ₃ ^? through the cage is rate controlling and within the cage, the electron transfer from N ₃ ^? ion to AuCl ₄ ^? is fast. The value of the rate determining constant k ₂ is 0.547 dm³ mol^?1 s^?1 and the equilibrium constant K _Cl for the cage formation is 5 dm³ mol^?1 at 25 °C. It is calculated that the minimum HN₃ concentration required before the reaction exhibits zero-order dependence in HN₃ is 0.31 mol dm^?3 when [H⁺] = 0.18 mol dm^?3 at 25 °C.     

On <Emphasis Type="Italic">Trans</Emphasis>-Resveratrol in Aqueous Solutions

A thermodynamic study on aqueous solutions of trans-Resveratrol (3,5,4′-trihydroxy-trans-stilbene) at 25.00 ± 0.02 °C, in 0.5 mol·dm^?3 NaCl, has been conducted. The protolysis equilibria and the complex formation between trans-Resveratrol and a metal(II), namely Mn²⁺ and Cu²⁺, have been investigated. The experimental method consists of potentiometric, spectrophotometric (absorption and emission) acid–base titrations. The pH range investigated is 2.5 ≤ pH ≤ 13 for the binary system, while for the ternary system it is 2.5 ≤ pH ≤ 6. The results of the graphical and numerical methods adopted indicate, for all the systems investigated, the formation of a predominant Me(II)–trans-Resveratrol mononuclear complex. UV–Vis absorption spectra and desorption/ionization time of flight mass spectra show the occurrence of hydrolytic species exhibiting a higher molecular weight than the Resveratrol molecule, becoming more evident as the pH and the time increased. Moreover, high performance liquid chromatography analysis and infrared spectroscopy of aqueous cis/trans-Resveratrol solutions upon excitation at 300 nm have highlighted a highly fluorescent compound, with absorption maximum at 250 nm, and a blue shift in the fluorescence emission that have not previously been reported.     

Effects of some nucleating agents on the supercooling of erythritol to be applied as phase change material

Nine nucleating agents, calcium pimelate (CaPi), bicyclic [1, 2, 2]heptane di-carboxylate (HPN-68), a commercially obtained aryl amide nucleating agent (TMB-5), calcium salt of hexahydrophthalic acid (HPN-20E), 1,3:2,4-di-p-methylbenzylidene sorbitol (MDBS) and sodium, potassium, magnesium and calcium salt of benzene-1, 3, 5-tricarboxylic acid (Na₃BTC, K₃BTC, Mg₃BTC₂ and Ca₃BTC₂, respectively), were applied to reduce the supercooling of erythritol, and their effects were investigated by cyclic differential scanning calorimetry (DSC). The results revealed that Na₃BTC and K₃BTC could not induce erythritol to crystallize under the experiment condition. MDBS could only make erythritol to crystallize at a temperature slightly higher than that of pure erythritol, and the effect was unstable. Mg₃BTC₂, Ca₃BTC₂ and HPN-68 could induce erythritol to crystallize at relatively high temperature, but the peak temperature of crystallizing (T _{p, cr}) and the phase change enthalpy of crystallizing (Δ_cr H) decreased greatly as the melting–crystallizing cycles increased. HPN-20E-doped erythritol crystallized at a high temperature with the T _{p, cr} of 69.3 °C at the first cycle, but the T _{p, cr} and Δ_cr H varied greatly during the melting–crystallizing cycles. CaPi and TMB-5 could induce erythritol to crystallize at a stable temperature with the T _{p, cr} of about 69 °C and 64 °C, respectively, and with a stable Δ_cr H of about 204 and 185 J g^?1, respectively, in all melting–crystallizing cycles. Hence, CaPi- and TMB-5-doped erythritol could be used as PCMs and applied in thermal energy storage in which the energy was absorbed at a high temperature and released at a lower but stable temperature.     

Effect of medium pH on chemical selectivity of oxalic acid biosynthesis by <Emphasis Type="Italic">Aspergillus niger</Emphasis> W78C in submerged batch cultures with sucrose as a carbon source

The pH of the medium is the key environmental parameter of chemical selectivity of oxalic acid biosynthesis by Aspergillus niger. The activity of the enzyme oxaloacetate hydrolase, which is responsible for decomposition of oxaloacetate to oxalate and acetate inside the cell of the fungus, is highest at pH 6. In the present study, the influence of pH in the range of 3–7 on oxalic acid secretion by A. niger W78C from sucrose was investigated. The highest oxalic acid concentration, 64.3 g dm^?3, was reached in the medium with pH 6. The chemical selectivity of the process was 58.6% because of the presence of citric and gluconic acids in the cultivation broth in the amount of 15.3 and 30.2 g dm^?3, respectively. Both an increase and a decrease of medium pH caused a decrease of oxalic acid concentration. The obtained results confirm that pH 6 of the carbohydrate medium is appropriate for oxalic acid synthesis by A. niger, but the chemical selectivity of the process described in this paper was high in comparison to values reported previously in the literature.     

Crystal and molecular structures and luminescence properties of [Eu(Cin)<Subscript>3</Subscript>]<Subscript><Emphasis Type="Italic">n</Emphasis></Subscript> complex

The crystal structure of europium cinnamate of the composition [Eu(Cin)₃] _n (Cin is cinnamic acid anion C₉H₇O ₂ ^? ) was determined by X-ray crystallography (a = 22.626(1) Å, c = 7.7330(7) Å, space group R3/c, Z = 3, ρ_calc = 1.448 g/cm³). The coordination polyhedron of Eu atoms is a distorted trigonal prism with three centered square faces. The structure is built of infinite polymeric chains [Eu(Cin)₃] _n running along the c axis and linked by van der Waals and π stacking interactions. Luminescent characteristics of the compound were determined.     

Citric acid production from raw glycerol by acetate mutants of Yarrowia lipolytica

Three acetate mutants of the yeast species Yarrowia lipolytica were screened using batch cultivation. The strain Y. lipolytica 1.31 was found to be the most suitable for citric acid production from raw glycerol, a by-product of biodiesel production from rapeseed oil. At the initial concentration of glycerol of 200 g dm⁻³, the citric acid production of 124.5 g dm⁻³, yield of 0.62 g g⁻¹, and productivity of 0.88 g dm⁻³ h⁻¹ were achieved. Presented at the 33rd International Conference of the Slovak Society of Chemical Engineering, Tatranské Matliare, 22–26 May 2006.     

Antimicrobial Property of Nanosilver Colloid Prepared by Electrical Spark Discharge Method on <Emphasis Type="Italic">Aspergillus niger</Emphasis>

This study employed an electrical spark discharge method (ESDM) to prepare a nano-Ag colloid as an antifungal solution. The solution was diluted to two concentrations, and the fungal medium prepared in this study was coated with Aspergillus niger. The nano-Ag colloid solution was mixed with A. niger in various concentrations and dripped onto 3M Petrifilm plates. Inhibited growth observed after several days confirmed the antifungal effect of the nano-Ag colloid on A. niger. Because direct washing produced inaccurate quantitation and yielded A. niger in an excessively high concentration, this study employed an inoculation loop method for A. niger quantitation. The concentrations of A. niger ranged from 10^?2 to 10^?7%. The optimal colony count was observed on day 2. During an experiment regarding the antifungal effect of the ESDM-prepared nano-Ag colloid on A. niger, 3M Petrifilm plates were employed to observe the growth of A. niger. The colony count of 10^?2% A. niger without nano-Ag colloid was approximately 60. After the nano-Ag colloid was added, the colony count substantially decreased to approximately 10. The colony count of 10^?7% A. niger was reduced to 11 or lower after the nano-Ag colloid was added. The results confirmed the antifungal effect of the nano-Ag colloid on the growth of A. niger.     

A novel metal chalcogenide: HgCd<Subscript>4</Subscript>S<Subscript>5</Subscript>

A novel metal chalcogenide HgCd₄S₅ (1) was synthesized from solid-state reactions and structurally characterized. Compound 1 crystallizes in the space group C222₁ of the orthorhombic system with four formula units in a cell: a = 12.5661(5) Å, b = 7.2551(5) Å, c = 10.7520(7) Å, V = 980.2(1) ų, Cd₄HgS₅, M _r = 810.49, D _c = 5.492 g/cm³, S = 1.010, μ(MoK _α) = 25.128 mm^?1, F(000) = 1408, R = 0.0611 and wR = 0.1495. Compound 1 is characterized by a 3-D cadmium sulfide framework structure with the infinite mercury sulfide chains located in the cavities.