Optical method for the determination of the oxygen-transfer capacity of small bioreactors based on sulfite oxidation.

The growth of microorganisms may be limited by operating conditions which provide an inadequate supply of oxygen. To determine the oxygen-transfer capacities of small-scale bioreactors such as shaking flasks, test tubes, and microtiter plates, a noninvasive easy-to-use optical method based on sulfite oxidation has been developed. The model system of sodium sulfite was first optimized in shaking-flask experiments for this special application. The reaction conditions (pH, buffer, and catalyst concentration) were adjusted to obtain a constant oxygen transfer rate for the whole period of the sulfite oxidation reaction. The sharp decrease of the pH at the end of the oxidation, which is typical for this reaction, is visualized by adding a pH dye and used to measure the length of the reaction period. The oxygen-transfer capacity can then be calculated by the oxygen consumed during the complete stoichiometric transformation of sodium sulfite and the visually determined reaction time. The suitability of this optical measuring method for the determination of oxygen-transfer capacities in small-scale bioreactors was confirmed with an independent physical method applying an oxygen electrode. The correlation factor for the maximum oxygen-transfer capacity between the chemical model system and a culture of Pseudomonas putida CA-3 was determined in shaking flasks. The newly developed optical measuring method was finally used for the determination of oxygen-transfer capacities of different types of transparent small-scale bioreactors.     

A Computer-aided noninterfering on-line technique for monitoring oxygen-transfer characteristics during fermentation processes

The role of computers in the monitoring and control of fermentation processes has increased steadfastly. The ultimate utility of the machines will not depend on the availability of online sensors but also on the availability of techniques that combine direct measurements, leading towards estimates of variable closely related to the microbial process or its control. In this article, a methodology for on-line and noninterfering evaluation of the volumetric mass-transfer coefficient K(l)a is developed. A detailed presentation of the procedure, called "the static method," is given. Its feasibility is proved through implementation of the method on an antibiotic fermentation process. These experiments indicate that operator actions meant to modify the oxygen-transfer conditions can be checked on-line. The quantitative value of the static method is ascertained by comparing the experimental results with K(l)a estimates obtained with the "gassing-out" method. A sensitivity analysis was carried out, revealing the need for temperature and pressure corrections and showing that the precision of the oxygen analyzer determines the precision of the static method.     

Measurement of oxygen solubility in fermentation media: a colorimetric method

Methods of measuring oxygen solubility in culture media are scarce, and those available are tedious to apply. A simple colorimetric assay was developed and applied to the analysis of oxygen solubility during alcoholic fermentation. The method was based on the consumption of oxygen by glucose oxidase activity and the production of the pink quinone of syringaldazine by coupled peroxidase activity. Color formation at 526 nm progressed through an optimum that was a linear function of the oxygen added to the assay. Sensitivity was maximized by operating at pH 7 and limiting the medium sample volume added. Each assay took 10-15 min to prepare and react. Reaction time was minimized by using abundant glucose and enzyme concentrations. Data obtained by the assay developed showed good agreement with published oxygen solubilities in water and selected media at various temperatures. Subsequent analyses of fermentation broths indicated falling sugar concentration to be primarily responsible for increases in oxygen solubility during fermentation. For example, during fermentations started with 230 g/L xylose or glucose, oxygen solubility could increase by 41% due to sugar consumption alone. This procedure can provide the solubility data needed to accurately calibrate in-line electronic probes for monitoring dissolved oxygen concentration during fermentation processes.     

Methodology for predicting oxygen transport on an intravenous membrane oxygenator combining computational and analytical models

A computational methodology for accurately predicting flow and oxygen-transport characteristics and performance of an intravenous membrane oxygenator (IMO) device is developed, tested, and validated. This methodology uses extensive numerical simulations of three-dimensional computational models to determine flow-mixing characteristics and oxygen-transfer performance, and analytical models to indirectly validate numerical predictions with experimental data, using both blood and water as working fluids. Direct numerical simulations for IMO stationary and pulsating balloons predict flow field and oxygen transport performance in response to changes in the device length, number of and balloon pulsation frequency. Multifiber models are used to investigate interfiber interference and length effects for a stationary balloon whereas a single fiber model is used to analyze the effect of balloon pulsations on velocity and oxygen concentration fields and to evaluate oxygen transfer rates. An analytical lumped model is developed and validated by comparing its numerical predictions with experimental data. Numerical results demonstrate that oxygen transfer rates for a stationary balloon regime decrease with increasing number of fibers, independent of the fluid type. The oxygen transfer rate ratio obtained with blood and water is approximately two. Balloon pulsations show an effective and enhanced flow mixing, with time-dependent recirculating flows around the fibers regions which induce higher oxygen transfer rates. The mass transfer rates increase approximately 100% and 80%, with water and blood, respectively, compared with stationary balloon operation. Calculations with combinations of frequency, number of fibers, fiber length and diameter, and inlet volumetric flow rates, agree well with the reported experimental results, and provide a solid comparative base for analysis, predictions, and comparisons with numerical and experimental data.     

Oxygen transfer in a multiple air-lift loop reactor

The Multiple Air-lift Loop reactor (MAL) is a new type of bioreactor, in which a series of airlifts with internal loops is incorporated into one vessel. As such, the MAL is an approximation of an aerated plug-flow fermenter. Gas/liquid oxygen transfer was studied as a function of the gas flow rate in a MAL. The second MAL-compartment in the series was investigated in particular, and a Rectangular Air-lift Loop reactor (RAL) was used as a reference. Both a dynamic and a steady-state method were used for the determination of the overall volumetric oxygen-transfer coefficient. Both methods gave the same results. The oxygen transfer coefficient in the second MAL-compartment was low compared to that of conventional internal-loop reactors. Wall effects probably caused bubble coalescence and a reduction in the oxygen transfer. For the RAL it was found that oxygen transfer was comparable to that in a bubble column.     

Non-steady-state O(2) diffusion in metmyoglobin solutions studied in a diffusion chamber

In this study, we studied the "passive" diffusion through myoglobin solutions by determining the oxygen diffusion coefficient (DO(2)) and the oxygen permeability (permeability O(2)) of metmyoglobin (metMb) solutions (3-33 g. 100 mL(-1)) at 25 degrees C. These oxygen diffusion parameters were determined in a diffusion chamber using a non-steady-state method and were also determined of albumin solutions (4-32 g. 100 mL(-1)) and distilled water for comparison. From these parameters, the oxygen solubility (alphaO(2)) could be calculated, because alphaO(2) = permeabilityO(2)/DO(2). Both DO(2) and permeabilityO(2) decreased with increasing metMb and albumin concentration. The values of DO(2), permeabilityO(2) and alphaO(2) of both metMb solutions and albumin solutions were comparable with literature values of methemoglobin (metHb) and serum protein solutions. The values of the metMb solutions can be used in following studies of facilitated oxygen diffusion through myoglobin solutions.     

NMR studies of water-gas interactions

Analysis of published data concerning the solubility of different gases in water as dependent on temperature was carried out. These dependences could be described by mono-or bi-exponential functions. Solubilities of nitrogen and oxygen are additive and depend on their percentage in the atmosphere over the liquid. The temperature dependence of oxygen and nitrogen dissolved in water corresponds to that in the atmospheric air. Measurements of water spin-lattice relaxation times, changing with the concentration of dissolved paramagnetic oxygen, showed that oxygen could be substantially but not completely eliminated by saturation with any gas. The best method is the contact with a water-immiscible liquid of gas capacity higher than water. However, all this leads to an unstable state of a gas-water system, converging to equilibrium.     

Evaluation of Drug Load and Polymer by Using a 96-Well Plate Vacuum Dry System for Amorphous Solid Dispersion Drug Delivery

It is well recognized that poor dissolution rate and solubility of drug candidates are key limiting factors for oral bioavailability. While numerous technologies have been developed to enhance solubility of the drug candidates, poor water solubility continuously remains a challenge for drug delivery. Among those technologies, amorphous solid dispersions (SD) have been successfully employed to enhance both dissolution rate and solubility of poorly water-soluble drugs. This research reports a high-throughput screening technology developed by utilizing a 96-well plate system to identify optimal drug load and polymer using a solvent casting approach. A minimal amount of drug was required to evaluate optimal drug load in three different polymers with respect to solubility improvement and solid-state stability of the amorphous drug-polymer system. Validation of this method was demonstrated with three marketed drugs as well as with one internal compound. Scale up of the internal compound SD by spray drying further confirmed the validity of this method, and its quality was comparable to a larger scale process. Here, we demonstrate that our system is highly efficient, cost-effective, and robust to evaluate the feasibility of spray drying technology to produce amorphous solid dispersions.     

Retrieving skin properties from in vivo spectral reflectance measurements

A previously developed inverse method was applied to in vivo normal‐hemispherical spectral reflectance measurements taken on the inner and outer forearm as well as the forehead of healthy white Caucasian and black African subjects. The inverse method was used to determine the thickness and melanin concentration in the epidermis, dermal blood volume fraction and oxygen saturation, and skin's spectral scattering coefficient. It was established that changes in melanin concentration due to racial difference and tanning, and differences in epidermal thickness and blood volume with anatomical location were detectable. The retrieved values were also consistent with independent measurements reported in the literature. The same method could be used for optical diagnosis of pathologies affecting the structure and pigmentation of human skin. (© 2011 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)     

Preferential hydration and solubility of proteins in aqueous solutions of polyethylene glycol

This paper is focused on the local composition around a protein molecule in aqueous mixtures containing polyethylene glycol (PEG) and the solubility of proteins in water + PEG mixed solvents. Experimental data from literature regarding the preferential binding parameter were used to calculate the excesses (or deficits) of water and PEG in the vicinity of β-lactoglobulin, bovine serum albumin, lysozyme, chymotrypsinogen and ribonuclease A. It was concluded that the protein molecule is preferentially hydrated in all cases (for all proteins and PEGs investigated). The excesses of water and deficits of PEG in the vicinity of a protein molecule could be explained by a steric exclusion mechanism, i.e. the large difference in the sizes of water and PEG molecules.

The solubility of different proteins in water + PEG mixed solvent was expressed in terms of the preferential binding parameter. The slope of the logarithm of protein (lysozyme, β-lactoglobulin and bovine serum albumin) solubility versus the PEG concentration could be predicted on the basis of experimental data regarding the preferential binding parameter. For all the cases considered (various proteins, various PEGs molecular weights and various pHs), our theory predicted that PEG acts as a salting-out agent, conclusion in full agreement with experimental observations. The predicted slopes were compared with experimental values and while in some cases good agreement was found, in other cases the agreement was less satisfactory. Because the established equation is a rigorous thermodynamic one, the disagreement might occur because the experimental results used for the solubility and/or the preferential binding parameter do not correspond to thermodynamic equilibrium.     

The bacterial degradation of fluoranthene and benzo[alpyrene.

A gas chromatographic method to determine fluoranthene and benzo[a]pyrene at concentrations close to the limit of their solubility in water was used to identify several species of pseudomonads able to degrade fluoranthene and benzol[a]pyrene. Degradation occurs most rapidly in cultures in the stationary phase, is heat sensitive, requires oxygen, and is enhanced in the presence of cyanide.     

Comparison of polarographic and chemical measurements of oxygen uptake in complex media: the example of lipoxygenase reaction.

A polarographic method using a Clark oxygen electrode was used to assess oxygen concentrations in model and complex media before and during the lipoxygenase-catalyzed oxidation of linoleic acid to hydroperoxides. The results were in good agreement with those obtained with the chemical determination of dissolved oxygen. The electrode correctly responded when the dissolved oxygen concentration was decreased by the addition of water activity depressors (sorbitol, sucrose, glucose). The influence of the medium components on the gas solubility was discussed. The linear relationship between partial pressure of oxygen (determined by polarographic method) and the oxygen concentration (determined by chemical method) indicated that the Clark oxygen electrode can be used to study enzyme reactions consuming or evolving oxygen in non-Newtonian media.     

Mass transfer and mixing rates in fermentation vessels

The effects on mass-transfer and overall mixing rates of varying impeller geometry and operating speed have been studied for flat-bladed turbines in laboratory fermentors, in aerated aqueous solutions, and in unaerated and aerated suspensions (1.6% w/v) of paper pulp. In the absence of suspended solid, oxygen absorption rates could be correlated directly with power input. In the pulp suspension, oxygen absorption at a given power input was influenced by impeller geometry and operating speed. The data for the three-phase system can be correlated by a dimensionless equation relating oxygen-transfer rates and mixing times to the geometrical and operating parameters of the impellers.     

A novel method of simulating oxygen mass transfer in two-phase partitioning bioreactors

An empirical correlation, based on conventional forms, has been developed to represent the oxygen mass transfer coefficient as a function of operating conditions and organic fraction in two-phase, aqueous-organic dispersions. Such dispersions are characteristic of two-phase partitioning bioreactors, which have found increasing application for the biodegradation of toxic substrates. In this work, a critical distinction is made between the oxygen mass transfer coefficient, k(L)a, and the oxygen mass transfer rate. With an increasing organic fraction, the mass transfer coefficient decreases, whereas the oxygen transfer rate is predicted to increase to an optimal value. Use of the correlation assumes that the two-phase dispersion behaves as a single homogeneous phase with physical properties equivalent to the weighted volume-averaged values of the phases. The addition of a second, immiscible liquid phase with a high solubility of oxygen to an aqueous medium increases the oxygen solubility of the system. It is the increase in oxygen solubility that provides the potential for oxygen mass transfer rate enhancement. For the case studied in which n-hexadecane is selected as the second liquid phase, additions of up to 33% organic volume lead to significant increases in oxygen mass transfer rate, with an optimal increase of 58.5% predicted using a 27% organic phase volume. For this system, the predicted oxygen mass transfer enhancements due to organic-phase addition are found to be insensitive to the other operating variables, suggesting that organic-phase addition is always a viable option for oxygen mass transfer rate enhancement.     

水/乙醇溶剂中阿奇霉素的热力学及多晶型研究

在278.2~308.2 K温度范围内,测定阿奇霉素在水/乙醇混合溶剂中的溶解度,根据固液平衡理论建立了该体系的溶解度修正模型。采用X线粉末衍射法和差示扫描量热法,对阿奇霉素在不同温度、不同体积比的水/乙醇混合溶剂中得到的晶体进行鉴别。同时利用溶解度数据估算了阿奇霉素在水/乙醇体系中的溶解热(-25.26~-16.11 k J/mol)、混合热(-9.94~-3.25 k J/mol)。通过溶液化学理论推导了阿奇霉素溶剂化平衡常数K与活度系数γ2的方程:γ2=1/(1+K),建立了溶剂化焓与温度、水/乙醇两者体积比(φ)之间的关系式,为ΔH=RTln(17.86exp(3.4φ)-1)。采用溶析结晶方法得到的6种阿奇霉素晶体,均属单斜晶系,但具有不同的晶胞参数且其密度和熔点也不同。同时发现温度越高,水/乙醇体积比越大,得到的晶体稳定性越差(晶体的熔点和密度降低)。在水/乙醇混合溶剂的溶析结晶体系中,产生阿齐霉素多晶型的现象与溶剂化作用的强弱有关。     

Oxygen-transfer strategy and its regulation effects in serine alkaline protease production by Bacillus licheniformis

The effects of oxygen transfer on the production and product distribution in serine alkaline protease (SAP) fermentation by Bacillus licheniformis and oxygen-transfer strategy in relation to the physiology of the bacilli were investigated on a defined medium with citric acid as sole carbon source in 3.5-dm(3) batch bioreactor systems. By forming a 3 x 3 matrix with the parameters air-inlet rates of Q(O)/V(R) = 0.2, 0.5, 1.0 vvm, and agitation rates of N = 150, 500, 750 min(-1), the effects of oxygen transfer were investigated at nine different conditions. The concentrations of the product SAP and by-products, i.e., neutral protease, alpha-amylase, amino acids, and organic acids, and SAP activities were determined throughout the bioprocess. Among the constant air-flow and agitation-rate fermentations, Q(O)/V(R) = 0.5 vvm, N = 750 min(-1) oxygen-transfer conditions produced maximum SAP activity that was 500 U cm(-3), at t = 37 h. With the increase in Q(O)/V(R) and/or N, Damk?hler number that is the oxygen-transfer limitation decreases; and the process passes from oxygen-transfer limited conditions to biochemical-reaction limited conditions. Further increase in SAP activity, A = 680 U cm(-3) was achieved by applying an oxygen-transfer strategy based on the analysis of the data obtained with the constant oxygen-transfer condition experiments, with a step increase in air-inlet rate, from Q(O)/V(R) = 0.2 to Q(O)/V(R) = 0.5 vvm at N = 750 min(-1) constant agitation rate at t = 24 h. Organic acids and amino acids that were excreted to the fermentation medium varied depending on the oxygen-transfer conditions. With the increase in oxygen-transfer rate acetic acid concentration increased; contrarily, with the decrease in the oxygen-transfer rate the TCA-cycle organic acids alpha-ketoglutaric and succinic acids, and gluconic acid were excreted to the fermentation broth; nevertheless, the application of the oxygen-transfer strategy prevented the increase in acetic acid concentration between t = 35-38 h. Under all the oxygen-transfer conditions, the amino acid having the highest concentration and the amino acid that was not excreted to the fermentation broth were lysine and asparagine, respectively; both of which belong to the aspartic acid-group amino acids. Further, this result indicates the requirement of the genetic regulation directed to the aspartic acid-group enzymes for the progress in SAP production in B. licheniformis.     

Study of isopropyl myristate microemulsion systems containing cyclodextrins to improve the solubility of 2 model hydrophobic drugs

The objectives of this project were to evaluate the effect of alkanols and cyclodextrins on the phase behavior of an isopropyl myristate microemulsion system and to examine the solubility of model drugs. Triangular phase diagrams were developed for the microemulsion systems using the water titration method, and the solubility values of progesterone and indomethacin were determined using a conventional shake-flask method. The water assimilation capacities were determined to evaluate the effective microemulsion formation in different systems. The alkanols showed higher microemulsion formation rates at higher concentrations. A correlation between the carbon numbers of the alkanol and water assimilation capacity in the microemulsions studied was observed; isobutanol and isopentanol produced the best results. The addition of cyclodextrins showed no effect or had a negative effect on the microemulsion formation based on the type of cyclodextrin used. Isopropyl myristate-based microemulsion systems alone could increase the solubility values of progesterone and indomethacin up to 3300-fold and 500-fold, respectively, compared to those in water. However, the addition of cyclodextrins to the microemulsion systems did not show a synergistic effect in increasing the solubility values of the model drugs. In conclusion, microemulsion systems improve the solubility of progesterone and indomethacin. But the two types of cyclodextrins studied affected isopropyl myristatebased microemulsion systems negatively and did not improve the solubilization of 2 model drugs.     

Deep understanding of structure-solubility relationship for a diverse set of organic compounds using matched molecular pairs

Aqueous solubility is an important biopharmaceutical property in drug discovery and development. Although it has been studied for decades, the impact on solubility by the substructures (or fragments) of compounds are still not fully understood and characterized. This study aims to obtain fragment-solubility relationships using matched molecular pairs, and to provide further insight and suggestions for chemists on structural modifications to improve solubility profiles of drug-like molecules. A set of 2794 compounds with measured intrinsic aqueous solubility (logS) was fragmented into rings, linkers, and R groups using a controlled hierarchical fragmentation method. Then matched molecular pairs that differ by only one chemical transformation (i.e., addition or substitution of fragments) were identified and analyzed. The difference in solubility for each matched molecular pair was calculated, and the impact of the corresponding chemical transformation on solubility was investigated. The final product of this study was a fragment-solubility knowledgebase containing relative contributions to solubility of various medicinal chemistry design elements (R-groups, linkers, and rings). Structural modifications that might improve solubility profiles, that is, addition/deletion/substitution of fragments, could be derived from this knowledgebase. This knowledgebase could be used as an expert tool in lead optimization to improve solubility profiles of compounds, and the analysis method could be applied to study other biological and ADMET properties of organic compounds.     

Oxygen solubility and permeability of carbohydrates

The saturated oxygen concentration in a series of aqueous solutions of sorbitol (up to 35% w/w) and maltitol (up to 50% w/w) was measured using colorimetric reagent vials based on Rhodazine D. The results indicate that the solubility of oxygen in low-water carbohydrates is considerably lower than its solubility in pure water. It was concluded that the low-oxygen solubility is a major factor contributing to the barrier properties of low-water content carbohydrates used in the encapsulation of flavours, lipids, peptides and other oxidisable species.     

Determination of aqueous solubility by heating and equilibration: A technical note

Summary and Conclusion  A modified shake-flask solubility method, where the equilibration time was shortened through heating, was used to determine the solubility of 48 different drugs and pharmaceutical excipients in pure water at room temperature. The heating process accelerates dissolution of the solid compound and frequently results in supersaturated solution. Seeding with the solid compound after heating and cooling to room temperature promotes precipitation of the solid compound in its original stable form. This modified shake-flask method generates reliable and reproducible solubility data. Published: January 13, 2006