Extraction of extracellular polymeric substances from the photosynthetic bacterium Rhodopseudomonas acidophila

Among the four methods for extracting extracellular polymeric substances (EPS) from Rhodopseudomonas acidophila (EDTA, NaOH, H₂SO₄, heating/centrifugation), EDTA extraction was found to be the most effective. The contents of the major components of EPS from R. acidophila, i.e., carbohydrate, protein and nucleic acid, were 6.5, 58.4 and 5.4 mg g^–1 dry cells, respectively. The optimum extraction time was 1–3 h and the EDTA dosage was approximately 2.8 g g^–1 dry cells. Under these conditions, no cell lysis was observed. The EPS content and the percentage of the three main components were greatly dependent on the extraction method. The intensity of absorption peaks for photosynthetic pigments in the UV–visible spectrum of bacteria remained unchanged prior to and after EDTA extraction; and no pigment peaks appeared in the EPS spectrum. This suggests that few cells were destroyed and lysis did not occur. UV–visible spectrum analysis, an easy and rapid technique, could be used to monitor cell lysis during EPS extraction from R. acidophila.     

Selection of organic solvents for in situ extraction of fermentation products fromClostridium thermohydrosulfuricum cultures

Summary Fifteen organic solvents were examined to determine their biocompatibility for in situ extraction of fermentation products from cultures of the thermophilic anaerobeClostridium thermohydrosul furicum. Five solvents (hexadecane, isooctane, kerosene, oleyl alcohol, Shellsol TD) were found to be non-toxic toClostridium thermohydrosul furicum. Interfacial tensions, phase separation and partition coefficients for ethanol of the biocompatible solvents were compared. With the exception of kerosene, these solvents showed good separation from the aqueous phase. Oleyl alcohol had the highest partition coefficient for ethanol (K_D=0.34 at 65°C) and appears to be suitable for extractive ethanol fermentation.     

Nitrogen assimilation in Rhodopseudomonas acidophila

Rhodopseudomonas acidophila strain 7050 assimilated ammonia via a constitutive glutamine synthetase/glutamate synthase enzyme system.Glutamine synthetase had a K _m for NH ₄ ⁺ of 0.38 mM whilst the nicotinamide adenine dinucleotide linked glutamate synthase had a K _m for glutamine of 0.55 mM. R. acidophila utilized only a limited range of amino acids as sole nitrogen sources: l-alanine, glutamine and asparagine. The bacterium did not grow on glutamate as sole nitrogen source and lacked glutamate dehydrogenase. When R. acidophila was grown on l-alanine as the sole nitrogen source in the absence of N₂ low levels of a nicotinamide adenine dinucleotide linked l-alanine dehydrogenase were produced. It is concluded, therefore, that this reaction was not a significant route of ammonia assimilation in this bacterium except when glutamine synthetase was inhibited by methionine sulphoximine. In l-alanine grown cells the presence of an active alanine-glyoxylate aminotransferase and, on occasions, low levels of an alanine-oxaloacetate aminotransferase were detected. Alanine-2-oxo-glutarate aminotransferase could not be demonstrated in this bacterium.Abreviations ADH alanine dehydrogenase - GDH glutamate dehydrogenase - GS glutamine synthetase - GOGAT glutamate synthase - MSO methionine sulphoximine     

5CN05 partitioning in an aqueous two-phase system: A new approach to the solubilization of hydrophobic drugs

Liquid–liquid extraction for the purification of molecules has been central to many advances in the pharmaceutical industry. These processes were developed based on the property that some polymer and/or micellar solutions present to separate into a concentrated phase and a diluted phase. Based on the differences in the physical and chemical environments of the two coexisting phases, and since both phases contain approximately 60–90% of water, liquid–liquid extraction provides a powerful alternative to both extract and solubilize a molecule. This paper examines the partition behavior of the synthetic drug, 2-[(3,4-dichlorine-benzylidene)-amino]-5,6-dihydro-4H-cyclopenta[b]thiophene-3-carbonitrile (5CN05), in an aqueous two-phase polymer system (ATPPS) and also in an aqueous two-phase micellar system (ATPMS). The results showed that both systems are favorable for extraction the 5CN05 drug high partition coefficient values (K_5CN05 > 200) and yield (Y_5CN05 > 99.48%) in the concentrated phase were achieved with the systems. However, the ATPPS generated a partition coefficient (K_5CN05) higher than the one obtained with ATPMS. The results suggest that both processes may be used for the extraction and concentration of molecules with hydrophobic characteristics, such as 5CN05. They also provide an optimal environment for the solubilization of such molecules, allowing for greater efficiency when purifying many classes of drugs.     

Production of extracellular polymeric substances from Rhodopseudomonas acidophila in the presence of toxic substances

A hydrogen-producing photosynthetic bacteria strain, Rhodopseudomonas acidophila, was used to investigate the production of extracellular polymeric substances (EPS) in the presence of toxic substances and the effect of toxicants on bacterial surface characteristics. Addition of the toxic substances including Cu(II), Cr(VI), Cd(II) and 2,4-dichlorophenol (2,4-DCP) stimulated the production of EPS but reduced the cell dry weight. At concentrations of 30 mg l⁻¹ Cu(II), 40 mg l⁻¹ Cr(VI), 5 mg l⁻¹ Cd(II) and 100 mg l⁻¹ 2,4-DCP, the EPS content increased by 5.5, 2.5, 4.0 and 1.4 times, respectively, than the control. These toxic substances also greatly influenced the proteins/carbohydrates ratio of EPS. The ratios in the presence of toxic substances were always higher than that of control. Furthermore, under toxic conditions, the increase in the protein content far exceeded than that of others in EPS, suggesting that extracellular proteins could protect cells against toxic substances. The toxic substances significantly changed the surface characteristics and flocculation ability of R. acidophila, such as surface energy, relative hydrophobicity and free energy of adhesion.     

Optimization of bovine serum albumin partition coefficient in aqueous two-phase systems

Partitioning of proteins in aqueous two-phase systems has been shown to provide a powerful method for separating and purifying mixtures of biomolecules by extraction. These systems are composed of aqueous solutions of either two water-soluble polymers, usually polyethylene glycol (PEG) and dextran (Dx), or a polymer and a salt, usually PEG and phosphate or sulfate. There are many factors which influence the partition coefficient K, the ratio of biomolecule concentration in the top phase to that in the bottom phase, in aqueous two-phase systems. The value of the partition coefficient relies on the physico-chemical properties of the target biomolecule and other molecules and their interactions with those of the chosen system. In this work, the partition behavior of pure bovine serum albumin in aqueous two-phase systems was investigated in order to see the effects of changes in phase properties on the partition coefficient K. The concentration of NaCl and pH were considered to be the factors having influence on K. Optimal conditions of these factors were obtained using the Box-Wilson experimental design. The optimum value of K was found as 0.0126 when NaCl concentration and pH were 0.14 M and 9.8, respectively, for a phase system composed of 8% (w/w) polyethylene glycol 3,350 - 9 (% w/w) dextran 37,500 - 0.05 M phosphate at 20 °C.     

Recovery of penicillin G from fermentation broth with reactive extraction in a mixer-settler

Summary In a laboratory countercurrent mixer-settler, penicillin was recovered from its fermentation broth by extraction with Amberlite LA-2 in n-butylacetate at pH 5.0 and reextracted from the ion-pair complex containing a solvent phase with a buffer at 7.2–7.5 with an overall degree of extraction above 90 %.Symbols A amine - AHP complex - c concentration - C partition coefficent - E degree of extraction - HP penicillin acid - K_G equilibrium constant - P, P^– penicillin acid anion Indices aq aqueous phase - org organic phase - A amine - AHP complex - G overall - HP free acid - P penicillin     

Relations between extraction protocols for activated sludge extracellular polymeric substances (EPS) and complexation properties of Pb and Cd with EPS: Part II. Consequences of EPS extraction methods on Pb2+ and Cd2+ complexation

To study the effect of extraction protocols on extracellular polymeric substances (EPS) metal binding ability, EPS from two activated sludges were extracted by eight extraction protocols: three chemical treatments, four physical treatments and a control. Two pK_a, for each EPS, were determined: pK_a1 may be specific for carboxyl and phosphoric and pK_a2 may be attributed to phenolic and amino functional groups according to EPS composition and IR spectra. EPS pK_a values could be affected by the presence of extraction reagents and/or the modifications of EPS by extraction reagents.Complexation study performed at pH 7 by a polarographic method has always showed a greater affinity of EPS for Pb²⁺ than for Cd²⁺. The complexation properties of EPS extracted by chemical methods were greatly modified. Concerning EPS extracted by physical methods, their complexation properties were close except for EPS obtained by heating. Standardized extraction methods must be established as a function of the aims of the EPS study.     

Chemoautotrophic growth of Rhodopseudomonas species with hydrogen and chemotrophic utilization of methanol and formate

A chemolithoautotrophic type of metabolism, which was hitherto unknown for purple nonsulfur bacteria, was demonstrated by growth experiments using Rhodopseudomonas capsulata Kb1 and Rhodopseudomonas acidophila 10050. These strains were able to grow in a mineral medium in the dark at the expense of H₂, O₂, and CO₂. A minimum doubling time of 9 h was obtained for R. capsulata under an atmosphere containing less than 15% oxygen; higher oxygen concentrations suppressed autotrophic but not chemoorganotrophic growth. Oxygen sensitivity of chemoautotrophically growing cells of R. acidophila was even more pronounced, whereas cells growing chemotrophically on methanol almost tolerated the oxygen concentration of air. Highest oxygen sensitivity of growth of R. acidophila was observed with formate as substrate. The growth yield of cultures grown semiaerobically in the dark on methanol was 0.23 g dry cell material per g methanol consumed.     

Interaction of indole and tryptophan derivatives with sodium dodecyl sulfate micelles measured with ultraviolet absorption and fluorescence quenching

The distribution of indole and tryptophan derivatives between sodium dodecyl sulfate (SDS) micellar and aqueous phases was analyzed using conventional methods of ultraviolet (UV) absorption spectroscopy and measurement of fluorescence quenching by succinimide. On the assumption of a simple pseudo-phase equilibrium between both phases the distribution coefficient was easily obtained by the measurement of the ratioR _pv of the absorbance intensity in the peak to that in the valley of the UV spectra or the fluorescence quenching constant K_sv. The possibilities and limitations of utilizing the ratio of the collisional quenching constant estimating from theK _sv value in the micellar phase to that in the aqueous phase for a measure of the polarity of the microenvironment around the tryptophan derivatives in the SDS micelle is discussed in comparison with theR _pv values for the UV spectra. The indole ring in the derivatives in the SDS micelle is localized near or on the micelle-water interface with its imino group directed toward the aqueous phase. Thus it can serve as a feasible model for interpreting the distribution coefficients andR _pv values obtained for the various indole and tryptophan derivatives.Abbreviations UV ultraviolet - SDS sodium dodecyl sulfate - ATEE N-acetyl-l-tryptophan ethyl ester - ATA N-acetyl-l-tryptophan-amide - CMC critical micelle concentration     

Differences in NMR Spectra between Some α- and γ-Glutamyl Dipeptides

A methanol-utilizing phototrophic bacterium, strain M402, was isolated from surface water of an acidic hot spring. The isolated strain was identified as Rhodopseudomonas acidophila from its morphological and physiological characters. Profiles of the utilization of non-aromatic compounds as carbon sources by this strain were in good agreement with those of some strains of R. acidophila reported by Pfennig [J. Bacteriol., 99, 597 (1969)]. However, strain M402 was found to be capable of utilizing vanillic acid, vanillin, vanillyl alcohol, ferulic acid, veratric acid, syringic acid, syringal-dehyde and benzyl alcohol as carbon sources under anaerobic-light conditions. Although Pfennig did not refer to these abilities of his strains, these notable characters of strain M402 seem to be additional new characters of R. acidophila.     

Characteristics of hexadecane partition by the growth medium of Acinetobacter sp.

The partition of n-hexadecane in the spent growth medium of Acinetobacter sp. HOI-N was determined by measuring the increase in the relative aqueous solubility of ³H-hexadecane as compared to controls. The amount of hexadecane partitioned was proportional to the protein concentration. The specific solubility of hexadecane (nmol/mg protein) was analyzed by least-squares fitting yielding an average slope of 0.6 with a standard deviation of 0.3, indicating either nonequilibrium of hexadecane or physical aggregation of protein. The amount of hexadecane partitioned was concentration dependent yielding optically clear microemulsions at hexadecane concentrations of less than 1.4mM and macroemulsions at hexadecane concentrations of 1.4mM or greater. Preliminary results indicated that hexadecane and partitioned by a lipoprotein complex.     

Analytical partitioning of poly(ethylene glycol)-modified proteins

Covalently grafting proteins with varying numbers (n) of poly(ethylene glycol) molecules (PEGs) often enhances their biomedical and industrial usefulness. Partition between the phases in aqueous polymer two-phase systems can be used to rapidly characterize polymer-protein conjugates in a manner related to various enhancements. The logarithm of the partition coefficient (K) approximates linearity over the range 0<n<x. However, x varies with the nature of the conjugate (e.g., protein molecular mass) and such data analysis does not facilitate the comparison of varied conjugates. The known behavior of surface localized PEGs suggests a better correlation should exist between log K and the weight fraction of polymer in PEG-protein conjugates. Data from four independent studies involving three proteins (granulocyte-macrophage colony stimulation factor, bovine serum albumin and immunoglobulin G) has been found to support this hypothesis. Although somewhat simplistic, ‘weight fraction’ based analysis of partition data appears robust enough to accommodate laboratory to laboratory variation in protein, polymer and phase system type. It also facilitates comparisons between partition data involving disparate polymer-protein conjugates.     

Reactive extraction of penicillin G in a pilot plant Karr-column

Summary Penicillin G was extracted from a model medium with a secondary amine (Amberlite LA-2) as carrier in n-butylacetate as solvent in a 7.6 m high pilot plant Karr-column at different stroke frequencies, throughput of the phases, concentrations of Penicillin G and carrier and ratios of the throughputs of the aqueous and organic phases. Up to penicillin concentrations of 30 gl^–1, throughputs of the aqueous phase of 100 lh^–1 and throughput ratios of the aqueous phase-to-organic phase of 3, very high degrees of extraction (99%) can be achieved with a penicillin loss below 1%.Symbols a specific interfacial area with regard to the volume of the continuous phase - C partition coefficient - cA, cA, i concentration of carrier (sec. amine) in the bulk at the interface - cAHP, cAHP, i concentration of complex in the bulk at the interface - cH proton concentration - cHP_a, cHP_a,i concentration of free acid in the bulk of the aqueous phase at the interface - cHP_o, cHP_{o, i} concentration of free acid in the bulk of the organic phase, at the interface - cP, cP, i concentration of acid anions in the bulk of the aqueous phase, at the interface - d₃₂ Sauter droplet diameter - E degree of extraction - f stroke frequency - KG reaction equilibrium constant - K_phys distribution coefficient - N number of stages in cascade - t mean residence time of the aqueous phase - _aq throughput of the aqueous phase - _o throughput of the organic phase - Z dimensionless longitudinal coordinate of the column with regard to its active length (4 m) - holdup of the organic phase     

Solvatochromic studies in polyethylene glycol–salt aqueous biphasic systems

The polarities of the co-existing phases of a polyethylene glycol (PEG)-2000–K₃PO₄ aqueous biphasic system (ABS) have been examined using Reichardt’s carboxylated pyridinium-N-phenoxybetaine dye as a probe. Using this probe, the polarities of these phases have been compared to those of conventional solvent extraction systems and micellar systems using values obtained from the literature. In general, these extraction systems are comparable in polarity to rather polar solvents. Data on the free energy of transfer of solvents suggests that this may be due to the failure of the probe to account for the real polarity of the salt-rich phase compared to the polymer-rich phase. Examination of the monophasic region of these systems suggests that the reason for this is that the probe is partitioned to a discreet solvent domain dominated by PEG, even though phase separation of the solution is not observed. The use of linear free energy relationships for the characterization of ABS is briefly discussed.     

Production,extraction, and thermodynamics protease partitioning from Aspergillus tamarii Kita UCP1279 using PEG/sodium citrate aqueous two-phase systems

Abstract

The protease from Aspergillus tamarii Kita UCP1279 extraction by aqueous two-phase PEG-Citrate (ATPS) systems, using a factorial design 2⁴, was investigated. Then, the variables studied were polyethylene glycol (PEG) molar mass (M_PEG), concentrations of PEG (C_PEG) and citrate (C_CIT), and pH. The responses analyzed were the partition coefficient (K), activity yield (Y) and purification factor (PF). The thermodynamic parameters of the ATPS partition were estimated as a function of temperature. ATPS was able to pre-purify the protease (PF = 1.6) and obtained 84% activity yield. The thermodynamic parameters ΔG°_m (?10.89?kJ mol^?1), ΔH_m (?5.0?kJ?mol^?1) and partition ΔS_m (19.74?J mol^?1 K^?1) showed that the preferential migration of almost all protein contaminants of the crude extract to the salt-rich phase, while the preferred protease was the PEG rich phase. The extracted enzyme presents optimum temperature and pH at range of 40–50?°C and 9.0–11.0, respectively. Moreover, the enzyme was identified as serine protease based on inhibition profile. ATPS showed the satisfactory performance as the first step for Aspergillus tamarii Kita UCP1279 protease pre-purification.     

Extractive partition of L-aspartase and fumarase fromEscherichia coli using aqueous polyethylene glycol-ammonium sulfate systems

Summary Inorganic sulfate salts are used to form aqueous two-phase systems with polyethylene glycol (PEG) for enzyme purification. Two enzymes, L-aspartase and fumarase produced byEscherichia coli are efficiently separated into different phases in spite of the high degree of similarity in molecular weight and amino acid sequence between them. The ratio of L-aspartase to fumarase in the PEG-rich phase is more than sixty (60) times the ratio before extraction. A high degree of purification in a single extraction step can be achieved by careful selections of PEG molecular weight, pH, cation of the salts, and sodium chloride levels. Cations of sulfate-containing salts in the following order: NH ₄ ⁺ >Na⁺>Mg²⁺ tend to increase the partition of L-aspartase in the PEG-rich phase. The maximal degree of enzyme purification is obtained by using PEG 4000 and ammonium sulfate as a phase system at a stable pH for both enzymes.     

Carotenoids of purple nonsulfur bacteria

Summary The carotenoid composition of some recently isolated new species of purple nonsulfur bacteria was investigated. All of the investigated R. tenue and R. purpureus strains and three of the R. acidophila strains synthesize the components of the rhodopinal series. It was shown that spirilloxanthin was present in all strains of R. acidophila; in three R. acidophila strains a very small amount of -carotene was found. Large amounts of the glucosides of rhodopin and rhodopinal were isolated from the cells of seven R. acidophila strains. Some taxonomic aspects are discussed.     

Partition of hexadecane to the cell surface of Candida tropicalis: Mechanism for the transport of water-insoluble substrates

The partition of hexadecane to the cell surface of Candida tropicalis was measured by incubating heat-inactivated cells with hexadecane-1-¹⁴C on a gyratory shaker. The free hexadecane was separated by centrifuging the cells through a 15% sucrose solution, and the partitioned hexadecane was quantified by scintillation spectrometry of the samples from the resulting cell sediment. Heat-inactivated cells did not take up hexadecane as determined by a membrane filtration technique involving organic solvent washing. The partitioning was a time-dependent process. The velocity increased by increasing the shake rate of te shaker. At 360 rpm and with baffled flasks, saturation of the cell surface with hexadecane was obtained after a 20 min incubation period. The amount of hexadecane partitioned depended on the initial hexadecane-to-cell concentration ratio. At a ratio of 5 μmol/mg cell protein the highest amount of hexadecane partitioned was measured at 2100 μmol/mg cell protein. At ratios higher than 6 μmol/mg cell protein the cells were no longer sedimentable by centrifugation. The partition of hexadecane to the cell surface was affected by removing the surface layer of the cell wall by Pronase treatment and by using detergents in the partition assay. Pronase treatment lowered the amount of hexadecane partitioned as a consequence of the removal of the lipophilic layer of the cell surface. Detergents influence the partition coefficient and also lowered the amount of hexadecane partitioning to the cell surface. At a low shaking intensity (280 rpm, unbaffled flasks), after Pronase treatment, and in the presence of detergents he uptake of hexadecane by the cells was limited by the partitioning.     

THE KINETICS OF PENETRATION : VI. SOME FACTORS AFFECTING PENETRATION

Some of the factors affecting penetration in living cells may be advantageously studied in models in which the organic salts KG and NaG diffuse from an aqueous solution A, through a non-aqueous layer B (representing the protoplasmic surface) into an aqueous solution C (representing the sap and hence called artificial sap) where they react with CO₂ to form KHCO₃ and NaHCO₃. Their relative proportions in C depend chiefly on the partition coefficients and on the diffusion constants in the non-aqueous layer. But the ratio is also affected by other variables, among which are the following: 1. Temperature, affecting diffusion constants and partition coefficients and altering the thickness of the unstirred layers by changing viscosity. 2. Viscosity (especially in the non-aqueous layers) which depends on temperature and the presence of solutes. 3. Rate of stirring, which affects the thickness of the unstirred layers and the transport of electrolyte in those that are stirred. 4. Shape and surface area of the non-aqueous layer. 5. Surface forces. 6. Reactions occurring at the outer surface such as loss of water by the electrolyte or its molecular association in the non-aqueous phase. The reverse processes will occur at the inner surface and here also combinations with acids or other substances in the "artificial sap" may occur. 7. Outward diffusion from the artificial sap. The outward movement of KHCO₃ and NaHCO₃ is small compared with the inward movement of KG and NaG when the concentrations are equal. This is because the partition coefficients³ of the bicarbonates are very low as compared with those of NaG and KG. Since CO₂ and HCO₃ ^- diffuse into A and combine with KG and NaG the inward movement of potassium and sodium falls off in proportion as the concentration of KG and NaG is lessened. 8. Movement of water into the non-aqueous phase and into the artificial sap. This may have a higher temperature coefficient than the penetration of electrolytes. 9. Variation of the partition coefficients with concentration and pH. Many of these variables may occur in living cells. (It happens that the range of variation in the ratio of potassium to sodium in the models resembles that found in Valonia.)