The reactive extraction of phenylalanine with Aliquat 336: buffer co-extraction equilibrium and mass transfer kinetics

The occurrence of significant co-extraction of buffer anions by the ion exchanger Aliquat 336 is unavoidable when high levels of system buffering is required. The co-extraction will result in inaccurate equilibrium and mass-transfer characterization of such a system unless its occurrence is taken into account, making process design and control difficult. A study of the equilibrium of phenylalanine extraction using Aliquat 336, a system where high levels of hydroxyl co-extraction occurs, was used as a model case to develop a method of accounting for co-extraction in mass-transfer modeling. Analysis of the equilibrium between bulk-aqueous-phase chloride and phenylalanine concentrations during mass transfer in a stirred-transfer cell showed there to be linear equilibrium relationships between the two parameters for a given extraction system of the form C(Cl,t) = alpha(C(A,t) - C(A,0)) for forward extraction and C(Cl,t) = epsilon C(A,t) + C(Cl,0) for backward extraction. The constants of proportionality of these relationships, or the "co-extraction constants," alpha and epsilon, were shown to be related to the selectivity of Aliquat 336 for the phenylalanine anion by the relationships alpha = -(1/S + 1) and epsilon; = -(1/S(-1) + 1). The linear equilibrium relationships were used to develop two-film theory mass-transfer models for both forward and backward extraction that account for co-extraction. These showed much higher accuracy in modeling stirred-transfer-cell data than the equivalent models which ignored co-extraction.     

New reactive extraction systems for separation of bio-succinic acid

Biotechnologically produced succinic acid has the potential to displace maleic acid and its uses and to become an important feedstock for the chemical industry. In addition to optimized production strains and fermentation processes, an efficient separation of succinic acid from the aqueous fermentation broth is indispensable to compete with the current petrochemical production processes. In this context, high molecular weight amines are known to be effective extractants for organic acids. For this reason, as a first step of isolation and purification, the reactive extraction of succinic acid was studied by mixing aqueous succinic acid solutions with 448 different amine–solvent mixtures as extraction agents (mixer-settler studies). The extraction agents consist either of one amine and one solvent (208 reactive extraction systems) or two amines and two solvents (240 reactive extraction systems). Maximum extraction yields of succinic acid from an aqueous solution with 423 mM succinic acid at pH 2.0 were obtained with more than 95% yield with trihexylamine solved in 1-octanol or with dihexylamine and diisooctylamine solved in 1-octanol and 1-hexanol. Applying these optimized reactive extraction systems with Escherichia coli fermentation broth resulted in extraction yields of 78–85% due to the increased ionic strength of the fermentation supernatant and the co-extraction of other organic acids (e.g., lactic acid and acetic acid), which represent typical fermentation byproducts.     

Reactive extraction of lactic acid using alamine 336 in MIBK: equilibria and kinetics

Lactic acid is an important commercial product and extracting it out of aqueous solution is a growing requirement in fermentation based industries and recovery from waste streams. The design of an amine extraction process requires (i) equilibrium and (ii) kinetic data for the acid-amine (solvent) system used. Equilibria for lactic acid extraction by alamine 336 in methyl-iso-butyl-ketone (MIBK) as a diluent have been determined. The extent to which the organic phase (amine +MIBK) may be loaded with lactic acid is expressed as a loading ratio, z=[HL](o)/[B](i,o). Calculations based on the stoichiometry of the reactive extraction and the equilibria involved indicated that more lactic acid is transferred to the organic phase than would be expected from the (1:1) stoichiometry of the reaction. The extraction equilibrium was interpreted as a result of consecutive formation of two acid-amine species with stoichiometries of 1:1 and 2:1. Equilibrium complexation constant for (1:1) and (2:1) has been estimated. Kinetics of extraction of lactic acid by alamine 336 in MIBK has also been determined. In a first study of its kind, the theory of extraction accompanied by a chemical reaction has been used to obtain the kinetics of extraction of lactic acid by alamine 336 in MIBK. The reaction between lactic acid and alamine 336 in MIBK in a stirred cell falls in Regime 3, extraction accompanied by a fast chemical reaction occurring in the diffusion film. The reaction has been found to be zero order in alamine 336 and first order in lactic acid with a rate constant of 1.38 s(-1). These data will be useful in the design of extraction processes.     

Factors influencing the potential use of Aliquat 336 for the in situ extraction of carboxylic acids from cultures of Pseudomonas putida

The use of extraction techniques to alleviate product inhibition in bioprocesses is one of a number of potential separation methods. However, the intimate contact of an organic phase with the broth implies that the organic components of this phase may be present in the aqueous phase at saturation levels. The quaternary amine Aliquat 336 (trioctyl/decylmethylammonium entity), dissolved in octan-1-ol showed no inhibition on the growth of Pseudomonas putida, at least with respect to molecular toxicity. Nevertheless, it is important to point out two main effects of Aliquat 336 associated with its ion exchange properties. It is able (1) to complex hydroxyl ions and therefore drastically lower the pH of the broth and (2) release its counter ion through these exchanges. Therefore, a strict control of the pH of the cultivation must be conducted, with the constraint that Ps. putida has an optimal pH growth of 7.4-7.5. The pH range tolerated by this strain is, however, between 5.0 and 9.0. In addition, the counter ion of Aliquat 336 needs to be carefully chosen and HSO4- should be preferred to Cl-.     

Fractionation of wheat proteins by counter-current distribution using aqueous two-phase systems containing propionic acid

Wheat proteins, soluble in diluted acid (glutenins), have been fractionated by counter-current distribution (CCD) using an aqueous two-phase system. The phase system is based on poly(ethylene glycol) and dextran but contains also 1% propionic acid and 6 mM magnesium sulfate. Approximately half of the bulk proteins partitioned to the upper phase while starch and other particles were recovered only into the lower phase. Whole wheat flour could be applied as sample for the CCD and 57 transfers were carried out. Starch and insoluble proteins remained stationary, while proteins followed the mobile phase to various degrees giving rise to a distribution pattern. The CCD pattern of the proteins showed distinct differences when various kinds of wheat flour were analysed. The patterns indicate that at least six subpopulations of proteins can be obtained by using two-phase extraction.     

Coordination chemistry of 2,2-bis(diphenylphosphinomethyl)propionic acid

The coordination chemistry of the diphosphine ligands 2,2-bis(diphenylphosphinomethyl)propionic acid, 1, and 2,2-bis(diphenylphosphinomethyl)propionate, 2, with copper(I), silver(I), gold(I), palladium(II) and platinum(II) is described. Structure determinations show that the carboxylic acid group in 1 can hydrogen bond to solvent molecules, to anions or to the carboxylic acid group of a neighboring complex, as in the complexes [MCl₂(1)] · 2DMSO (M = Pd or Pt), [Pt(1)₂](OTf)₂ or [Pd(NCMe)₂(1)](OTf)₂, respectively. The tridentate diphosphine-carboxylate ligand 2 forms oligomeric or polymeric complexes, such as [{Ag(2)}_n], [{PdCl(2)}_n] or [{PtMe(2)}_n].     

Fed-batch fermentation with and without on-line extraction for propionic and acetic acid production byPropionibacterium acidipropionici

Fed-batch propionic and acetic acid fermentations were performed in semi-defined laboratory medium and in corn steep liquor withPropionibacterium acidipropionici strain P9. On average, over four experiments, 34.5 g/l propionic acid and 12.8 g/l acetic acid were obtained in about 146 h in laboratory medium with 79 g/l glucose added over five feeding periods. The highest concentration of propionic acid, 45 g/l, was obtained when the glucose concentration was not allowed to drop to zero. In corn steep liquor 35 g/l propionic acid and 11 g/l acetic acid were produced in 108 h from 59.4 g/l total lactic acid provided as seven feedings of corn steep liquor. Extractive fed-batch fermentations were conducted in semi-defined medium using either flat-sheet-supported liquid membranes or hollow-fiber membrane extraction to remove organic acids from the culture medium. As operated during the course of the fermentation, these systems extracted 25% and 22% of the acetic acid and 36.5% and 44.5% of the propionic acid, respectively, produced in the fermentation. Total amounts of acids produced were about the same as in comparable nonextractive fermentations: 30–37 g/l propionic acid and 13 g/l acetic acid were produced in 150 h. Limitations on acid production can be attributed to limited substrate feed, not to failure of the extraction system.Journal paper J-16303 of the Iowa Agriculture and Home Economics Experiment Station, Ames, Iowa. Project 3122.     

Characterization of propionic acid bacteria using matrix-assisted laser desorption/ionization (MALDI) mass spectrometry

It was shown that matrix-assisted laser desorption/ionization (MALDI) mass spectrometry could be used for the diagnostic characterization of propionic acid bacteria (PABs). The spectra of proteins (whole PAB cells) with a molecular mass of 3000 to 11 000 were obtained and analyzed using three matrices: sinapinic (SA), 2,5-dihydroxibenzoic (DHB), and α-cyano-4-hydroxycinnamic acid (HCCA). The MALDI spectra of PAB revealed the protein peaks characteristic of (1) the genus Propionibacterium (3496, 5386, 5605, 10 470), (2) the groups of species sharing the common composition of their cell walls and fatty acids, and (3) a species (four species were investigated). Exemplified by the P. shermanii strains (the collection and mutant ones) producing and not producing vitamin B₁₂, the possibility of using MALDI profiles for strain differentiation was confirmed. The MALDI profiles of the propionic acid cocci of the genus Luteococcus differ substantially from the profiles of PAB strains of the genus Propionibacterium, which is an additional proof of the validity of whole-cell MALDI spectra for generic differentiation of bacteria. Our investigation shows that the bacterial groups determined using the MALDI profiles correlate with the phylogenetic 16S rRNA gene groups, thus demonstrating the high resolution of this method for the differentiation of intraspecific differences (subspecies, strains).     

Preparation of biocompatible chitosan grafted poly(lactic acid) nanoparticles

Chitosan grafted poly(lactic acid) (CS-g-PLA) copolymer was synthesized and characterized by FT-IR and elemental analysis. The degree of poly(lactic acid) substitution on chitosan was 1.90 ± 0.04%. The critical aggregation concentration of CS-g-PLA in distilled water was 0.17 mg/ml. Three methods of preparing CS-g-PLA nanoparticles (diafiltration method, ultrasonication method and diafiltration combined with ultrasonication method) were investigated and their effect was compared. Of the three methods, diafiltration combined with ultrasonication method produced nanoparticles with optimal property in terms of size and morphology, with size ranging from 133 to 352 nm and zeta potential from 36 to 43 mV. Also, the hemolytic activity and cytotoxicity of the CS-g-PLA based nanoparticles was tested, and results showed low hemolysis rate (<5%) and no significant cytotoxicity effect of these nanoparticles.     

Metalloinitiation routes to biocompatible poly(lactic acid) and poly(acrylic acid) stars with luminescent ruthenium tris(bipyridine) cores

Poly(lactic acid) (PLA) and poly(acrylic acid) (PAA) biomaterials with luminescent ruthenium tris(bipyridine) centers couple drug delivery and imaging functions. Hydrophobic [Ru(bpyPLA2)3](PF6)2 (1) was generated from [Ru[bpy(CH2OH)2]3](PF6)2 in bulk monomer using 4-(dimethylamino)pyridine as the catalyst. The bromoesters, [Ru[bpy(CH2OR)2]3](PF6)2, [Ru[bpy(C13H27)2][bpy(CH2OR]2](PF6)2 (4), and [Ru[bpy(PLAOR)2]3]2+ (9) (R=COCBr(CH3)2), served as initiators for tert-butyl acrylate (tBA) polymerization. Conversion of PtBA to PAA via hydrolysis affords water soluble materials, [Ru(bpyPAA2)3]2+ (7) and [Ru[bpy(C13H27)2](bpyPAA2)2]2+ (8) and the amphiphilic star polymer [Ru[bpy(PLA-PAA)2]3)](PF6)2 (11), which is soluble in a H2O/CH3CN (1:1) mixture. Luminescence excitation and emission spectra of the Ru polymers were in agreement with the parent [Ru(bpy)3]2+ chromophore (lambdaex=468, lambdaem=621 nm). Lifetimes of tau approximately 700 ns in both air and nitrogen atmospheres are typical for most materials; however, the amphiphilic star block copolymer 11 is quenched by oxygen to some degree. Thermal analysis shows the expected glass transitions for the polymeric ruthenium complex materials.     

3-(p-hydroxyphenyl)propionic acid as a new fluorogenic reagent for amine oxidase assays

A detailed procedure of a new and extremely sensitive fluorometric assay for amine oxidases is presented. Hydrogen peroxide, produced by the oxidase reaction, reacted with 3-(p-hydroxyphenyl)propionic acid in the presence of peroxidase to yield a fluorescent compound by which enzyme activity could be determined. The enzyme reaction was terminated by NaOH solution, which increased the fluorescence intensity three- to fivefold. The detection limit thus obtained was as little as 0.02 nmol. The alkalinization also contributed to stopping the enzyme reaction and to the clarification of assay mixtures containing turbid enzyme preparations.     

A high-throughput screening method for improved R-2-(4-hydroxyphenoxy)propionic acid biosynthesis

Bioprocess and Biosystems Engineering - R-2-(4-hydroxyphenoxy)propionic acid (R-HPPA) is a key intermediate of the enantiomerically pure phenoxypropionic acid herbicides. R-HPPA could be...     

Biocatalytic acylation of sugar alcohols by 3-(4-hydroxyphenyl)propionic acid

Enzymatic synthesis of aromatic esters of four different sugar alcohols (xylitol, arabitol, mannitol, and sorbitol) with 3-(4-hydroxyphenyl)propionic acid was performed in organic solvent medium, using immobilized Candida antarctica lipase (Novozyme 435), and molecular sieves for control of the water content. The influence of reaction parameters on the conversion has been investigated, including reaction time, temperature, alcohol/acid molar ratio, and enzyme amount. The highest conversions (94% for xylitol, 98% for arabitol, 80% for mannitol, and 93% for sorbitol) were obtained in pure tert-butanol at 60 °C and 72 h reaction time, 0.3 alcohol/acid molar ratio, and 0.5 g/mol enzyme/substrate ratio. The isolated new sugar alcohols esters were identified by different spectral analyses. MALDI-TOF MS analysis showed the formation of monoesters, diesters, and small quantities of triesters for all investigated sugar alcohols. The catalytic efficiency of the enzyme was higher for the pentitol substrates, decreasing in the following order: arabitol > xylitol > sorbitol > mannitol. These new compounds could have interesting applications in food, pharmaceutical and cosmetic formulations.     

Microbial production of propionic acid and vitamin B12 using molasses or sugar

With a cell concentration of 125 g dry biomass 1^–1 and a dilution rate of 0.1 h^–1,Propionibacterium acidipropionici produces 30 g propionic acid 1^–1 from sugar with a productivity of 3 g 1^–1 h^–1. The yield of propionic acid is approx. 0.36–0.45 g propionic acid g^–1 sucrose and is independent of the dilution rate and cell concentration. Acetic acid is an unwanted by-product in the production of propionic acid. The concentration of acetic acid only increases slightly when the cell concentration is increased. A two-stage fermentation process was developed for the conversion of sugar or molasses of various types to propionic acid and vitamin B₁₂. By fermentation of blackstrap molasses (from sugar beet and sugar cane) in the first fermentation stage 17.7 g propionic acid 1^–1 with a yield of 0.5 g propionic acid g^–1 carbohydrate was produced with a dilution rate of 0.25 h^–1. In the second stage 49 mg vitamin B₁₂ 1^–1 was produced at a dilution rate of 0.03 h^–1.     

The Effect of a Quaternary Amine (Aliquat 336) on Growth and Photosynthesis of the Green Alga Chlorella emersonii, and the Effect on Photosynthesis in Isolated Spinach Chloroplasts

A quaternary amine, Aliquat 336, inhibits the growth of the green alga Chlorella emersonii, ¹⁴C-fixation of the alga is also inhibited. The effect and the site of action of the compound was studied by using isolated spinach chloroplasts. The carbon dioxide dependent oxygen evolution of the chloroplasts is inhibited directly upon the addition of the amine and the oxygen evolution is replaced by an oxygen uptake. By investigating some electron transport reactions in the chloroplasts we were able to show that Aliquat 336 affects the electron transport on the level of photophosphorylation. The results from the in vivo and the in vitro experiments thus show that the quaternary amine affects the photosynthetic process. Aliquat 336 is a solvent extractant used in several industrial processes for extraction of metals from aqueous solutions. Aliquat 336 could be considered a presumptive water pollutant as the compound could enter a recipient water body and thus affect photosynthesis.