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.     

Resistance of Lactobacillus casei in plastic-composite-support biofilm reactors during liquid membrane extraction and optimization of the lactic acid extraction system

Lactic acid fermentations were performed with plastic-composite-support (PCS) disks in solvent-saturated media with Lactobacillus casei subsp. rhamnosus (ATCC 11443). The PCS disks contained 50% (w/w) polypropylene, 35% (w/w) ground soybean hulls, 5% (w/w) yeast extract, 5% (w/w) soybean flour, and 5% (w/w) bovine albumin. Bioassays were performed by growing L. casei in solvent-saturated media after soaking the PCS disks. Eighteen different solvent and carrier combinations were evaluated. Overall, L. casei biofilm fermentation demonstrated the same lactic acid production in solvent-saturated medium as suspended cells in medium without solvents (control). To evaluate PCS solvent-detoxifying properties, two bioassays were developed. When solvent-saturated medium in consecutive equal volumes (10 mL then 10 mL) was exposed to PCS, both media demonstrated lactic acid fermentation equal to the control. However, when solvent-saturated medium with two consecutive unequal volumes (10 mL then 90 mL) was exposed to PCS, some degree of toxicity was observed. Furthermore, iso-octane, tributylphosphate (TBP), and Span 80 were optimized for recovery as 91%, 5%, and 4% (v/v), respectively, with a 1:1 ratio of 1.2 M Na(2)CO(3) stripping solution. Also, recovery by emulsion liquid extraction in the hollow-fiber contactor was minimal due to low recovery at pH 5.0 and incompatibility of the solvent and hollow-fiber material. These results suggest that PCS biofilm reactors can benefit lactic acid fermentation by eliminating the toxic effect from solvent leakage into the fermentation medium from liquid-liquid extractive integrated fermentations.     

Rapid screening of solvents and carrier compounds for lactic acid recovery by emulsion liquid extraction and toxicity on Lactobacillus casei (ATCC 11443)

This paper describes a rapid method to identify the best solvent and carrier compound combinations with the highest extraction capability and the lowest microbial toxicity characteristics for product recovery from microbial fermentation. The extraction system has an aqueous phase, and an emulsion phase, which was a blend of sodium carbonate and organic phase [91% (v/v) organic solvent, 5% (v/v or wt/v) carrier compound, and 4% (v/v) surfactant Span 80]. Alamine 336, or tri-n-octylamine in n-heptane; Alamine 336, Alamine 304, or tributyl phosphate in hexane; and Alamine 304 or tributyl phosphate in iso-octane; Alamine 304 or Amberlite in xylene demonstrated high lactic acid extraction. For determination of bacterial toxicity of selected solvent and carrier compounds, Lactobacillus casei subsp. rhamnosus (ATCC 11443) was grown in LAF medium containing one of the selected organic solvent, carrier compound, and Span 80 in 250 ml flask at 37 °C and 125 rpm. Samples were collected regularly during 48 hour incubation, and measured for changes in cell density by absorbance at 620 nm, cell count using a fluorescent dye with flow cytometry, and lactic acid, and glucose concentrations by HPLC. Hexadecane:tributyl phosphate, n-dodecane:tri-n-octylamine, and kerosene:tri-n-octylphosphine oxide demonstrated the least microbial toxicity among the tested blends with excess solvent media. Whereas, hexanes:Alamine 304 and xylenes:Alamine 304 were nontoxic in solvent saturated media.This revised version was published online in October 2005 with corrections to the Cover Date.     

Extractive fermentation for butyric acid production from glucose by Clostridium tyrobutyricum

A novel extractive fermentation for butyric acid production from glucose, using immobilized cells of Clostridium tyrobutyricum in a fibrous bed bioreactor, was developed by using 10% (v/v) Alamine 336 in oleyl alcohol as the extractant contained in a hollow-fiber membrane extractor for selective removal of butyric acid from the fermentation broth. The extractant was simultaneously regenerated by stripping with NaOH in a second membrane extractor. The fermentation pH was self-regulated by a balance between acid production and removal by extraction, and was kept at approximately pH 5.5 throughout the study. Compared with conventional fermentation, extractive fermentation resulted in a much higher product concentration (>300 g/L) and product purity (91%). It also resulted in higher reactor productivity (7.37 g/L. h) and butyric acid yield (0.45 g/g). Without on-line extraction to remove the acid products, at the optimal pH of 6.0, the final butyric acid concentration was only approximately 43.4 g/L, butyric acid yield was 0.423 g/g, and reactor productivity was 6.77 g/L. h. These values were much lower at pH 5.5: 20.4 g/L, 0.38 g/g, and 5.11 g/L. h, respectively. The improved performance for extractive fermentation can be attributed to the reduced product inhibition by selective removal of butyric acid from the fermentation broth. The solvent was found to be toxic to free cells in suspension, but not harmful to cells immobilized in the fibrous bed. The process was stable and provided consistent long-term performance for the entire 2-week period of study.     

Toxicity of organic extraction reagents to anaerobic bacteria

Various forms of liquid-liquid extraction systems are being developed to separate products, such as ethanol and volatile fatty acids (VFA), from fermentation liquids, since distillation is energetically expensive. Continuous extraction is advantageous, as product inhibition of the fermentation is minimized. However, some extraction solvents may be toxic or inhibitory to microorganisms.Thirty organic chemicals were examined by means of a small scale (60 mL) batch fermentation bioassay procedure for their toxicity to a commercial inoculum (Methanobac, W.B.E. Ltd.), which was a mixed culture of facultatively anaerobic, acid-producing bacteria. Gas production, pH change of medium, and the concentrations of ethanol, VFA, and lactic acid were measured after 75 h growth. The optimum experimental conditions for toxicity testing were alfalfa as substrate (2 g), a buffered nutrient medium (pH 6.8), "Methanobac" inoculum (10 mL), and test chemicals at levels between 10 and 100 muL/mL.Thirteen chemicals were nontoxic, and included the paraffins (C(6)-C(12)), phthalates, organophosphorus compounds, Freon 113 (1,1,2-trichloro-1,2,2-trifluoro ethane), Aliquat 336 (tricaprylylmethyl ammonium chloride), di-isoamyl ether, and trioctylamine. Other amine extractants were partially toxic. Alcohols (C(5)-C(12)), ketones (C(5)-C(8)), benzene derivatives, isoamyl acetate, and di-isopropyl ether were toxic. Generally, the chemicals were not toxic unless present at levels in excess of that expected to be required to saturate the aqueous phase.Total gas production was a good indicator of toxicity even within 24 h, but the presence of homofermentative (nongas producing) lactic acid bacteria complicated interpretation."Methanobac" inoculum was compared with an inoculum derived from a rumen culture for four test chemicals. The results were essentially the same. However, the toxicity of a chemical to bacteria is likely to vary considerably between bacterial species.     

Emulsion liquid membrane extraction of lactic acid from aqueous solutions and fermentation broth

Studies on the batch extraction of lactic acid using an emulsion liquid membrane system are reported. The membrane phase consists of the tertiary amine carrier Alamine 336 and the surfactant Span 80 dissolved in n-heptane/paraffin and aqueous solutions of sodium carbonate in the internal phase. The effects of internal phase reagent, extraction temperature, and initial external phase pH on the extraction efficiency and the emulsion swelling are examined. A statistical factorial experiment on extraction from clarified lactic acid fermentation broth was carried out to obtain knowledge of the performance of the extraction system from a broth. The extraction efficiency from the fermentation broth is found to be lower as compared to aqueous solutions of pure lactic acid. The effect of pH and the presence of other ionic species on selectivity are discussed. (c) 1993 John Wiley & Sons, Inc.     

Utilization of rice bran as nutrient source for fermentative lactic acid production

To reduce nutrient cost for lactic acid production, rice bran, one of agricultural wastes, was chosen as a nutrient source in this study. Although rice bran is rich in protein and vitamins, the use of rice bran without any treatment was inefficient in lactic acid production. Rice bran was treated by acid-hydrolysis before it was put in experiment, when it was hydrolyzed at initial pH 1, 30 g/L rice bran could provide a productivity to that degree of about 8 g/L YE, showing such a desirable result that the use of rice bran as nutrient source would be a solution for reducing nutrient cost. However, the addition of hydrolyzed rice bran prolonged lag phase of fermentation, especially, in the fermentation with rice bran hydrolyzed at initial pH 0.5, a prolonged lag phase of about 40 h was observed. According to the quantitative determination of thiamine, pyridoxine, organic nitrogen and carbon, the prolongation of lag phase might be the result from the destruction of B vitamins and excessive hydrolysis of protein. To shorten the lag phase, combining hydrolyzed rice bran with yeast extract (YE) of small amount was considered to be a solution. When 3g/L YE was combined with 30 g/L rice bran hydrolyzed at initial pH 1, obtained was a productivity 1.6 times higher than that of the control fermentation with 15 g/L YE.     

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-.     

Facilitated transport of lactic acid in a stirred transfer cell

The application of liquid membrane extraction to the recovery of lactic acid from model systems and fermentation media was investigated. An experimental study of the facilitated transport of lactic acid using ALIQUAT 336 as a mobile carrier in a stirred transfer cell is reported. The effect of stirring speed, initial lactic acid concentration, carrier concentration, and NaCl as a reagent in the acceptor phase are considered. (c) 1994 John Wiley & Sons, Inc.     

Extractive fermentation for lactic acid production

Lactic acid extractive fermentation was demonstrated using Alamine 336 in oleyl alcohol at acidic pH. The use of an efficient extraction system was possible through employment of the cell immobilization procedure. Process modeling was performed to relate the various process parameters such as flow rate, concentration, and pH. In experiments with 15% Alamine 336/oleyl alcohol, the bioreactor operation resulted in a higher productivity (12 g/L gel h) compared to that of a control fermentation (7 g/L gel h). Strategies for optimizing the extractive fermentation process were proposed considering both productivity and product recovery.     

三相流化床中固定化米根霉萃取发酵生产L-乳酸

以TRPO／磺化煤油为萃取剂,在2L三相流床反应器中进行了固定化米根霉原位萃取和异位萃取发酵生产L-乳酸的实验,结果表明,发酵液中的pH值能被控制在3．5左右．产酸速率高达每小时．每1L固定化颗粒产生11gL-乳酸。提出了一个数学模型用以描述萃取发酵中L-乳酸的积累及在各相的分配情况。模型计算曲线与实验值符合良好。     

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.     

Effective lactic acid production by two-stage extractive fermentation

For effective microbial lactic acid production using Lactobacillus delbrueckii, two-stage extractive fermentation was examined. Extractants were screened from the viewpoints of a high distribution coefficient for lactic acid and less toxicity toward the microorganism. Even if the extractant showed some toxicity toward the microorganism, it was found that a reduction of toxicity was possible by back-extraction using oleyl alcohol. As a result, 40% Alamine 336 diluted with oleyl alcohol, and oleyl alcohol, were selected as the extractant and the back-extractant, respectively. After two-stage extraction by these extractants, the growth rate was improved by the removal of lactic acid. This method was then applied to continuous extractive fermentation using a jar-fermentor. During 4-h extraction, lactic acid accumulation in the broth was significantly suppressed, while the cell growth and glucose consumption rates were also found not to be reduced. After 24 h, the cell concentration attained an OD₆₆₀ of 14, which corresponded to a level 1.25 times higher than that of the control culture without extraction. Total lactic acid productivity was 1.4 times level compared with the control culture.     

Improving the membrane permeability of sialic acid derivatives

The potential of boronic acids to improve the bioavailability of carbohydrate derived drugs was investigated through the study of the transport of four sialic acid derivatives through a lipophilic supported liquid membrane at departure phase pH's of 7.4, 8.5 and 10.0. It was found that facilitated transport did occur in most cases, but interestingly, and in contrast to that observed with monosaccharides such as d-fructose, the lipophilic ammonium salt, Aliquat 336, promoted fluxes than those of the boronic acid. The triol side chain of the sialic acid derivatives, combined with the amide at C5, appears to represent a previously unrecognised chloride binding domain which promotes extraction of these compounds into membranes containing Aliquat 336, leading to fluxes greater than those produced by boronic acids.     

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.     

反相高效液相色谱法测定肠球菌FQ15发酵液中的有机酸

目的研究肠球菌FQ15发酵液中的有机酸在不同发酵时间的变化趋势。方法建立反相高效液相色谱法测定此株益生菌发酵液中有机酸的主要成分及其在发酵不同时间的变化趋势。结果丙酸、丙酮酸、乳酸、乙酸的浓度都是在菌体生长处于衰亡期时达到最大值,乳酸随发酵时间的延长浓度明显下降,而乙酸在发酵后期含量呈上升趋势。结论此方法重现性好,精密度高,为研究微生物合成中有机酸种类及变化趋势提供了一种可供参考的快捷分析手段。     

Extraction of organic acids from kiwifruit juice using a supported liquid membrane process

In order to extract or remove organic acids from kiwifruit juice, we evaluated their separation and transport rates through supported liquid membranes (SLMs). The liquid membrane consisted of an organic solution composed of a carrier (Aliquat 336/Alamine 336) and a linear alcohol (oleyl alcohol) and was loaded on a microporous polypropylene support (commercial grade Celgard 2500/2400). These SLMs were evaluated (i) in a batch cell to determine the permeability and (ii) in a continuous spiral membrane module to study the effects of various process parameters – flow of feed and strip solutions, membrane composition, recycling mode of operation and kiwifruit juice at natural pH. It was observed that there exists an optimum for each system: pH?2.5–?3.0 for Alamine 336/oleyl alcohol and pH?4.5 for Aliquat 336/oleyl alcohol. At this pH?the flux rates of citric acid and malic acid was greater (6–8 times) than that of quinic acid. The flux rates decreased (greatly for citric acid) with the flow rate of feed and strip solutions and increased (considerably for citric acid) with the SLM composition . The recycling of feed and strip solutions significantly improved the removal efficiency. The SLM system retained its performance over a period of a few days. The SLM process allowed extraction of the above three organic acids (ascorbic acid was removed in trace amounts) from kiwifruit juice at a rate of a few percent (5%) in a single-pass processing.     

Simultaneous saccharification and co-fermentation of crystalline cellulose and sugar cane bagasse hemicellulose hydrolysate to lactate by a thermotolerant acidophilic Bacillus sp

Polylactides produced from renewable feedstocks, such as corn starch, are being developed as alternatives to plastics derived from petroleum. In addition to corn, other less expensive biomass resources can be readily converted to component sugars (glucose, xylose, etc.) by enzyme and/or chemical treatment for fermentation to optically pure lactic acid to reduce the cost of lactic acid. Lactic acid bacteria used by the industry lack the ability to ferment pentoses (hemicellulose-derived xylose and arabinose), and their growth and fermentation optima also differ from the optimal conditions for the activity of fungal cellulases required for depolymerization of cellulose. To reduce the overall cost of simultaneous saccharification and fermentation (SSF) of cellulose, we have isolated bacterial biocatalysts that can grow and ferment all sugars in the biomass at conditions that are also optimal for fungal cellulases. SSF of Solka Floc cellulose by one such isolate, Bacillus sp. strain 36D1, yielded l(+)-lactic acid at an optical purity higher than 95% with cellulase (Spezyme CE; Genencor International) added at about 10 FPU/g cellulose, with a product yield of about 90% of the expected maximum. Volumetric productivity of SSF to lactic acid was optimal between culture pH values of 4.5 and 5.5 at 50 degrees C. At a constant pH of 5.0, volumetric productivity of lactic acid was maximal at 55 degrees C. Strain 36D1 also co-fermented cellulose-derived glucose and sugar cane bagasse hemicellulose-derived xylose simultaneously (SSCF). In a batch SSCF of 40% acid-treated hemicellulose hydrolysate (over-limed) and 20 g/L Solka Floc cellulose, strain 36D1 produced about 35 g/L lactic acid in about 144 h with 15 FPU of Spezyme CE/g cellulose. The maximum volumetric productivity of lactic acid in this SSCF was 6.7 mmol/L (h). Cellulose-derived lactic acid contributed to about 30% of this total lactic acid. These results show that Bacillus sp. strain 36D1 is well-suited for simultaneous saccharification and co-fermentation of all of the biomass-derived sugars to lactic acid.     

Extractive lactic acid fermentation in poly (ethyleneimine)-based aqueous two-phase system

The potential of an aqueous two-phase system composed of a polycation, poly(ethyleneimine) (PEI), and an uncharged polymer, (hydroxyethyl) cellulose (HEC), for extractive lactic acid fermentation was tested. Batch fermentation with 20 g/L glucose in two-phase medium using Lactococcus lactis without external pH control resulted in 3-4 times higher amount of lactate and biomass produced as compared to that in a conventional one-phase medium. Lactic acid was preferentially partitioned to the PEI-rich bottom phase. However, the cells which favored the HEC-rich top phase in a fresh two-phase medium were partitioned to a significant extent to the bottom phase after fermentation. Addition of phosphate buffer or pH adjustment to 6.5 after fermentation caused fewer cells to move to the bottom phase. With external pH control, fermentation in normal and two-phase medium showed no marked differences in glucose consumption and lactic acid yield, except that about 1.3 times higher cell density was obtained in the two-phase broth, especially at initial glucose concentrations of 50-100 g/L. Use of higher concentration of phosphate during batch fermentation in the two-phase medium with 50 g/L sugar provided a 15% higher yield of lactic acid, but the growth rate of cells was nearly half of the normal, thus affecting the productivity. Continuous fermentation with twice the normal phosphate concentration resulted in higher cell density, product yield, and productivity in two-phase medium than in monophasic medium. (c) 1996 John Wiley & Sons, Inc.     

Fermentative lactic acid production with a metabolically engineered yeast immobilized in photo-crosslinkable resins

In this study, the immobilization technique involving photo-crosslinkable resin gels was used for lactic acid production. Saccharomyces cerevisiae OC-2T T165R, a metabolically engineered yeast that produces optically pure l(+)-lactic acid, was immobilized in hydrophilic photo-crosslinked resin gels as a biocatalyst. Three resin gels, TEP 1, TEP 2 and TEP 3, were examined and all of them showed high performance as to lactic acid production. Resin gel TEP 1, which exhibited the highest productivity among the resin gels was used for 15 consecutive batch fermentations without decreases in productivity and mechanical deformation, indicating that it was a suitable carrier for long-term lactic acid fermentation. Moreover, the use of the immobilization technique can improve the productivity of the metabolically engineered yeast in the fermentation with or without extraction, showing promise for using the immobilized engineered yeast for lactic acid production.