Application of metabolically engineered Saccharomyces cerevisiae to extractive lactic acid fermentation

In this study, Saccharomyces cerevisiae OC-2T T165R, metabolically engineered to produce optically pure L(+)-lactic acid, was used to develop a high performance extractive fermentation process. Since the transgenic yeast could produce lactic acid efficiently even at lower than pH 3.5, high extractive efficiency was achieved when tri-n-decylamine (TDA), a tertiary amine, was used as the extractant. Separation of microorganisms by means of a hollow fiber module could not only improve the total amount of lactic acid produced but also increase the lactic acid concentration in the solvent. Moreover, pH had a significant effect on extractive fermentation. The highest rate of recovery of lactic acid could be obtained on pH-uncontrolled fermentation (pH 2.5); however, the lowest amount of lactic acid was produced. Taking into account the trade-off between the fermentation and extraction efficiencies, the optimum pH value was considered to be 3.5, with which the largest amount of lactic acid was produced and the highest lactic acid concentration in the solvent was obtained. The results show promise for the use of the transgenic yeast for extractive fermentation.     

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.     

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。     

Production of ethanol by coupling fermentation and solvent extraction

Summary A new technology of fermentation is proposed. The inhibitor product is removed continuously by coupling fermentation and solvent extraction. Applied to ethanol fermentation this technology is suitable to any case where the terminal product is inhibitory.The proposed technology uses both plug flow reactor and liquid-liquid extraction to achieve continuously the extractive fermentation of ethanol. The solvent used for liquid-liquid extraction is dodecanol. A new reactor was used. It is a column packed with a porous material . The fermentation broth is pulsed (a) to increase the interfacial area between the liquid medium and the dodecanol, and (b) to: decrease the gas hold up.Alcoholic fermentations were performed on glucose syrup at 35°C using Saccharomyces cerevisiae, with adsorbed cells as reference, with adsorbed cells and extractive fermentation. The results show that the fermentation is substantially improved. By this new method the ethanol productivity was multiplied by 5 and a solution of 407 g/l of glucose was totally fermented with a yeast which cannot normally transform more than 200 g/l glucose.     

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.     

A Mathematical model for ethanol production by extractive fermentation in a continuous stirred tank fermentor

Extractive fermentation is a technique that can be used to reduce the effect of end product inhibition through the use of a water-immiscible phase that removes fermentation products in situ. This has the beneficial effect of not only removing inhibitory products as they are formed (thus keeping reaction rates high) but also has the potential for reducing product recovery costs. We have chosen to examine the ethanol fermentation as a model system for end product inhibition and extractive fermentation and have developed a computer model predicting the productivity enhancement possible with this technique together with other key parameters such as extraction efficiency and residual glucose concentration. The model accommodates variable liquid flowrates entering and leaving the system, since it was found that the aqueous outlet flowrate could be up to 35% lower than the inlet flowrate during extractive fermentation of concentrated glucose feeds due to the continuous removal of ethanol from the fermentation broth by solvent extraction. The model predicts a total ethanol productivity of 82.6 g/L h if a glucose feed of 750 g/L is fermented with a solvent having a distribution coefficient of 0.5 at a solvent dilution rate of 5.0 h(-1). This is more than 10 times higher than for a conventional chemostat fermentation of a 250 g/L glucose feed. The model has furthermore illustrated the possible trade-offs that exist between obtaining a high extraction efficiency and a low residual glucose concentration.     

Separation of lactic acid from fermented broth by reactive extraction

The separation of lactic acid from complex fermentation broth was examined. Liquid–liquid extraction using reversible chemical complexation for reactive extraction was chosen to be the separation method. Over 50% yield of lactic acid was obtained from fermented broth in a single extraction step, when using the tertiary amine as the extractant, 1-dekanol as the diluent and trimethylamine (TMA) as the stripping solution. The effect of complex media on the extraction behaviour has hardly been examined previously.     

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.     

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     

Present state and perspective of downstream processing of biologically produced 1,3-propanediol and 2,3-butanediol

1,3-Propanediol and 2,3-butanediol are two promising chemicals which have a wide range of applications and can be biologically produced. The separation of these diols from fermentation broth makes more than 50% of the total costs in their microbial production. This review summarizes the present state of methods studied for the recovery and purification of biologically produced diols, with particular emphasis on 1,3-propoanediol. Previous studies on the separation of 1,3-propanediol primarily include evaporation, distillation, membrane filtration, pervaporation, ion exchange chromatography, liquid–liquid extraction, and reactive extraction. Main methods for the recovery of 2,3-butanediol include steam stripping, pervaporation, and solvent extraction. No single method has proved to be simple and efficient, and improvements are especially needed with regard to yield, purity, and energy consumption. Perspectives for an improved downstream processing of biologically produced diols, especially 1,3-propanediol are discussed based on our own experience and recent work. It is argued that separation technologies such as aqueous two-phase extraction with short chain alcohols, pervaporation, reverse osmosis, and in situ extractive or pervaporative fermentations deserve more attention in the future.     

Liquid–liquid extraction of fermentation inhibiting compounds in lignocellulose hydrolysate

Several compounds that are formed or released during hydrolysis of lignocellulosic biomass inhibit the fermentation of the hydrolysate. The use of a liquid extractive agent is suggested as a method for removal of these fermentation inhibitors. The method can be applied before or during the fermentation. For a series of alkanes and alcohols, partition coefficients were measured at low concentrations of the inhibiting compounds furfural, hydroxymethyl furfural, vanillin, syringaldehyde, coniferyl aldehyde, acetic acid, as well as for ethanol as the fermentation product. Carbon dioxide production was measured during fermentation in the presence of each organic solvent to indicate its biocompatibility. The feasibility of extractive fermentation of hydrolysate was investigated by ethanolic glucose fermentation in synthetic medium containing several concentrations of furfural and vanillin and in the presence of decanol, oleyl alcohol and oleic acid. Volumetric ethanol productivity with 6 g/L vanillin in the medium increased twofold with 30% volume oleyl alcohol. Decanol showed interesting extractive properties for most fermentation inhibiting compounds, but it is not suitable for in situ application due to its poor biocompatibility. Biotechnol. Bioeng. 2009;102: 1354–1360. © 2008 Wiley Periodicals, Inc.     

小檗胺结晶因素的研究

研究了小檗胺结晶与提取溶剂、萃取剂加入量及盐析的关系,得出提高工业生产小檗胺结晶得率的方法．     

Production of D-phenylglycine from racemic (D,L)-phenylglycine via isoelectrically-trapped penicillin G acylase

Solvent selection for extractive fermentation for propionic acid was conducted with three systems: Alamine 304-1 (trilaurylamine) in 2-octanol, 1-dodecanol, and Witcohol 85 NF (oleyl alcohol). Among them, the solvent containing 2-octanol exhibited the highest partition coefficient in acid extraction, but it was also toxic to propionibacteria. The most solvent-resistant strain among five strains of the microorganism was selected. Solvent toxicity was eliminated via two strategies: entrapment of dissolved toxic solvent in the culture growth medium with vegetable oils such as corn, olive, or soybean oils; or replacement of the toxic 2-octanol with nontoxic Witcohol 85 NF. The complete recovery of acids from the Alamine 304-1/Witcohol 85 NF was also realized with vacuum distillation.     

Extractive fermentation by Zymomonas mobilis and the use of solvent mixtures

Summary A systematic and comprehensive screening of 1330 solvents, individually and as mixtures, was undertaken with the aim of improving on present extractive fermentation technology. The screening resulted in the selection of a solvent mixture of 5% by volume 4-heptanone and 95% Adol 85 NF (primarily oleyl alcohol). The performance of this solvent mixture in an extractive fed-batch fermentation configuration was compared with that of pure Adol 85 NF. The solvent mixture had a distribution coefficient 12% higher than that of pure Adol 85 NF and had no significant inhibitory effect on the fermentation under typical operating conditions.     

Effects of organic phase, fermentation media, and operating conditions on lactic Acid extraction

Lactic acid has extensive uses in the food, pharmaceutical, cosmetic and chemical industry. Lately, its use in producing biodegradable polymeric materials (polylactate) makes the production of lactic acid from fermentation broths very important. The major part of the production cost accounts for the cost of separation from very dilute reaction media where productivity is low as a result of the inhibitory nature of lactic acid. The current method of extraction/separation is both expensive and unsustainable. Therefore, there is great scope for development of alternative technology that will offer efficiency, economic, and environmental benefits. One of the promising technologies for recovery of lactic acid from fermentation broth is reactive liquid-liquid extraction. In this paper the extraction and recovery of lactic acid based on reactive processes is examined and the performance of a hydrophobic microporous hollow-fiber membrane module (HFMM) is evaluated. First, equilibrium experiments were conducted using organic solutions consisting of Aliquat 336/trioctylamine (as a carrier) and tri-butyl phosphate (TBP)/sunflower oil (as a solvent) The values of the distribution coefficient were obtained as a function of feed pH, composition of the organic phase (ratio of carrier to solvent), and temperature (range 8-40 degrees C). The optimum extraction was obtained with the organic phase consisting of a mixture of 15 wt % tri-octylamine (TOA) and 15% Aliquat 336 and 70% solvent. The organic phase with TBP performed best but is less suitable because of its damaging properties (toxicity and environmental impact) and cost. Sunflower oil, which performed moderately, can be regarded as a better option as it has many desirable characteristics (nontoxic, environment- and operator-friendly) and it costs much less. The percentage extraction was approximately 33% at pH 6 and at room temperature (can be enhanced by operating at higher temperatures) at a feed flow rate of 15-20 L/h. These results suggest that the hollow-fiber membrane process yields good percentage extraction at the fermentation conditions and its in situ application could improve the process productivity by suppressing the inhibitory effect of lactic acid.     

EXTRACTIVE FERMENTATION OF GIBBERELLIC ACID WITH FREE AND IMMOBILIZED Gibberella fujikuroi

Gibberelic acid fermentation using extractive methods was carried out in the presence of corn oil and Alamine 336. Gibberella fujikuroi fungus (NRRL 2278) was used to produce gibberellic acid. Oleyl alcohol was a diluting agent for Alamine 336. The effects of oleyl alcohol (100%, v/v), corn oil (5–25%, v/v), the concentration of Alamine 336 in oleyl alcohol, and feeding air were examined in this study. According to the results, oleyl alcohol was not effective on the production. On the other hand, oleyl alcohol solutions containing 15–30% (v/v) Alamine 336 showed effects as a toxic substance. In order to reduce solvent toxicity, corn oil was used. Addition of corn oil increased the concentration of gibberellic acid 1.3-fold compared to the control. Then the effects of immobilization and co-immobilization on extractive gibberelic acid fermentation were investigated. The highest total gibberellic acid concentration of 158.9 mg/L was produced with immobilized cells and feeding air by using extractive fermentation. The yield of gibberellic acid increased about 2.6-fold compared with the shake-flask fermentation (60.5 mg/L) without organic solutions.     

Ethanol production in a microporous hollow-fiber-based extractive fermentor with immobilized yeast

Microporous-membrane-based extractive product recovery in product-inhibited fermentations allows in situ recovery of inhibitory products in a nondispersive fashion. A tubular bioreactor with continuous strands of hydrophobic microporous hollow fibers having extracting solvent flowing in fiber lumen was utilized for yeast fermentation of glucose to ethanol. Yeast was effectively immobilized on the shell side in small lengths of chopped microporous hyrophilic hollow fibers. The beneficial effects of in situ dispersion-free solvent ex (oleyl alcohol and dibutyl phthalate) were demonstrated for a 300 g/L glucose substrate feed. Outlet glucose concentration dropped drastically from 123 to 41 g/L as solvent/ substrate flow ratio was increased from 0 to 3 at 9 mL/h of substrate flow rate with oleyl alcohol as extracting solvent. The significant productivity increase with in situ solvent extraction became more evident as solvent/ substrate flow ratio increased. A model of the locally integrated extractive bioreactor describes the observed fermentor performance quite well.     

用非常规培养方法提高乳酸菌产酸率的研究

以德氏乳酸杆菌为研究对象,考察了八种有机溶剂分别加入培养基对细菌生长及产酸的影响。结果表明,采用油醇和三辛胺混合溶剂时,既能降低对细胞生长的毒性,又保持了较强的萃取能力。对悬浮细胞发酵和萃取整合的方法和固定化细胞发酵和萃取整合的方法进行了比较,表明这两种方法均较常规培养方法提高乳酸产率60％以上。     

Design and evaluation of a continuous semipartition bioreactor for in situ liquid‐liquid extractive fermentation

Extractive fermentation (or in situ product removal (ISPR)) is an operational method used to combat product inhibition in fermentations. To achieve ISPR, different separation techniques, modes of operation and physical reactor configurations have been proposed. However, the relative paucity of industrial application necessitates continued investigation into reactor systems. This article outlines a bioreactor designed to facilitate in situ product extraction and recovery, through adapting the reaction volume to include a settler and solvent extraction and recycle section. This semipartition bioreactor is proposed as a new mode of operation for continuous liquid‐liquid extractive fermentation. The design is demonstrated as a modified bench‐top fermentation vessel, initially analysed in terms of fluid dynamic studies, in a model two‐liquid phase system. A continuous abiotic simulation of lactic acid (LA) fermentation is then demonstrated. The results show that mixing in the main reaction vessel is unaffected by the inserted settling zone, and that the size of the settling tube effects the maximum volumetric removal rate. In these tests the largest settling tube gave a potential continuous volumetric removal rate of 7.63 ml/min; sufficiently large to allow for continuous product extraction even in a highly productive fermentation. To demonstrate the applicability of the developed reactor, an abiotic simulation of a LA fermentation was performed. LA was added to reactor continuously at a rate of 33ml/h, while continuous in situ extraction removed the LA using 15% trioctylamine in oleyl alcohol. The reactor showed stable LA concentration of 1 g/L, with the balance of the LA successfully extracted and recovered using back extraction. This study demonstrates a potentially useful physical configuration for continuous in situ extraction.