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.     

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.     

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.     

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.     

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.     

In-situ recovery of butanol during fermentation

End product inhibition can be reduced by the in situ removal of inhibitory fermentation products as they form. Extractive fermentation, in which an immiscible organic solvent is added to the fermentor in order to extract inhibitory products, was applied to the acetone-butanol fermentation. Six solvents or solvent mixtures were tested in batch extractive fermentations: kerosene, 30 wt% tetradecanol in kerosene, 50 wt% dodecanol in kerosene, oleyl alcohol, 50 wt% oleyl alcohol in a decane fraction and 50 wt% oleyl alcohol in benzyl benzoate. The best results were obtained with oleyl alcohol or a mixture of oleyl alcohol and benzyl benzoate. In normal batch fermentation of Clostridium acetobutylicum, glucose consumption is limited to about 80 kg/m³ due to the accumulation of butanol in the broth. In extractive fermentation using oleyl alcohol or a mixture of oleyl alcohol and benzyl benzoate, over 100 kg/m³ of glucose can be fermented. Removal of butanol from the broth as it formed also increased the rate of butanol production. Maximum volumetric butanol productivity was increased by as much as 60% in extractive fermentation compared to batch fermentation. Butanol productivities obtained in extractive fermentation compare favorably with other in situ product removal fermentations.     

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.     

In situ extractive fermentation of acetone and butanol

The productivity of the acetone-butanol fermentation was increased by continuously removing acetone and butanol from the fermentation broth during fed-batch culture. Whole broth containing viable cells of Clostridium acetobutylicum was cycled to a Karr reciprocating plate extraction column in which acetone and butanol were extracted into oleyl alcohol flowing counter-currently through the column. By continuously removing these toxic metabolites from the broth, end product inhibition was reduced, and a concentrated feed solution containing 300 g/L glucose was fermented at an overall butanol productivity of 1.0 g/L h, 70% higher than the productivity of normal batch fermentation. The continuous extraction process provides flexible operation and lends itself to process scale-up.     

Effects of extractive fermentation on butyric acid production byClostridium acetobutylicum

Summary The addition of an oleyl alcohol extractant to a batch fermentation of glucose byClostridium acetobutylicum resulted in a concentration profile that was distinctly different from the non-extractive control fermentation. The concentration of butyric acid increased and subsequently decreased in the control fermentation. The concentration of butyric acid increased but did not subsequently decrease in the oleyl alcohol extractive fermentation. The production of butyric acid was found to have been prolonged into the solventogenic phase in the oleyl alcohol extractive fermentation. Butyric acid was continually replenished from glucose while it was being converted to butanol. Supplementation of exogenous acetic and butyric acids, the metabolic uncoupler carbonyl cyanide 3-chlorophenylhydrazone, or decanol to the oleyl alcohol extractive fermentation helped to reinstate the normal butyric acid concentration profile. These findings are discussed with respect to the effects of these additives on the pH ofC. acetobutylicum and its importance with regard to the production of butyric acid.     

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.     

Separation of dilute aqueous butanol and acetone solutions by pervaporation through liquid membranes

A simultaneous extraction-stripping process is proposed for separating volatile products from fermentation broths, it is based on pervaporation through a liquid membrane supported with a hydrophobic porous membrane. The liquid membrane prepared with oleyl alcohol was selected as the most suitable for separating volatile products resulting from acetone-butanol fermentation. The separation performance and stability of the oleyl alcohol liquid membrane were investigated by using dilute aqueous butanol and acetone solutions. The oleyl alcohol liquid membrane was found to be superior by far in both selectivity and permeability of butanol to the better known silicone rubber membrane in its high selectivity for alcohols. Using the oleyl alcohol liquid membrane, the dilute aqueous butanol solutions of around 4 g/L obtained in acetone-butanol fermentation could be concentrated up to 100 times. The stability of this liquid membrane was also quite good as long as the surface tension of the feed solution was less than the critical surface tension of the support membrane. No change in the separation performance was found after the continuous usage in a long period of 100 h.     

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。     

Extractive lactic acid fermentation using ion-exchange resin

Lactic acid fermentation is an end-product-inhibited reaction. The restriction imposed by lactic acid on its fermentation can be avoided by extractive fermentation techniques. Studies were performed by attaching an ion-exchange resin packed column with a 2-L fermentor for separation of lactic acid. The fermentation, in a conventional batch mode, resulted in a lactic acid yield of 0.828 g . g(-1) and a lactic acid productivity of 0.313 g . L(-1) . h(-1). However, these could be further enhanced to 0.929 g . g(-1) and 1.665 g . L(-1) . h(-1) by extractive fermentation techniques. The effect of temperature on extractive fermentation was remarkable and has been included in this work.     

In-situ recovery of butanol during fermentation

End-product inhibition in the acetone-butanol fermentation was reduced by using extractive fermentation to continuously remove acetone and butanol from the fermentation broth. In situ removal of inhibitory products from Clostridium acetobutylicum resulted in increased reactor productivity; volumetric butanol productivity increased from 0.58 kg/(m³h) in batch fermentation to 1.5 kg/(m³h) in fed-batch extractive fermentation using oleyl alcohol as the extraction solvent. The use of fed-batch operation allowed glucose solutions of up to 500 kg/m³ to be fermented, resulting in a 3.5- to 5-fold decrease in waste water volume. Butanol reached a concentration of 30–35 kg/m³ in the oleyl alcohol extractant at the end of fermentation, a concentration that is 2–3 times higher than is possible in regular batch or fed-batch fermentation. Butanol productivities and glucose conversions in fed-batch extractive fermentation compare favorable with continuous fermentation and in situ product removal fermentations.List of Symbols C _g kg/m³ concentration of glucose in the feed - C _w dm³/m³ concentration of water in the feed - F(t) cm³/h flowrate of feed to the fermentor at time t - V(t) dm³ broth volume at time t - V _i dm³ initial broth volume - V _si dm³ volume of the i-th aqueous phase sample - effective fraction of water in the feed Part 1. Bioprocess Engineering 2 (1987) 1–12     

Enhancement of Butanol Formation by Clostridium acetobutylicum in the Presence of Decanol-Oleyl Alcohol Mixed Extractants

Extractive fermentation has been proposed to enhance the productivity of fermentations that are end product inhibited. Unfortunately, good extractants for butanol, such as decanol, are toxic to Clostridium acetobutylicum. The use of mixed extractants, namely, mixtures of toxic and nontoxic coextractants, was proposed to circumvent this toxicity. Decanol appeared to inhibit butanol formation by C. acetobutylicum when present in a mixed extractant that also contained oleyl alcohol. However, maintenance of the pH at 4.5 alleviated the inhibition of butanol production and the consumption of butyrate during solventogenesis. A mixed extractant that contained 20% decanol in oleyl alcohol enhanced butanol formation by 72% under pH-controlled conditions. The production of acetone and acetoin was also increased, even though these two products were not extractable. The enhancement of butanol formation was not limited by the toxicity of decanol. Supplementation of glucose and butyrate in the extractive fermentation yielded a 47% increase in butanol. The enhancement of butanol formation appeared to be dependent on the presence of dissolved decanol in the broth but was not observed unless an organic phase was present to extract butanol. A mechanism for the effects of decanol on product formation is proposed.     

In situ recovery of 2,3-butanediol from fermentation by liquid–liquid extraction

End-product conversion, low product concentration and large volumes of fermentation broth, the requirements for large bioreactors, in addition to the high cost involved in generating the steam required to distil fermentation products from the broth largely contributed to the decline in fermentative products. These considerations have motivated the study of organic extractants as a means to remove the product during fermentation and minimize downstream recovery. The aim of this study is to assess the practical applicability of liquid–liquid extraction in 2,3-butanediol fermentations. Eighteen organic solvents were screened to determine their biocompatibility, and bioavailability for their effects on Klebsiella pneumoniae growth. Candidate solvents at first were screened in shake flasks for toxicity to K. pneumoniae. Cell density and substrate consumption were used as measures of cell toxicity. The possibility of employing oleyl alcohol as an extraction solvent to enhance end product in 2,3-butanediol fermentation was evaluated. Fermentation was carried out at an initial glucose concentration of 80 g/l. Oleyl alcohol did not inhibit the growth of the fermentative organism. 2,3-Butanediol production increased from 17.9 g/l (in conventional fermentation) to 23.01 g/l (in extractive fermentation). Applying oleyl alcohol as the extraction solvent, about 68% of the total 2,3-butanediol produced was extracted. An erratum to this article can be found at     

固定化细胞利用离子交换树脂萃取发酵乳酸的研究

本文提出了利用海藻酸钙凝胶包埋固定化乳酸菌生产乳酸,用离子交换树脂从发酵液中分离出乳酸的新方法。该法成功地消除了产物乳酸对乳酸菌生长和产物乳酸形成的抑制作用,使发酵时间由１２０小时缩短到９６小时,乳酸的体积生产率由０．３２８ｇ／Ｌ·ｈ提高到０．４３２ｇ／Ｌ·ｈ。     

Improvement of welan gum production and redistribution of metabolic flux under pH control process in Alcaligenes sp. CGMCC2428

Batch fermentations of welan gum from Alcaligenes sp. CGMCC2428 at pH values of 5.5 ～ 7.0 were studied. Based on the kinetic analysis, a pH control process for improving welan production was developed. By maintaining the culture pH at 7.0, the process significantly improved the maximal welan concentration and productivity to reach 25.1 ± 0.65 g/L and 0.42 ± 0.003 g/L/h, respectively, compared with those in native pH processes where pH value would decrease from 7.0 to 5.1 (18.5 ± 0.72 g/L and 0.28 ± 0.002 g/L/h). This improvement may be due to the increased metabolic flux of glucose-1-phosphate to welan induced by pH control process. Furthermore, scale-up fermentation under controlled pH was implemented using 300-L fermentor. The highest welan concentration of 27.5 ± 0.97 g/L was obtained. This simplified process proved effective in industry fermentation for high welan production.     

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.     

Theoretical development and performance evaluation for extractive fermentation using multiple extractants

Mathematical formulation was made for the performance evaluation of extractive fermentation using multiple solvents. Two types of solvent-supplying strategies were considered. One is to add multiple solvents simultaneously and the product is removed at one time. Another is to add them one by one consecutively. Computer simulation was made for batch, fed-batch, and repeated fed-batch operation of acetone-butanol fermentation to show the power of the approach. The result shows that the significant performance improvement in terms of the productivity and the product concentration is expected when two extractants such as oleyl alcohol and benzyl benzoate are used as compared with the case of using only one solvent.