A novel extractive fermentation process for propionic acid production

Propionic acid is the major product from lactose fermentation, with acetic acid and carbon dioxide as byproducts. Propionic acid is a strong inhibitor...
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Biotechnol. h g . 1992, 8, 104-110

A Novel Extractive Fermentation Process for Propionic Acid Production from Whey Lactose Vivian P. Lewis and Shang-Tian Yang* Department of Chemical Engineering, The Ohio State University, 140 West 19th Avenue, Columbus, Ohio 43210

An extractive fermentation process was developed to produce propionate from lactose. The bacterium Propionibacterium acidipropionici was immobilized in a spiral wound, fibrous matrix packed in the reactor. Propionic acid is the major product from lactose fermentation, with acetic acid and carbon dioxide as byproducts. Propionic acid is a strong inhibitor to this fermentation. A tertiary amine was used to selectively extract propionic acid from the bioreactor, hence enhancing reactor productivity by over 100%. We also speculate that by selectively extracting propionic acid, lactose metabolism can be directed to yield more propionate and less byproducts. Other advantages of extractive fermentation include better pH control (by removing acid products) and a purer product. The propionic acid present in the extractant can be easily stripped with small amounts of an alkaline solution, resulting in a concentrated propionate salt. The extractant was also regenerated in this stripping step. Thus, the process is energy-efficient and economically attractive.

Introduction Propionic acid is an important mold inhibitor. Its calcium, sodium, and potassium salts are widely used as food and feed preservatives. To date, commercial production of propionic acid is almost entirely by petrochemical routes (Playne, 1985). However, there has been increasinginterest in producing propionic acid from whey lactose and other cheap biomass using propionibacteria (Blanc & Goma, 1987a; Bodie et al., 1987; Border et al., 1987;Boyaval and Corre, 1987;Cavin et al., 1985;Clausen and Gaddy, 1981; Crespo et al., 1990; Datta, 1981; Emde and Schink, 1990; Hendricks et al., 1985; Hsu and Yang, 1991; Lewis, 1991; O’Brien et al., 1990). Propionic acid bacteria have long been used in the dairy industry. These bacteria play important roles in the development of the characteristic flavor and eye production in Swiss-type cheeses. Propionibacteria are Grampositive, nonspore-forming,rod-shaped, facultative anaerobes. The optimum pH range for growth is between 6 and 7, and at pH C 4.5 there is practically no growth (Hsu & Yang, 1991). Like most organic acid fermentations, the propionic acid fermentation is inhibited by acidic pHs and the major fermentation product, propionic acid (Blanc and Goma, 1987b; Ibragimova et al., 1969; Neronova et al., 1967). The conventional fermentation technology for propionate production is thus limited by low fermentation rate and low product concentration. Furthermore, the fermentation is heterogeneous; i.e., propionate is produced along with other byproducts. This not only results in a low product yield but also renders product purification difficult and expensive. Consequently, the conventional fermentation route for propionic acid production is inefficient and it competes with difficulty with petrochemical routes. Presently, only small amounts of propionate are produced by fermentation of whey and are used as a natural product in foods for the labeling purpose. In order to make the fermentation route economically viable, it is necessary to develop novel fermentation processes that use highly efficient bioreactors and separations techniques.

* Corresponding author. 8756-7938/92/3008-0104$03.00/0

Recently, integrated fermentation-separation systems have been successfully used to reduce end-product inhibition and, thus, to improve the overall process efficiency (Daugulis, 1988; Roffler et al., 1984). To date, the most studied process has been the extractive fermentation for ethanol production. Several extractive fermentation systems also have been studied for organic acid production (Bar and Gainer, 1987; Yabannavar and Wang, 1991). Extractive fermentation removes the inhibitory, acidic product from the reactor and, therefore, provides better pH control on the reactor and results in higher reaction rates. Also, products are present in relatively pure and concentrated forms. Thus, savings in the downstream recovery and purification costs can be realized. However, all prior extractive fermentation studies dealt with homofermentative products, such as lactic, acetic, and citric acids. To our knowledge, no extractive fermentation has been studied for heterofermentative products such as propionic acid. Successful development of an extractive fermentation process requires careful selection of a highly efficient and nontoxic solvent for extraction. Conventional solvents such as alcohols, ketones, ethers, and aliphatic hydrocarbons are not efficient when applied to dilute, carboxylic acid solutions because of the low aqueous activity of carboxylic acids resulting in low distribution coefficients (Kertes and King, 1986). Furthermore, most organic solvents are toxic to bacteria; they will either inhibit or stop bacterial growth. Extractive fermentation using conventional extractants usually suffers from solvent toxicity and a low separation factor, and it may require formidably expensivesolvent regeneration procedures. To date, no extractive fermentation has been developed for industrial applications. Recently, several aliphatic amines have been used successfullyto extract carboxylicacids (Wardell and King, 1978; Ricker et al., 1980; Kertes and King, 1986; Wennersten, 1983;Tamada and King, 1990;Yang et al., 1991). The strong amine interactions with the acid allow for formation of acid-amine complexes and thus provide for high distribution coefficients. In addition, the high affinity of the organic base for the acid gives selectivity for the

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acid over nonacidic components in the mixture. A tertiary amine, Alamine 336, has been shown to pose only slight toxicity to immobilized homolactic bacteria and was used successfully in extractive fermentation to improve the lactic acid production rate (Yabannavarand Wang, 1991b). In this work, we used an Alamine 336/2-octanol mixture as the extractant in an extractive fermentation process for propionate production from whey lactose. This extractant has higher distribution coefficients for propionic acid than for acetic acid (Yang et al., 1991). Thus, propionic acid is preferentially extracted from the mixed acid products present in the fermentation broth. By properly designing the extractor, it is possible to selectively remove propionic acid and thus to produce a pure product, even though the fermentation is heterogeneous. We also speculated that by selectively extracting propionic acid, lactose metabolism can be directed toward propionic acid, instead of acetic acid or other byproducts, to yield more propionate. The results from a prototype laboratory unit are reported in this article. Effects of pH, product inhibition, and solvent toxicity on propionic acid fermentation were also studied and are discussed in this paper.

Materials and Methods Culture and Media. Propionibacterium acidipropionici ATCC 4875 was used in this work. A synthetic lactose medium was used as the feed to the bioreactor. This medium contained the following (per liter): 10 g of yeast extract (Difco), 5 g of Trypticase (BBL), 0.25 g of KzHP04,0.05 g of MnS04, and 16g of lactose. The medium was prepared in two parts: the basal medium (without lactose) and a concentrated lactose solution. These two solutions were adjusted to pH 7.0 by the addition of NaOH solution. They were then mixed aseptically, after heat sterilization at 121 "C and 15 psig for 20 min. In some kinetic studies, lactate was used to replace lactose as the carbon source for cell growth. To study the pH effect, the medium pH was adjusted by the addition of 4 N HC1 or 4 N NaOH. For a study of the effects of acetic and propionic acids, the media also contained various amounts of propionic acid (0-20 g/L) or acetic acid (0-9 g/L). For a study of the solvent toxicity, the media were prepared from distilled water with different degrees of solvent saturation. All media were purged with N2 before being autoclaved. Fermentation Kinetics. Inhibition kinetics were studied to evaluate the effects of pH, propionic and acetic acids, and solvent toxicity on cell growth. Unless otherwise noted, these kinetic studies were carried out at 30 "C, in 18-mL serum tubes, and lactate was used as the carbon source in the growth medium to minimize pH change due to growth. (About 1 mol of propionic and acetic acids would form from each mol of lactate fermented.) Each tube containing 10mL of the medium was inoculated with 0.5 mL of 1-day-old culture. Cell growth was monitored by direct measurement of the optical density a t 660 nm (ODm) in the serum tube using a spectrophotometer (Sequoia-Turner, Model 340). The specific growth rate (p) was determined from the slope of the semilogarithmicplot of OD versus time. Only the initial growth rate data were used to evaluate the effects of pH, acetate, propionate, and solvent. All experiments were at least duplicated to get consistent and statistically meaningful results. Extraction. The amine extractant, Alamine 336 (from Henkel Corp.), was mixed with a diluent, 2-octanol, at various proportions to determine the optimal extractant composition for propionic and acetic acids extraction. Acid solutions (pH 3.0) containing 30 g/L propionic or acetic acid or both were mixed with an equal volume of the exN

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Figure 1. The immobilized cell bioreactor and extractor used in ex situ extractive fermentation.

tractant. After equilibrium was reached at 30 "C, phase separation was facilitated by centrifugation. The distribution coefficient at equilibrium was determined from the acid concentrations in the organic and aqueous phases. Organic acids in the aqueous phase were determined by high-performance liquid chromatography (HPLC) or gas chromatography (GC). The organic phase was backextracted with an alkaline solution first, and then, this solution was analyzed to determine the amounts of acids present in the extractant (organic phase). A material balance was performed to check that the total amounts of the organic acids before and after extraction were equal. Immobilized Cell Bioreactor. The bioreactor used for extractive fermentation was made of a 2-in. glass column packed with spiral wound, cotton towel as the matrix for cell immobilization. This packing design allows good contact between two different moving phases (such as gasliquid or solvent-aqueous two phases). The reactor packing length was 12 in., and the reactor working volume was 600 mL. These reactors were autoclaved twice and then filled with sterilized growth medium and inoculated with 50 mL of cells. The cells in the bioreactor were allowed to grow for 2-3 days before a continuous feed was started. These reactors were fed with a lactate medium for several weeks to build up the cell mass in the reactor. The lactate medium was used to avoid a significant pH drop in the reactor. After the reactor packing has been saturated with the cells, the reactor feed was switched to the lactose medium. Extractive Fermentation. Extractive fermentation was studied first with external extraction and then with in situ extraction. The experimental apparatus used for the ex situ extraction process is shown in Figure 1. The extraction column was made of a 1-in.glass column packed with 0.2541. ceramic saddles. The packing length was 18 in. Both the bioreactor and the extractor were controlled at 30 "C. The extractant used was 40% (w/w) Alamine 336 in 2-octanol. Before use, fresh extractant was contacted with the medium to wash off any water-soluble impurities in the solvent and to saturate the solvent with the ingredients present in the medium. After each extraction use, the extractant was regenerated by contacting it with 1N NaOH solution to strip propionic and acetic acids from the solvent. The regenerated extractant was then reused for the next extraction. The extractive fermentation was operated at the following conditions: feed rate, 0.3 L/d; dilution rate, 0.5 d-l; recycle rate, 1.2 L/d;extractant flow rate, 1.68L/d. At appropriate time intervals, samples were collected from the bioreactor outlet and two extractor outlet streams (aqueous and organic phases). For the in situ extraction process, the extractant was fed directly into the reactor

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from the reactor bottom. All other operating conditions remained unchanged except that there was no recirculation. Both the aqueous and organic phases were collected from the outlet at the top of the reactor. All aqueous samples were analyzed for pH and cell concentration immediately. These samples were then stored in a freezer for future HPLC analysis. Organic-phase samples were back-extracted with 0.33 N NaOH, and the resulting aqueous samples were also stored for future HPLC analysis. Assay Methods. A high-performanceliquid chromatograph was used in analyzing organic compounds including lactose and propionic and acetic acids in the fermentation broth. A Waters HPLC system (6000Apump,710B WISP injector, 481 variable wavelength detector, and R410 refractometer) and a Bio-Rad HPX-87H column (at 46 "C) were used; 0.013 N HzS04, a t a flow rate of 0.6 mL/min, was used as the eluant. Samples containing low concentrations of volatile compounds (e.g., propionic and acetic acids) were determined using a gas chromatograph (GC) equipped with a flame ionization detector (FID) and a fused silica megabore column (DBWAX, 15 m X 0.534 mm; J & W Scientific). The carrier gas was N2 at a flow rate of 30 mL/min. The operating temperatures were injector, 110 "C; column, 110 "C; and detector, 160 "C. Ethanol or butyric acid was used as an internal standard in determining the acid concentration in the sample. Suspended cell growth was monitored by the optical density (OD) at 660 nm using a spectrophotometer. OD was measured in a 1.5-mL polystyrene cuvette with a light path of 10 mm. Immobilized cell growth in the fibrous matrix was examined by using a scanning electronic microscope (SEM).Detailed procedures can be found elsewhere (Lewis, 1991).

Rssults Fermentation Kinetics. Effect of pH. Figure 2 shows the effect of pH on the specific growth rate ( p ) . When lactose was the growth substrate, this propionibacterium has an optimum pH at -7 and p decreases with a decreasing pH. Note that p dropped drastically a t pH below 5. Effect ofPropionic Acid. Propionic acid is known to inhibit the propionic acid fermentation. Figure 3 shows that at all pH values studied, p dropped dramatically with an increasing propionate concentration. However, this decreasing trend leveled off when the propionate concentration was greater than -10 g/L. Effect of Acetic Acid. At pH 6.15, acetic acid did not affect p significantly (Figure 4). However, as also shown in Figure 4, slight acetate inhibition was found a t a lower pH value, but the effect was not as dramatic as that found with propionate. Solvent Toxicity. It is known that the organic solvent used for extraction usually imposes some degree of toxicity

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to the microorganism. The toxicity of the extractant (Alamine 336/2-octanol mixture) dissolved in the growth medium was studied first with batch cultures of suspended cells. As shown in Figure 5, the extractant inhibits the cell growth, especially at concentrations higher than 60 % saturation. However, even at 100% saturation, the bacterium still has significant growth. The solvent toxicity was also tested with immobilized cells in the continuous process. The reactor was operated under plug-flow conditions and fed with media containing -45 g/L lactate and solvent at different degrees of saturation. Steady-state concentrations of lactate, propionate, and acetate in the effluent were monitored and are shown in Table I. As shown in this table, generally there were more substrate left and less products produced when more solvent was present, indicating that fermentation was inhibited by the solvent. Nevertheless, even at 1005% saturation the solvent toxicity on the fermentation was not severe. As also shown in Table I, the Ypand Y, values remained almost unchanged at all levels of solvent saturation. Thus, the presence of solvent did not affect product yields from the lactate fermented, although the total acid production could be lower at higher solvent concentrations due to slower reaction rates.

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Table I. Effects of Solvent Toxicity on Propionic Acid Fermentation Ypn Y," solvent concn lactic propionic acetic (% saturation) acid (g/L) acid (g/L) acid (g/L) (g/g) (g/g) 0.68 0.22 6.13 16.75 19.33 0 5.96 0.67 0.22 18.40 25 17.62 0.62 0.20 17.34 5.47 50 17.19 0.73 0.23 13.83 4.32 75 26.00 0.69 0.22 15.08 4.84 100 23.00 Ypis propionic acid yield, and Y. is acetic acid yield; both are based on the amount of lactate fermented.

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Extraction Kinetics. It is known that the diluent 2octanol can greatly increase the extracting power of the amine extractant by providing more solvating ability to the solvent (Yang et al., 1991). The optimal composition of the Alamine/2-octanol mixture was studied first to obtain the highest distribution coefficient (Kd) for propionic acid. Figure 6 shows that the optimal wt % of Alamine 336 in 2-octanol was -40%, where Kd reached maximum values for both propionic and acetic acids. The Kd values were about the same for extraction with propionic or acetic acid alone (Figure 6a,b) or in a mixture (Figure 6c). However, the Kd values for propionic acid

are much higher than those for acetic acid, suggesting that this extractant can extract propionic acid better. The amine extractants generally extract much better with longer carboxylicacids with large hydrophobic alkyl group (Yang et al., 1991). Extractive Fermentation. When lactose was the carbon source in continuous, immobilized cell fermentations, the reactor performance deteriorated with time if no pH control was provided. After running for a week, only a small amount of lactose was fermented. This was attributed to low pH and propionic acid inhibition. As shown in Figure 7,when the reactor was operated under plug-flow conditions, the reactor pH dropped quickly with propionic acid production and it almost reached the lower pH limit at the first 25% of the reactor length. Ex Situ Extraction. Ex situ extraction of propionic acid with amine extractant was performed to demonstrate the advantages of extractive fermentation over conventional fermentations. As shown in Figure 8a, with extraction, the lactose concentration in the bioreactor outlet stream decreased from 11g/L to -6 g/L, indicating that more lactose was fermented under extractive fermentation conditions. This improvement was due to the reduced level of propionic acid and a proper pH value maintained in the bioreactor. As shown in Figure 8b, the concentration of propionic acid in the bioreactor reduced from 3.3 g/L to -2.0 g/L. The propionic acid concentration in the recycle stream after extraction was less than 1 g/L, and significant amounts of propionic acid produced were extracted into the organic phase. The concentrations of acetic acid in various streams are shown in Figure 8c. It is clear that the solvent extraction only removed a small portion of the acetic acid produced and that most acetic acid remained in the recycle stream. The total propionic acid production in terms of grams per liter can be calculated by doing the material balance either around the bioreactor using the two aqueous-phase propionic acid concentrations as given by pa(g/L) = + 4(p1 - p 2 ) or around the system using the two outlet concentrations of propionic acid (aqueous and organic phases) as given by

Pb(g/L) = PI+ 5.6P3 PI,Pz, and P3 denote propionic acid concentrations in the reactor outlet, recycling stream entering the reactor, and organic-phase outlet from the extractor, respectively. In the above two equations, the number 4 is the ratio between the recycle rate and feed rate, and 5.6 is the ratio between the extractant flow rate and the feed rate. Acetic acid production was also calculated similarly.

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Figure 8. Performance of the ex situ extractive fermentation: (a) the concentration of lactose in the reactor outlet; (b) concentrationsof propionic acid in the reactor outlet (Pl),in the aqueous phase of the extractor outlet (P2), and in the organic phase of the extractor outlet (P3); (c) concentrations of acetic acid in the reactor outlet (Al), in the aqueous phase of the extractor outlet (A2), and in the organic phase of the extractor outlet (A3).

As shown in Figure 9, both calculation methods gave about the same result. Without extraction, propionic acid production was only -3 g/L, and with extraction it increased to -7 g/L (Figure 9a). Similarly, acetic acid production increased from 0.9 g/L (without extraction) to - 2 g/L (Figure 9b). Hence, with extraction, both propionic acid and acetic acid production increased by more than 100%. The weight ratio of propionic acid to acetic acid (PIA)remained unchanged a t -3.3 with or without extraction. On the basis of the amount of lactose fermented, the propionic and acetic acid yields from the extractive fermentation were 0.73 (g/g) and 0.22 (g/g), respectively. These yield values were significantly higher than those previouslyfound for batch fermentations with lactose (Hsu and Yang, 1991). Also, on the basis of the balanced dicarboxylic acid pathway (Playne, 1985), the theoretical yield values are 0.55 (gig) for propionic acid and 0.22 (gig) for acetic acid, and the PIA ratio is -2.5. One advantage of using extractive fermentation for propionic acid production is to obtain a purer product. As shown in Figure 10, the propionic acid to acetic acid ratio (PIA)in the extractant was -4.0, and the PIA ratio in the

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aqueous phase was 2.0 before extraction and 1.2 after extraction. Hence, propionic acid was preferentially recovered and purified to some degree from the product mixture by the extraction. This separation and purification effect can be further enhanced by optimizing the design and operating conditions of the extractor. In Situ Extraction. Fermentation with in situ extraction was also studied, and the results are shown in Figure 11. As shown in this figure, the lactose concentration in the outlet stream decreased with time from 11 g/L to -4.5 g/L. However, the total propionic acid production increased only initially; it then decreased to a lower level (Figure 12). At the same time, both glucose and galactose increased from 0 to -3.6 g/L (Figure 11). Glucose and galactose were not found in previous fermentation experiments with ex situ extraction or no extraction. This indicates that the direct contact of the solvent with the cells might have caused some cells to lyse and release 8-galactosidase. Consequently, the reactor productivity was low. However, the reactor still maintained production, indicating that there were viable cells

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using solvents that are essentially insoluble in water. The solubility of Alamine 336 in water is less than 5 ppm. In this work, the dissolved solvent did not pose any major problem to the fermentation, especially for the case of ex situ extraction. It is noted that, with a recycle ratio of 4, the solvent concentration in the bioreactor should be below 80% saturation. On the contrary, phase toxicity seemed to be the major cause of decreased propionic acid production from the in situ extractive fermentation. Thus, cells should be protected from directly contacting the solvent to allow the in situ extractive fermentation to work. Effects of pH. For extractive fermentation, the reactor performance is highly dependent on the pH. Both the fermentation and extraction are highly pH-dependent. The distribution coefficient, &, is greatly affected by the solution pH (Yanget al., 1991). It is known that the amine extractant only extracts the undissociated acids. The distribution coefficient & is essentially zero at pH 7.0. It increases with a decrease in the pH value and reaches its maximum value at pH 4.0 (Yang et al., 1991). On the other hand, cells grow better at pH values higher than 5.0, with an optimum around pH 7. Thus, a higher pH favors the fermentation, whereas a lower pH favors the extraction. Therefore, it is essential to optimize the reactor pH profile to facilitate both fermentation and extraction. Without extraction, the pH value of the outlet stream from the bioreactor was -4.3, which is close to the pH limit for cell growth. With ex situ extraction, this pH value increased to -4.8. The outlet stream from the extractor had a pH value of -6.0. The & values reported in Figure 6 were obtained at an equilibrium pH of less than 4.0. Since Kd decreases with an increasing pH, the Kd values in the extractive fermentation would be lower than those shown in Figure 6. It is noted here that the conditions chosen to operate the bioreactor in this study were not anywhere near the optimal conditions for these bioreactors either with or without extraction. The objective of this study was to demonstrate the concept and advantages of extractive fermentation for propionic acid production. For practical purpose, the lactose concentration in the feed stream should be -5% and the residual lactose concentration in the effluent stream should be close to zero to minimize waste disposal problems. Further work is needed to optimize the reactor and extractor before a process can be developed for industrial uses. Extractive Fermentation Process. An extractive separation and recovery process normally involves two steps: extraction and solvent regeneration. The amine extractant only extracts undissociated acid and does not extract organic acids at basic conditions. This allows solvent regeneration through back-extraction with an alkaline solution. Therefore, the extractant can be easily regenerated by strippingwith a small volume of an alkaline solution. In the mean time, a concentrated organic salt solution is obtained. This allows efficient propionate salt production by using an extractive fermentation process. Figure 13 shows a conceptual process flowsheet for propionate production from whey permeate. Our preliminary economic analysis indicates that for a typical dairy plant processing 1000 000 lbs of whey per day, about 40 000 lbs of calcium propionate can be produced at a cost of --$0.15/lb. The cost analysis was based on 95 7% conversion of lactose a t a retention time of 24 h, which have been found to be adequate for the bioreactor fed with 4 % lactate without the effects of extraction (Lewis, 1991). With the effects of extraction,a better bioreactor performance may be realized. The extractive fermentation process for propionate production is thus

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Figure 11. Performance of the in situ extractive fermentation: (a) outlet concentrations of lactose, glucose, and galactose; (b) outlet concentrations of propionic acid in the aqueous phase (Pl) and in the organic phase (P3).

Time (hours)

Figure 12. Overall propionic acid production from the in situ extractive fermentation.

present in the reactor. These cells probably were inside the fibrous matrix and thus were protected from the solvent.

Discussion Solvent Toxicity. Solvent toxicity can exert on the microorganisms at both the molecular level and the phase level. Toxicity at the phase level comes from the direct contact of the solvent phase with the cells, which may block nutrient diffusion from the medium to cells due to solvent coating and may disrupt the cell wall due to increased surface tension. Toxicity at the molecular level comes from the dissolved organic solvent which can inhibit enzymes or modify cell membrane permeability. Phase toxicity can be eliminated by using hydrophobic membranes (Cho and Shuler, 1986)or cell immobilization (Yabannavar and Wang, 1991a). Solvent toxicity at the molecular level can be reduced to a minimal degree by

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L i m e (CaO)

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Figure 13. A conceptual extractive fermentation process for propionate production from whey lactose.

economically attractive. However, further research and development are necessary to optimize and scale-up the process.

Conclusion The feasibility and advantages of using the extractive fermentation process for propionic acid production from lactose have been demonstrated in this work. The mixture of Alamine 336/2-octanol has a high extraction coefficient, is generally not toxic to the propionic bacterium, and can be used for ex situ extraction during propionic acid fermentation. Over a 100% increase in reactor productivity was attained. The improved reactor productivity was attributed to proper pH control and reduced product inhibition through the removal of propionic acid by solvent extraction. Propionic acid yields were enhanced, and a purer product can be obtained by using the extractive fermentation process.

Acknowledgment This work was supported in part by the Ohio Department of Transportation and the U.S. Department of Energy Innovative Concept Program. The financial support to S.-T.Y. from the Du Pont Young Faculty Award is also gratefully acknowledged.

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