Extractive Lactic Acid Fermentation Using Ion-Exchange Resin

Aradhana Srivastava, Pradip K. Roychoudhury,* and Vikram Sahai Biochemical Engineering Research Centre, Indian Institute of Technology, Hauzkhas, New Delhi 110016, India Received June 11, 1991/Accepted September 19, 1991

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-' and a lactic acid productivity of 0.313 g . L-' . h-'. However, these could be further enhanced to 0.929 g . g-' and 1.665g . L-' * h- ' by extractive fermentation techniques. The effect of temperature on extractive fermentation was remarkable and has been included in this work. Key words: lactic acid Lactobacillusdelbreuckii extractive fermentation product inhibition packed-column and ionexchange resin
INTRODUCTION

dustrial point of view, this process seems to be unimpressive because of its high maintenance and operational cost." However, lactic acid fermentation with in situ separation by ion-exchange resin involves considerably less operational and maintenance cost. Therefore, the present studies have been carried out on the lactic acid fermentation by using an ion-exchange resin. It has been observed by us that the extractive fermentation is very much dependent on the temperature of operation. In this article the effect of temperature on lactic acid extractive fermentation using ion-exchange resin has therefore also been included.
MATERIALS AND METHODS

Microorganism and lnocula The lactic acid production has received much attention due to its numerous uses in food and biochemical indusLactobacillus delbreuckii NRRL-B445 was used for the tries.33The substrates such as ~ h e y , ' - ~ . ' soybean * 2 present studies. The organism was maintained in glu~~~~ ~~~~ milk,21corn,I3 sulfite waste liquor,I5and potatoes* have cose-yeast extract medium" by periodic transfer. Stabs been used for lactic acid production. However, the comwere incubated at 37C for 24 h in an anaerobic chammercial success of its production is yet to be realized. ber and then stored at 4C. The anaerobic environThe main reason being the high product recovery cost ment was maintained by using C 0 2 . Stab cultures were and the complex nature of the biological p r o ~ e s s . ~ ~ *transferred to the seed culture medium containing (all ~' The end product inhibiti~n'~-'~ lactic acid causes sevby in g * L-I) sucrose, 100; yeast extract, 30; sodium suleral problems in which the most important ones are low fate, 2; sodium succinate, 2; KH2P04, 0.2; K2HP04, lactate formation rate and its recovery from fermenta0.2; M g S 0 4 . 7 H 2 0 , 0.3; MnS0, H 2 0 , 0.03; and tion broth. Moreover, the lactic acid itself is esterified FeS04 . 7H20, 0.03. The medium was incubated at and forms polylactic acid." Therefore, an integrated ap37C for 24 h in an anaerobic environment. The seed proach with separation of product has gained a lot of culture transfer was repeated twice in order to adapt the interest in this fermentation process.36One possible apculture to the fermentation environment. A 10% (v/v) proach is extractive fermentation which involves ferinoculum was added into the fermentor. mentation with in situ separation of lactic acid. Considerable insight into these problems can be gained via the techniques of extractive lactic acid ferBatch Fermentation mentation. Studies have been carried out by several auThe batch fermentation of lactic acid was carried out in thorS4,5,11,12.14.16,19,20,27,29-31,35,37-39 using either liquid-liquid a 2-L bioreactor (Bioengineering AG) with 1.6 L workextraction or membrane separation techniques. The soling volume. Batch studies were carried out by using the vent extraction technique is not preferred because it following medium composition (all in g * L-I) sucrose, causes several physical, chemical, and biochemical prob300; yeast extract, 30; sodium sulfate, 2; sodium succilems on the catalytic activity of the ~ e l l s . ~ ~ , ~ * it Although nate, 2; KH2P04,0.2; K2HP04,0.2; MgS04 . 7H20, has been found by Vickroy et al.34that continuous lactic 0.3; MnS04 * H 2 0 , 0.03; and FeSO, * 7H20, 0.03. The acid fermentation with membrane separations provides pH was controlled at 6.0 by automatic addition of 2.5N lactic acid productivity of 100 g . L-' . h-I, from an inNaOH. The anaerobic environment was maintained by * To whom all correspondence should be addressed. sparging nitrogen for 2-3 min after every 4-h interval of
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fermentation. The fermentation runs were carried out at different temperatures ranging from 37C to 45C. The impeller speed was adjusted at 500 rpm.
Resin and Its Preparation

An anion-exchange resin Amberlite IRA-400 was used for lactate separation. The resin has chloride ions on its surface, which have high electronegativity as compared to lactate ions. Therefore, the resin with chloride ions had to be replaced with hydroxyl ions for lactate separation .5.9,28.31 This can be done by saturating the resin packed column (RPC) with NaOH solution. The following steps are involved in saturation. First, the resin packed column is thoroughly washed with distilled water. Second, the column is treated with 2.5N NaOH solution until the chloride ions stop coming out through the column. Third, the column is washed with distilled water to remove the alkali. The wet RPC is then steam sterilized before it is connected with the fermentor.
Extractive Fermentat ion

cess was started by switching on the recycle pump (Pl) which allowed the broth containing cells to pass through the RPC. The effluent of the column (flow rate 5 mL * min-') was recycled back to the reactor. The anionexchange resin in the column selectively removes the lactate ions from the fermentation broth passing through it. As long as the pump is on, the pH of the broth within the fermentor increases due to continuous removal of lactate ions. On the other hand, the pH begins to decrease due to the production of lactic acid when the pump switches off. Thus recycling through the column permitted the control of pH in the bioreactor by automatic switching of the recycling pump connected to a pH controller. The pH was controlled at 6.0 k0.1. The recycle pump started whenever the pH fell down to 5.9. The pump stopped when the pH went up to 6.1. The extractive fermentation was carried out at different temperatures ranging between 37C and 45C.
Assay Methods

Dry Cell Weight

Extractive lactic acid fermentations were carried out in a 2-L bioreactor with 1.6 L working volume. The bioreactor was connected to a column (Size 4.0 x 21.0 cm, Capacity 120 g) packed with the anion-exchange resin (Amberlite IRA-400) for the lactic acid separation (Fig. 1).The system was started as a batch process, and the pH was controlled at 6.0 by automatic addition of 2.5N NaOH solution. In about 24 h, when the lactic acid concentration reached 18-19 g L-', the separation pro-

A known volume of sample was centrifuged. The centrifuged cells were then washed with isotonic solution (0.85% sodium chloride solution) and transferred into previously weighed aluminum foil cups. The isotonic solution was used to protect the bacterial cells from osmotic shock. The cells were dried at 70C for 24 h for estimating the dry cell mass. The amount of salt added in the form of isotonic solution was deducted for estimation of dried cell mass. For calculation of dry cell weight,

Figure 1. Schematic diagram of extractive lactic acid fermentation: F, 2-L fermentor, Bioengineering AG; NG, nitrogen gas; GF, gas filter; C, RPC,size 4.0 x 21.0 cm, amount 120 g; PHC, pH controller; AC, acid solution; AL, alkali solution; PI, peristatic pump for recycling the broth; Pz, peristatic pump for alkali; P3, peristatic pump for acid.

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a factor of 4.5 g * L-' per unit optical density value was used which was determined from a standard curve.
Sucrose and Lactic Acid

The sucrose and lactic acid in the fermentation broth were estimated by high-performance liquid chromatography (HPLC) (Waters Associates, USA) using a SUGAR-PAK column (mobile phase triple distilled water with 50 mg L-' Ca-EDTA solution, flow rate 0.4 mL * min-', temperature 9VC) and differential refractometer R401 as detector.
Adsorbed Lactic Acid

The adsorbed lactic acid on the resin surface was estimated by the following procedure, The RPC saturated with lactic acid was washed with distilled water to remove the unadsorbed lactic acid from the column. The adsorbed lactic acid was recovered by eluting the column with 2.5N hydrochloric acid. The eluted lactic acid was estimated by HPLC after dilution.
RESULTS AND DISCUSSIONS
Batch versus Extractive Fermentation

The conventional batch fermentation profiles obtained are given in Figure 2. It can be observed that the cell concentration reaches its maximum level (19 g * L-') after 120 h of fermentation. However, the cell growth rate decreases considerably after 24 h of fermentation due to accumulation of lactic acid. The sucrose utilization rate is also inhibited at this concentration. The overall conversion of sucrose is observed to be 85.7%

within 196 h of fermentatin. Further increase in the sucrose conversion as well as the lactic acid productivity can only be possible if the restriction imposed by the lactic acid is removed. This can be achieved by simultaneous removal of lactic acid from the fermentor. The results of the studies carried out with extractive fermentation are shown in Figure 3. The concentrations of lactic acid and cells reach to a maximum level of 80.2 g . L-' and 30.5 g * L-', respectively, in the broth within the 60 h of fermentation. It has been observed that the removal of lactic acid from the broth decreases its inhibition on fermentation. The decrease in lactic acid concentration in the fermentor following startup of recycling operation is due to its production rate being lower than the adsorption rate. The lactic acid concentration in the broth begins to increase after 26 h as the rate of production exceeds the rate of adsorption. The lactic acid concentration steadily rises further up to 30 h of fermentation to a value of 45.3 g * L-'. The adsorption rate, which is dependent upon the lactic acid concentration, also increases steadily. By 30 h, the adsorption rate of lactic acid exceeds its production rate, resulting in a decrease in lactic acid concentration in the broth, as is evident from Figure 3. Thereafter, by 36 h, the lactic acid concentration in the broth begins to increase due to the fact that the column begins to approach the saturation point causing the adsorption rate to slow down significantly. The recirculation was carried out until the column became saturated with lactate ions. The adsorption of lactic acid on the ion-exchange resin packed column during the fermentation is shown in Table I. It can be observed from this table that the column became saturated with lactate ions within 36 h of its operation. The total adsorbed lactic acid, estimated by eluting the column after fermentation, was found to be 39.08 g.

Time, h

Figure 2. Batch lactic acid fermentation: initial sucrose 105.75 g; initial cell concentration 2.08 g; temperature 39C; pH 6.0. (A) Sucrose concentration. (A) Lactic acid concentration. (0)Dry cell concentration.

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I 2 O t loor

lLO

Time, h

Figure 3. Lactic acid production by extractive fermentation: initial sucrose 107.6 g; initial cell concentration 1.9 g; temperature 39C; pH 6.0. (A) Sucrose concentration. (A)Lactic acid concentraDry cell concentration. tion. (0)

Table I1 shows the comparative study of batch and extractive fermentation processes. It is evident that extractive fermentation reduces the fermentation time by 69.4% when compared with the conventional batch process. The direct consequence of this time reduction is indicated by a 5.32-fold increase in the overall lactic acid productivity. Simultaneous removal of lactic acid from fermentation broth increases the lactic acid yield by 12.13% and cell yield by 36.4% as compared to that of the batch process. This increased yield reveals that in extractive fermentation more substrate can be diverted
Table I. Adsorption of lactic acid by time of operation in ion-exchange resin packed column at 39C and pH 6.0 +0.1. Time, h 0 2 3
4

c,/co

8 12 24 36 48
a

0.000 0.000 0.388 0.518 0.720 0.798 0.863 0.920 0.923

Ratio of outlet to inlet lactic acid concentration in column.

Table 11. Comparative study of batch and extractive lactic acid fermentation. Parameters Time, h Sucrose conversion, % Cell yield, g . g-' Product yield, g . g-' Productivity, g . L-' . h-' Specific productivity, h-' Batch 196 85.7 0.192 0.828 0.313 0.016 Extractive fermentation 60 100 0.262 0.929 1.665 0.054

for lactic acid formation. The specific lactic acid productivity also increases by 3.3-fold when compared with that of the batch process. In extractive lactic acid fermentation, the final cell concentration in the broth was 30.5 g * L-' (Fig. 3). However, it has been observed by the batch studies (Fig. 2) that the growth is very much affected when the lactic acid concentration exceeds 18-19 g . L-'. The increase in cell concentration in extractive fermentation at higher lactic acid concentration is presumably due to the following. After 24 h, when the pH in the bioreactor is controlled by column operation, the column containing the broth operates in a semicontinuous manner with feeding and withdrawals take place everytime the pH falls to 5.9 and last until the pH rises to 6.1. Between any two consecutive feeding and withdrawals, the column stays in the batch mode. During this batch phase, most likely, concentration of the lactic acid in the broth within the column goes well below the inhibition level due to adsorption of lactic acid, thereby permitting the cell growth to take place in the column itself. However, even if the concentration of lactic acid in the liquid broth bulk, within the column, is higher than normal inhibition levels, one can expect a gradient of lactic acid concentration across the liquid film around the resin particles where the concentration of lactic acid on the particle side is considerably lower than the inhibition levels as long as the resin has a finite capacity to adsorb lactic acid. In such a situation, it can be expected that the cell growth would occur in the liquid film near the surface of resin particles where inhibition conditions are less predominant. The growing cells are repelled into the liquid bulk and eventually appear in the bioreactor during the recycling operations. Presently, experiments are in progress to substantiate the explanations given

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for cell growth in the presence of inhibitory levels of lactic acid concentration. The investigation outcome will be presented in a subsequent communication.
Resin Toxicity to L. delbreuckii Cells

tation (34.6 g) carried out at the same temperature and pH. Therefore, it can be inferred that the physical adsorption of L . delbreuckii has practically very little effect on lactic acid adsorption.
Effect of Temperature on Extractive Fermentation

Studies of resin toxicity on L. delbreuckii cells were carried out in 500-mL flasks in triplicate with 300 mL of sucrose-yeast extract medium containing 5 g of resin. For control, similar experiments were done without the resin. All the flasks were inoculated with microorganisms. The experiments were carried out at 37C for 24 h. It has been observed that the presence of resin does not affect the cell growth (Table 111). However, very little cell adsorption on the resin surface was observed (Fig. 4).
Effect of Cell Adsorption on Extractive Fermentation

This effect was studied by plotting the breakthrough curve for pure lactic acid adsorption (Fig. 5). The curve was obtained by using pure lactic acid concentration of 90 g . L-' at the inlet of the adsorption column (4 x 21 cm) at 45C and pH 6.0. It is clear from Figure 5 that the column capacity for lactic acid adsorption is 36.3 g, which is close to that obtained from extractive fermen-

The studies were carried out at different temperatures ranging from 37C to 45C. The results obtained are given in Table IV, The yield of lactic acid is found to be almost constant at different temperatures. However, the yield of cell mass in the reactor does not remain constant and shows irregular changes (within 18%) with temperature. This is, perhaps, due to irregular cell adsorption on the resin. The overal sucrose utilization rate increases with an increase in temperature up to 39C. However, the rate decreases with an increase in temperature between 39C to 45C as higher temperature decreases the rate of adsorption. The optimum growth temperature for lactic acid producing organisms is 37"C, whereas for lactic acid production, it is 44-45"C.7,'2,32,34 Therefore, the observed yield of lactic acid at 37C is less as compared to that at higher temperatures. In the present system, the two processes, i.e., lactic acid production and its adsorption on RPC, are temperature dependent. The lower temperature favors adsorption rate, whereas higher temperature favors its production rate

Table 111. Resin toxicity to L. delbrueckii cells.

Fermentation condition Without resin With resin

Temperature (0C)
31 31

Resin amount Initial pH

(9
5.0

Optical density (1 OD = 4.5 g . L-I) 0.958 0.929

6.0 6.0

Figure 4. (a) Electron scanning micrograph of resin without cells (stereo scan 360, Cambridge Instruments). Pure resin Amberlite IRA400 before used for extractive fermentation studies. Conditions: EHT, 20.0 kV; WD, 22 mm length corresponds to 5.0 y m length on resin surface ( m a g n i f i c a t i o n x 4 , 4 0 0 ) . ( b ) E l e c t r o n s c a n n i n g micrograph o f resin w i t h c e l l s (stereo s c a n 3 6 0 , Cambridge Instruments). Resin Amberlite IRA-400 after used for extractive fermentation studies. Conditions: EHT, 20.0 kV, WD, 22 mm length corresponds to 5.0 g m length on resin surface (magnification x4, 400).

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Breok through copocity Total copocity

= 25.929
= 36.28 g

Figure 5. Breakthrough curve for lactic acid adsorption. Inlet lactic acid concentration 90.0 g . L-I; temperature 39C; p H 6.0; resin amount 120 g. (El) ratio of outlet to inlet lactic acid concentration.
Table IV. Lactic acid production by extractive fermentation at different temperatures.

Fermentation temperature (OOC)

37 38 39 40 41 43 45
a

Lactic acid yield (g . g-')

0.84 0.92 0.93 0.91 0.89 0.88 0.90

Cell mass yield (g . g-I)

0.24 0.22 0.26 0.25 0.22 0.24 0.22

Overall cell productivity (g . L-'. h-')a

0.267 0.400 0.469 0.385 0.263 0.210 0.200

Overall lactic acid productivity (g . L-'. h-l)a

0.928 1.222 1.665 1.397 1.025 0.892 0.899

Substrate utilization rate (g . L-' . h-I)

1.09 1.97 1.79 1.54 1.21 0.92 0.91

Specific lactic acid productivity (h-9

0.008 0.015 0.016 0.013 0.009 0.008 0.008

Overall productivity =

g . L-' component produced fermentation time ' permeate by L. helveticus. Appl. Microbiol. Biotechnol. 32: 398-402. Aesclimann, A., Stocker, U.V. 1989. The production of lactic acid fermentation of whey permeate by L. helveticus. Biotechnol. Lett. 11: 195-200. Aesclimann, A., Stasi, L. D., Stockar, U.V. 1990. Continuous production of lactic acid from whey permeate by L. helveticus in two chemostat in series. Enz. Microb. Technol. 12: 926-932. Boyaval, P., Corre, C., Terre, S. 1987. Continuous lactic acid fermentation with concentrated product recovery by ultrafiltration and electrodialysis. Biotechnol. Lett. 9: 207-212. Brink, L. E. S., Tramper, J. 1985. Optimization of organic solvent in multiphase biocatalysis. Biotechnol. Bioeng. 27: 12581269. Brown, P. R., Krstulovic, A. 1978. Ion exchange chromatography. pp. 197-217 In: E.S. Perry and A. Weisserberger (eds.), Separation and purification. 3rd edition. Wiley, New York. Buyukgungor, H., Aksu, Z., Kutsal, T., Cagler, A. 1984. Production of lactic acid by free and immobilised L. delbreuckii cells in stirred and column reactor. Eur. Congr. Biotechnol. 3rd 2: 163-168. Cordon, T. C., Treadway, R. H., Walsh, M. D., Osborne, M. F. 1950. Lactic acid from potatoes. Ind. Eng. Chem. 42: 1833-1836. Dorfner, K. 1973. Ion exchange types. pp. 15-94 In: A. F. Coers (ed.), Ion-exchangers-properties and applications. Ann Arbor Science Publishers, Ann Arbor, Michigan.

in the range 37OC-45"C. Lactic acid productivity optimizes at 39C due to the combined effect of the two temperature-dependent processes.
CONCLUSION

2.

3.

It has been demonstrated that the sucrose utilization rate is significantly enhanced by extractive fermentation. The inhibition of lactic acid on fermentation limits lactic acid productivity. This has been circumvented through the removal of lactic acid from the system by adsorption techniques. This results in substantial increase (5.32-fold) in the lactic acid productivity as compared to the conventional batch process. The direct contact of cells with the resin in the column permits cell growth even when the concentration of lactic acid in the bioreactor is above inhibition levels.
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5.

6.

7.

8.
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