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Clarification of lactic acid fermentation broths
Separation and Purification Technology 22-23 (2001) 393– 401 www.elsevier.com/locate/seppur
Clarification of lactic acid fermentation broths Stephen Milcent, He´le`ne Carre`re * Institut National de la Recherche Agronomique, Laboratoire de Ge´nie et Microbiologie des Proce´de´s Alimentaires, CBAI INA-PG, 78850 Thi6er6al-Grignon, France
Abstract This work is focused on the clarification of fermentation broths in order to optimise a batch downstream process for the recovery and purification of lactic acid produced by fermentation. Lactic acid was produced in a 75-l fermentor and the clarification was achieved using a 0.15 m2 filtration unit. Beet molasses was used as carbon source for fermentation and was inoculated with freeze-dried Lactobacillus delbrueckii ssp. lactis. The protein supplement was selected according to the fermentation and filtration performance. In batch processes, a lag time may occur between production and clarification; thus we investigated the effects of two stabilisation processes. Permeate rates were identical for the filtration of fresh, frozen or sterilised broths. pH was found to be a key parameter in fermentation and filtration. During fermentation, it was set at the constant value of 6.2. Lower pH values led to lower filtration rates whereas higher pH values first improved and then decreased filtration rates. The optimum pH range was between 7 and 8. The effects of the classic operating filtration parameters (pore diameter, temperature, cross-flow velocity, transmembrane pressure, permeate flux) were also investigated. Critical fluxes depending on axial velocity were identified. © 2001 Elsevier Science B.V. All rights reserved. Keywords: Cross-flow filtration; Fermentation broth; Lactobacillus delbrueckii spp. lactis; Beet molasses; Critical flux
1. Introduction Lactic acid and lactates are used widely in the food industry as acidulants, preservatives or flavour enhancers. They are mainly used in food production ( 80%), and in pharmaceuticals, cosmetics, the textile and leather industry [1]. These markets are expected to grow considerably with the use of polylactic acid as biodegradable polymer [2]. The industrial production of lactic acid is * Corresponding author. Tel.: +33-1-30815486; fax: + 331-30815597. E-mail address: [email protected] (H. Carre`re).
achieved by chemical reaction ( 50%) and by fermentation for the remainder [3]. But the proportion of lactic acid of biological origin is due to increase as its natural grade makes it more attractive as a food additive and as L-lactic acid isomer is used as the monomer to produce biodegradable materials. Chemical synthesis always leads to the optically inactive racemic mixture whereas selectivity can be obtained by fermentation using appropriate bacteria. However, the biological process requires the use of an efficient and economic downstream process to recover lactic acid and isolate it from various impurities in the fermentation broth [4]. In classic industrial biopro-
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cesses [5], calcium carbonate is added to the broth to maintain a low free lactic acid concentration in order to prevent the inhibition of bacteria. The hot calcium lactate solution is then clarified with filter aids. It is subsequently crystallised and converted into lactic acid by adding sulphuric acid, which results in high quantities of gypsum (calcium sulfate) as wastes. They are eliminated by filtration and thorough washing of the cake. Further purification is achieved by treatment by activated carbon, ion exchange resins, solvent extraction or by esterification followed by distillation and hydrolysis. However, as environment is a major concern to industrials, a new recovery and purification process for lactic acid produced by fermentation in order to limit waste production was developed. It includes broth clarification by cross-flow filtration, treatment by chelating resins, concentration of lactate salts by electrodialysis, conversion of lactate salts into free acid by bipolar membrane electrodialysis and ion exchange treatment [6]. This paper focuses on the first unit operation of this process: the separation of the biomass from the fermentation broth by crossflow filtration which, contrary to the conventional process, requires no adjuvant. To separate microorganisms from fermentation broths, filtration membranes are used more often in continuous processes (membrane bioreactors) [7]. In that case, cells are recycled in the bioreactor and the permeate is continuously drawn off. In this study, we used a batch process, and did not reuse bacteria after filtration. We did not pay attention to cell viability. In this paper, we present some experiments collected to study the effects of operating parameters on the filtration performance. A preliminary work was devoted to the selection of some of the fermentation medium components with respect to membrane fouling.
2. Material and methods
for fermentation as it is a cheap material widely used in industrial processes [5,8]. The molasses, supplied by Ets Legrand, Arras, France, contained 600 g/l of sucrose. The microorganism Lactobacillus delbrueckii ssp. lactis was used for the fermentation of sucrose [5]. We used a freezedried strain (Ezal LB 120) supplied by Texel (Dange´ Saint Romain, France). The nitrogen and vitamin sources were chosen in order to obtain the best performance in fermentation and filtration. The fermentation process performance was evaluated using the CINAC method described by Spinnler and Corrieu [9]. Yeast extracts (ref. A5990404, lot 0198316, Biofit, Labosi Oulchy-leChaˆteau, France), malt sprout extracts (ref. 1899, lot 459133, Biofit, Osi Bio Paris, France) and corn steep solids (C-8160, lot 123H0551, Sigma Chemical, Saint Louis, MO) were tested. The filtration rates of solutions of each component in water were compared.
2.1.2. Fermentations The fermentation medium was composed of molasses diluted in water in order to have 60 l of a sucrose solution at 120 g/l. Yeast extracts were added at a concentration of 15 g/l. This medium was heat treated at 102°C during 30 min at a stirring rate of 200 rpm in a 75-l bioreactor (LSL Biolaffite). The reactor was cooled down to 44°C and this temperature was maintained during all the fermentation process. The pH was adjusted to 6.2 by adding sulphuric acid (95%) and the medium was inoculated with 20 g of freeze-dried bacteria. The broth was stirred at 200 rpm and pH was maintained at 6.2 by automatic addition of ammonium hydroxide (32% vol/vol). The fermentation broth thus contained ammonium lactate which had to be converted into lactic acid during the a further step of the purification process. The fermentation process was stopped after 140 h when addition of ammonium hydroxide had stopped.
2.1. Lactic acid production 2.2. Cross-flow filtration experiments 2.1.1. Selection of the fermentation medium components Beet molasses was used as the carbon source
Cross-flow filtration experiments were carried out in a feed and bleed pilot plant (Fig. 1)
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equipped with a tubular ceramic (Al2O3, TiO2) membrane (K01BX Kerasep from Orelis, France). The membrane was 1.178 m long and it had seven channels with an inner diameter of 5.75 mm which corresponded to a total filtration area of 0.1491 m2. For most experiments, we used a membrane with a nominal pore size of 0.1 mm. Membranes with an average pore diameter of 0.8 mm and a molecular weight cut-off of 300 kDa were also tested. Temperature was regulated with a heat exchanger on the retentate circulation loop. The positive displacement gear pump (P02) equipped with a speed variator made it possible to
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set different cross-flow velocities within a 1.5 –4.5 m/s range. The motor valves V1 (DN15 valve, type 698/15/D, Gemu, Germany) and V2 (DN10 valve, type 618/008/D, Gemu) made it possible to regulate either the transmembrane pressure or the permeate flux. Transmembrane pressure was calculated from the retentate and permeate absolute pressures at the membrane inlet and outlet using absolute pressure transmitters Philips (Germany), type P21, calibrated 0–10 bar, accuracy 90.5% of full scale. Cross-flow velocity was calculated from circulating flow rate measurements (magnetic
Fig. 1. The filtration unit. T1 and T2, feed tanks, respectively, equipped with a heating jacket and a serpentine where some vapour can circulate; W1 and W2, waste outlets; PO1, feed pump; PO2, circulation pump; V1 and V2, motor valves (DN10 and DN15); TS1–TS3, temperature probes; P1–P4, absolute pressure transmitter; P5, differential pressure transmitter; FM1– FM3, electromagnetic flowmeters (feed, retentate and permeate, respectively).
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Table 1 Steady-state permeate flux for the filtration of solutions with different nitrogen sources and of diluted molasses on a 0.1 mm Kerasep membranea
Malt sprouts Yeast extracts Corn steep Molasses a
Concentration (g/l)
Viscosity v (Pa s)
Steady-state permeate flux J (l/h m2)
Jv (Pa s l/h m2)
3 3 3 175
10−3 10−3 10−3 1.4×10−3
365 310 140 85
0.365 0.310 0.140 0.119
Temperature, 20°C; transmembrane pressure, 1 bar; cross-flow velocity, 4 m/s.
flowmeter FOXBORO, USA model 800H-WCR accuracy 1%). Permeate flow rate was measured by a magnetic flowmeter ALTOMETER, KRONE, Holland, model K280, 0–150 l/h, accuracy 1%. Temperatures were measured with platinum probes PT100, accuracy 9 0.1°C. Each sensor signal (4 – 20 mA) was converted by OPTO 22 cards and computerised using a DOS software programme developed by our laboratory. Measurements were made every 10 s and data were recorded every min, which means that each experimental value was the arithmetical average of six measurements. The permeate was reintroduced in the feed tank in order to operate with a constant bulk concentration. After each experiment, the membrane was first rinsed with water. It was then cleaned with an Ultrasil 25 F (Henkel) solution (5 g/l) heated to 50°C and rinsed for neutralisation with high-purity water. Lastly it was cleaned with a nitric acid solution (10 g/l) heated to 85°C and rinsed once more for neutralisation with high-purity water.
phase was 0.01 M H2SO4 and the flow rate was 0.6 ml/min. The viscosity of the permeate was determined by measuring the time of a sample flowing (under gravity) in a capillary viscometer (Prolabo, UF viscometer, 04 902.515).
3. Results and discussion
3.1. Selection of the nitrogen source Table 1 shows the filtration rates of the solutions containing the three tested nitrogen sources using a 0.1 mm membrane. The filtration rate was higher with yeast extracts or malt sprouts than with corn steep solids. Furthermore, yeast extracts were more suitable for fermentation and were selected for the lactic acid production process. However, the comparison of the fluxes obtained during the filtration of dilute beet molasses (Table 1) and supplement solutions showed that the highest flux decrease was observed with beet molasses.
2.3. Analysis The bacterial concentration was obtained by the dry cell weight, determined by filtering the cells (0.2 mm) and drying the filter at 105°C for 24 h. The concentrations of sucrose and ammonium lactate were quantified using high performance liquid chromatography (Waters Associates, Milford, MA). Each sample was filtered (0.2 mm). The HPLC analysis was carried out using a cation exchange column (Aminex Ion exclusion HPX 97 H, Biorad, Richmond, CA) at 35°C. The mobile
3.2. Repeatability of the filtered fermentation broth When filtering real complex media, it is important to know the repeatability of the product treated. The results of HPLC analysis of broths obtained after fermentation are described in Table 2. As sucrose and lactate ammonium were not retained by the membranes used, there is no reason to assume that a change in their concentration affects the filtration rate, except if the
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viscosity changed. Here the permeate viscosity was found to be the same in all the experiments: vp = 0.8290.03 mPa s at 48°C. The bacterial concentration was also found to be constant in all the runs, according to the measurement accuracy: C = 2.690.3 g/l. Thus, we can assume that the Table 2 Sucrose and ammonium lactate concentrations of filtered fermentation broths (mean of ten fermentation broths)
Sucrose Ammonium lactate
Concentration (g/l)
Standard deviation (g/l)
13.9 90.4
8.8 15.2
Table 3 Protein amount in fresh and sterilised broths according to the Bradford method
Proteins (g/l)
Fresh broth
Sterilised broth
0.95
1.03
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small differences in the initial product composition did not influence the results obtained during filtration runs. As a stabilisation process or a lag time between fermentation and filtration can be required, we compared the performance of filtration of a fresh fermentation broth, a frozen (at − 20°C) broth and a sterilised (120°C, 20 min) broth. As filtration fluxes were similar in the three experiments, it can be concluded that the fermentation broths can be preserved by freezing. However, lower fluxes could have been expected for the filtration of the sterilised broth as dead bacteria liberated intracellular proteins which could cause additional fouling. However, the analysis of the proteins (Bradford method) in the fresh and sterilised broths showed that the increase of the protein amount was very small (Table 3). In fact, most of the proteins present in the broths were introduced with yeast extracts and they led to lower fouling than some components of the molasses (Table 2).
3.3. Influence of pore size
Fig. 2. Cross-flow filtration of a lactic acid fermentation broth on different Kerasep membranes. Permeate flux variation with time. Table 4 Membrane resistances to water transfer Membrane
Kerasep 0.8 mm
Kerasep 0.1 mm
Kerasep 300 kDa
Rm (m−1)
6.00×1010
3.27×1011
4.86×1011
According to Matson [10], microfiltration membranes or ultrafiltration membranes with molecular weight cut-off values of at least 100 –300 kDa can be used in cell harvesting. Fig. 2 shows a variation of the permeate flux with time during the filtration of fermentation broths on three Kerasep membranes with average pore size of 0.1 mm, 0.8 mm and a molecular weight cut-off of 300 kDa. These membranes made it possible to clarify thoroughly the broths: no microorganism was detected in the permeate by dry weight analysis and by microscope observation (objectives 40× and 100× ). The resistance of the membranes to water transfer is shown in Table 4. The 0.8 mm membrane, even with the lower intrinsic resistance, led to the lowest permeate flux. This may be due to the blocking of some pores by bacteria. Bacteria were rod shaped and their average size, determined by image analysis, was 19 0.3 mm in diameter and 891 mm long. Thus, the 0.8 mm membrane was not appropriate for this clarification process. The 0.1 mm membrane, which led to the highest permeate flux, was selected for this study.
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3.4. Influence of transmembrane pressure
Fig. 3. Cross-flow filtration of a lactic acid fermentation broth on a 0.1 mm Kerasep membrane. Permeate flux variation with time as a function of transmembrane pressure.
Fig. 3 shows a variation of the permeate flux with time and transmembrane pressure. As soon as the transmembrane pressure was increased, the flux immediately increased and then decreased. Nevertheless the steady-state flux was not reached within the observation times of 150 min. But the difference between the fluxes observed 150 min after the variation of the transmembrane pressure is low: 21% in the 0.5 –2 bar transmembrane pressure range. Results presented in the literature concerning such studies evoke pressure independent steady state permeate flux. For example, during the filtration of a Bacillus subtilis broth, Tanaka et al. [11] observed an almost pressure-independent flux. In the filtration of a Lactobacillus hel6eticus broth [12], the steady-state permeate flux increased with transmembrane pressure only with low values below 0.3 bar. According to the critical pressure equal to 0.35 bar for a cross-flow velocity of 3 m/s (Fig. 7b), we can conclude that our results are in agreement with reference [12]. The steady-state flux is transmembrane pressure independent above 0.35 bar.
3.5. Influence of the cross-flow 6elocity
Fig. 4. Cross-flow filtration of a lactic acid fermentation broth on a 0.1 mm Kerasep membrane. Permeate flux variation with time as a function of cross-flow velocity. (a) Decrease in cross-flow velocity, (b) increase in cross-flow velocity.
Fig. 4 shows an example of permeate flux variation with time and cross-flow velocity. The steadystate fluxes were reached within a time of about one hour. Decreasing the velocity from 4 to 2 m/s led to a reduction of the permeate flux by 27%. These results agree with the usual expectation that increasing cross-flow velocity increases the permeate flow rate, as observed by Shimizu et al. [13] for the filtration of rod shaped Bacillus caldolyticus. However, the increase of cross-flow velocity did not lead to an increase of the flux (Fig. 4b): irreversible fouling occurred. On the other hand, some authors reported a decrease of the permeate flux as cross-flow velocity increased. This can be explained by an increase of the cartridge pressure drop, resulting in higher transmembrane pressures and thereby in a cake layer compaction and a lower permeability (microfiltration of a Bacillus polymyxa broth [14]). Another explanation is that the increase in the specific resistance of the cake
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layer is due to the shear-induced arrangement of the cells parallel to the flow direction, as shown in the microfiltration of L. delbrueckii or B. subtilis broths [11].
constant, the maximum variation is 13% of the mean value. In fact, within the temperature range (25 –70°C) we studied, the ratio of the fluxes at different temperatures is directly linked to that of the permeate viscosity at these temperatures.
3.6. Influence of temperature
3.7. Influence of medium pH
Fig. 5 shows the variation of the permeate flux and of the total resistance to mass transfer with time and temperature. The shape of the permeate flux and temperature plots is almost parallel. The effect of temperature on the filtration rate is thus immediate and the hydraulic resistance is almost
At the end of fermentation, pH was equal to 6.2. The pH of the filtered broth was modified by adding sulphuric acid (95%) or ammonium hydroxide (32%). Steady-states permeate fluxes versus pH are plotted in Fig. 6. Decreasing the pH values below 6.2 decreased the filtration fluxes; whereas the pH increase first improved, and then decreased the filtration rate. The optimum pH range was between 7 and 8. There were large differences between the steady-state fluxes obtained with different pH values. The flux maximal value was twice as large as the minimal value. Thus, pH is a key parameter which has to be taken into account when optimising the clarification process of fermentation broths by microfiltration.
3.8. Influence of permeate flux, identification of critical fluxes Fig. 5. Cross-flow filtration of a lactic acid fermentation broth on a 0.1 mm Kerasep membrane. Permeate flux and total resistance to mass transfer variation with time as a function of temperature.
Fig. 6. Cross-flow filtration of a lactic acid fermentation broth on a 0.1 mm Kerasep membrane. Steady-state permeate flux vs. broth pH.
Fig. 7 shows two examples of filtration runs performed with a stepwise increased or decreased permeate flux. When considering the variation of the transmembrane pressure, two series of permeate flux values were distinguished. At low permeate flux values, transmembrane pressure was almost constant whereas it increased with time with high permeate fluxes. At the highest values, the sharp pressure increase indicated that it was impossible to run filtration under these conditions. The maximum value of the permeate flux at which the transmembrane pressure was stable with time is named ‘critical flux’, it is a function of the cross-flow velocity: for 6= 4 m/s
Jcrit = 50 l/h m2
for 6= 3 m/s
Jcrit = 20 l/h m2
These critical fluxes did not correspond to the strong form of critical flux defined by Field et al.
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4. Conclusions Lactic acid fermentation broths were properly clarified with a 0.1 mm pore diameter membrane and permeate fluxes were high enough. Among the fermentation broth components, the molasses led to the highest permeate flux drop. Nevertheless, we identified critical fluxes that were function of cross-flow velocity. Operating filtration at fluxes below these critical values made it possible to extend filtration times by reducing the formation of the bacterial cell cake at the membrane wall. However, irreversible fouling due to, among others, the adsorption of some of the molasses components always occurred. The broth pH, which is generally determined by the fermentation conditions, was also found to affect the filtration performance. In this case, the pH should be adjusted between the values of 7 and 8 before filtration, as the bacteria are not reused in the batch process.
Acknowledgements Fig. 7. Controlled permeate flux cross-flow filtration of a lactic acid fermentation broth on a 0.1 mm Kerasep membrane. Transmembrane pressure variation with time as a function of permeate flux. (a) 6= 4 m/s, (b) 6 =3 m/s.
[15] or Howell [16] (equivalent to the corresponding clean water flux) because fouling due to, among others adsorption of some molasses components, always occurred. Low permeate flux values made it possible to control only the formation of the bacterial cell cake at the membrane wall. However, the existence of such critical fluxes is very interesting for improving filtration industrial processes as it makes it possible to extend operating times. Such critical fluxes, or more precisely a critical value of the permeate flux ratio on the wall shear stress (J/~w), have already been reported for the microfiltration of a L. hel6eticus broth [12].
The authors would like to acknowledge the Orelis (Saint Maurice de Beynost, France) and Texel (Dange´ Saint Romain, France) companies for their kind gifts of the membranes and of the microorganisms. We are grateful to Drs C. Be´al and E. Latrille (LGMPA-INRA Grignon) for their helpful advice in the improvement of the fermentation process. We are also indebted to G. Rigou (INRA’s translation unit, Jouy-en-Josas) for revising the English version of the manuscript.
References [1] T.B. Vick Roy, in: M. Moo-Young (Ed.), Comprehensive Biotechnology, vol. 3, Pergamon Press, Oxford, 1985, pp. 761– 776. [2] R.L. Evangelista, Z.L. Nikolov, Appl. Biochem. Biotechnol. 57/58 (1996) 471. [3] S. Tejayadi, M. Cheryan, Appl. Microbiol. Biotechnol. 43 (1995) 242. [4] A.S. Kertes, C.J. King, Biotch. Bioeng. 28 (1986) 269. [5] J.S. Kascak, J. Kominek, M. Roehr, in: H.J. Rehm, G. Reed, A. Puhler, P. Stadler (Eds.), Biotechnology,
S. Milcent, H. Carre`re / Separation/Purification Technology 22-23 (2001) 393–401
[6]
[7]
[8] [9] [10]
Product of Primary Metabolism, Ch. 8, vol. 6, VCH, Weinheim, Germany, 1996. M. Bailly, H. Roux de Balmann, P. Aimar, F. Lutin, 2nd European Congress of Chemical Engineering, Montpellier, France, 5 – 7 October, 1999. P. Boyaval, C. Lafforgue-Delorme, G. Goma, in: G. Daufin, F. Rene´, P. Aimar (Eds.), Les Se´parations par Membrane dans les Proce´de´s de l’Industrie Alimentaire, Ch. 13, Tec&Doc Lavoisier, Paris, 1998. Y. Goksungur, U. Guvenc¸, J. Chem. Tech. Biotechnol. 69 (1997) 399. H.E. Spinnler, G. Corrieu, J. Dairy Res. 56 (1989) 755. S.L. Matson, in: R.D. Noble, S.A. Stern (Eds.), Mem-
[11] [12] [13] [14] [15] [16]
.
401
brane Separations Technology, Principles and Applications, Ch. 8, Elsevier, Amsterdam, 1995. T. Tanaka, K. Usui, K. Kouda, K. Nakanishi, J. Chem. Eng. Jpn. 29 (1996) 973. P. Boyaval, C. Lavenant, G. Ge´san, D. Daufin, Biotechnol. Bioeng. 49 (1996) 78. Y. Shimizu, K.I. Shimodera, A. Watanabe, J. Fermentation Bioeng. 76 (1993) 493. N. Nagata, K.J. Herouvis, D.M. Dziewulski, G. Belfort, Biotechnol. Bioeng. 34 (1989) 447. R.W. Field, D. Wu, J.A. Howell, B.B. Gupta, J. Membr. Sci. 100 (1995) 259. J.A. Howell, J. Membr. Sci. 107 (1995) 165.
Clarification of lactic acid fermentation broths Stephen Milcent, He´le`ne Carre`re * Institut National de la Recherche Agronomique, Laboratoire de Ge´nie et Microbiologie des Proce´de´s Alimentaires, CBAI INA-PG, 78850 Thi6er6al-Grignon, France
Abstract This work is focused on the clarification of fermentation broths in order to optimise a batch downstream process for the recovery and purification of lactic acid produced by fermentation. Lactic acid was produced in a 75-l fermentor and the clarification was achieved using a 0.15 m2 filtration unit. Beet molasses was used as carbon source for fermentation and was inoculated with freeze-dried Lactobacillus delbrueckii ssp. lactis. The protein supplement was selected according to the fermentation and filtration performance. In batch processes, a lag time may occur between production and clarification; thus we investigated the effects of two stabilisation processes. Permeate rates were identical for the filtration of fresh, frozen or sterilised broths. pH was found to be a key parameter in fermentation and filtration. During fermentation, it was set at the constant value of 6.2. Lower pH values led to lower filtration rates whereas higher pH values first improved and then decreased filtration rates. The optimum pH range was between 7 and 8. The effects of the classic operating filtration parameters (pore diameter, temperature, cross-flow velocity, transmembrane pressure, permeate flux) were also investigated. Critical fluxes depending on axial velocity were identified. © 2001 Elsevier Science B.V. All rights reserved. Keywords: Cross-flow filtration; Fermentation broth; Lactobacillus delbrueckii spp. lactis; Beet molasses; Critical flux
1. Introduction Lactic acid and lactates are used widely in the food industry as acidulants, preservatives or flavour enhancers. They are mainly used in food production ( 80%), and in pharmaceuticals, cosmetics, the textile and leather industry [1]. These markets are expected to grow considerably with the use of polylactic acid as biodegradable polymer [2]. The industrial production of lactic acid is * Corresponding author. Tel.: +33-1-30815486; fax: + 331-30815597. E-mail address: [email protected] (H. Carre`re).
achieved by chemical reaction ( 50%) and by fermentation for the remainder [3]. But the proportion of lactic acid of biological origin is due to increase as its natural grade makes it more attractive as a food additive and as L-lactic acid isomer is used as the monomer to produce biodegradable materials. Chemical synthesis always leads to the optically inactive racemic mixture whereas selectivity can be obtained by fermentation using appropriate bacteria. However, the biological process requires the use of an efficient and economic downstream process to recover lactic acid and isolate it from various impurities in the fermentation broth [4]. In classic industrial biopro-
1383-5866/01/$ - see front matter © 2001 Elsevier Science B.V. All rights reserved. PII: S1383-5866(00)00124-6
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cesses [5], calcium carbonate is added to the broth to maintain a low free lactic acid concentration in order to prevent the inhibition of bacteria. The hot calcium lactate solution is then clarified with filter aids. It is subsequently crystallised and converted into lactic acid by adding sulphuric acid, which results in high quantities of gypsum (calcium sulfate) as wastes. They are eliminated by filtration and thorough washing of the cake. Further purification is achieved by treatment by activated carbon, ion exchange resins, solvent extraction or by esterification followed by distillation and hydrolysis. However, as environment is a major concern to industrials, a new recovery and purification process for lactic acid produced by fermentation in order to limit waste production was developed. It includes broth clarification by cross-flow filtration, treatment by chelating resins, concentration of lactate salts by electrodialysis, conversion of lactate salts into free acid by bipolar membrane electrodialysis and ion exchange treatment [6]. This paper focuses on the first unit operation of this process: the separation of the biomass from the fermentation broth by crossflow filtration which, contrary to the conventional process, requires no adjuvant. To separate microorganisms from fermentation broths, filtration membranes are used more often in continuous processes (membrane bioreactors) [7]. In that case, cells are recycled in the bioreactor and the permeate is continuously drawn off. In this study, we used a batch process, and did not reuse bacteria after filtration. We did not pay attention to cell viability. In this paper, we present some experiments collected to study the effects of operating parameters on the filtration performance. A preliminary work was devoted to the selection of some of the fermentation medium components with respect to membrane fouling.
2. Material and methods
for fermentation as it is a cheap material widely used in industrial processes [5,8]. The molasses, supplied by Ets Legrand, Arras, France, contained 600 g/l of sucrose. The microorganism Lactobacillus delbrueckii ssp. lactis was used for the fermentation of sucrose [5]. We used a freezedried strain (Ezal LB 120) supplied by Texel (Dange´ Saint Romain, France). The nitrogen and vitamin sources were chosen in order to obtain the best performance in fermentation and filtration. The fermentation process performance was evaluated using the CINAC method described by Spinnler and Corrieu [9]. Yeast extracts (ref. A5990404, lot 0198316, Biofit, Labosi Oulchy-leChaˆteau, France), malt sprout extracts (ref. 1899, lot 459133, Biofit, Osi Bio Paris, France) and corn steep solids (C-8160, lot 123H0551, Sigma Chemical, Saint Louis, MO) were tested. The filtration rates of solutions of each component in water were compared.
2.1.2. Fermentations The fermentation medium was composed of molasses diluted in water in order to have 60 l of a sucrose solution at 120 g/l. Yeast extracts were added at a concentration of 15 g/l. This medium was heat treated at 102°C during 30 min at a stirring rate of 200 rpm in a 75-l bioreactor (LSL Biolaffite). The reactor was cooled down to 44°C and this temperature was maintained during all the fermentation process. The pH was adjusted to 6.2 by adding sulphuric acid (95%) and the medium was inoculated with 20 g of freeze-dried bacteria. The broth was stirred at 200 rpm and pH was maintained at 6.2 by automatic addition of ammonium hydroxide (32% vol/vol). The fermentation broth thus contained ammonium lactate which had to be converted into lactic acid during the a further step of the purification process. The fermentation process was stopped after 140 h when addition of ammonium hydroxide had stopped.
2.1. Lactic acid production 2.2. Cross-flow filtration experiments 2.1.1. Selection of the fermentation medium components Beet molasses was used as the carbon source
Cross-flow filtration experiments were carried out in a feed and bleed pilot plant (Fig. 1)
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equipped with a tubular ceramic (Al2O3, TiO2) membrane (K01BX Kerasep from Orelis, France). The membrane was 1.178 m long and it had seven channels with an inner diameter of 5.75 mm which corresponded to a total filtration area of 0.1491 m2. For most experiments, we used a membrane with a nominal pore size of 0.1 mm. Membranes with an average pore diameter of 0.8 mm and a molecular weight cut-off of 300 kDa were also tested. Temperature was regulated with a heat exchanger on the retentate circulation loop. The positive displacement gear pump (P02) equipped with a speed variator made it possible to
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set different cross-flow velocities within a 1.5 –4.5 m/s range. The motor valves V1 (DN15 valve, type 698/15/D, Gemu, Germany) and V2 (DN10 valve, type 618/008/D, Gemu) made it possible to regulate either the transmembrane pressure or the permeate flux. Transmembrane pressure was calculated from the retentate and permeate absolute pressures at the membrane inlet and outlet using absolute pressure transmitters Philips (Germany), type P21, calibrated 0–10 bar, accuracy 90.5% of full scale. Cross-flow velocity was calculated from circulating flow rate measurements (magnetic
Fig. 1. The filtration unit. T1 and T2, feed tanks, respectively, equipped with a heating jacket and a serpentine where some vapour can circulate; W1 and W2, waste outlets; PO1, feed pump; PO2, circulation pump; V1 and V2, motor valves (DN10 and DN15); TS1–TS3, temperature probes; P1–P4, absolute pressure transmitter; P5, differential pressure transmitter; FM1– FM3, electromagnetic flowmeters (feed, retentate and permeate, respectively).
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Table 1 Steady-state permeate flux for the filtration of solutions with different nitrogen sources and of diluted molasses on a 0.1 mm Kerasep membranea
Malt sprouts Yeast extracts Corn steep Molasses a
Concentration (g/l)
Viscosity v (Pa s)
Steady-state permeate flux J (l/h m2)
Jv (Pa s l/h m2)
3 3 3 175
10−3 10−3 10−3 1.4×10−3
365 310 140 85
0.365 0.310 0.140 0.119
Temperature, 20°C; transmembrane pressure, 1 bar; cross-flow velocity, 4 m/s.
flowmeter FOXBORO, USA model 800H-WCR accuracy 1%). Permeate flow rate was measured by a magnetic flowmeter ALTOMETER, KRONE, Holland, model K280, 0–150 l/h, accuracy 1%. Temperatures were measured with platinum probes PT100, accuracy 9 0.1°C. Each sensor signal (4 – 20 mA) was converted by OPTO 22 cards and computerised using a DOS software programme developed by our laboratory. Measurements were made every 10 s and data were recorded every min, which means that each experimental value was the arithmetical average of six measurements. The permeate was reintroduced in the feed tank in order to operate with a constant bulk concentration. After each experiment, the membrane was first rinsed with water. It was then cleaned with an Ultrasil 25 F (Henkel) solution (5 g/l) heated to 50°C and rinsed for neutralisation with high-purity water. Lastly it was cleaned with a nitric acid solution (10 g/l) heated to 85°C and rinsed once more for neutralisation with high-purity water.
phase was 0.01 M H2SO4 and the flow rate was 0.6 ml/min. The viscosity of the permeate was determined by measuring the time of a sample flowing (under gravity) in a capillary viscometer (Prolabo, UF viscometer, 04 902.515).
3. Results and discussion
3.1. Selection of the nitrogen source Table 1 shows the filtration rates of the solutions containing the three tested nitrogen sources using a 0.1 mm membrane. The filtration rate was higher with yeast extracts or malt sprouts than with corn steep solids. Furthermore, yeast extracts were more suitable for fermentation and were selected for the lactic acid production process. However, the comparison of the fluxes obtained during the filtration of dilute beet molasses (Table 1) and supplement solutions showed that the highest flux decrease was observed with beet molasses.
2.3. Analysis The bacterial concentration was obtained by the dry cell weight, determined by filtering the cells (0.2 mm) and drying the filter at 105°C for 24 h. The concentrations of sucrose and ammonium lactate were quantified using high performance liquid chromatography (Waters Associates, Milford, MA). Each sample was filtered (0.2 mm). The HPLC analysis was carried out using a cation exchange column (Aminex Ion exclusion HPX 97 H, Biorad, Richmond, CA) at 35°C. The mobile
3.2. Repeatability of the filtered fermentation broth When filtering real complex media, it is important to know the repeatability of the product treated. The results of HPLC analysis of broths obtained after fermentation are described in Table 2. As sucrose and lactate ammonium were not retained by the membranes used, there is no reason to assume that a change in their concentration affects the filtration rate, except if the
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viscosity changed. Here the permeate viscosity was found to be the same in all the experiments: vp = 0.8290.03 mPa s at 48°C. The bacterial concentration was also found to be constant in all the runs, according to the measurement accuracy: C = 2.690.3 g/l. Thus, we can assume that the Table 2 Sucrose and ammonium lactate concentrations of filtered fermentation broths (mean of ten fermentation broths)
Sucrose Ammonium lactate
Concentration (g/l)
Standard deviation (g/l)
13.9 90.4
8.8 15.2
Table 3 Protein amount in fresh and sterilised broths according to the Bradford method
Proteins (g/l)
Fresh broth
Sterilised broth
0.95
1.03
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small differences in the initial product composition did not influence the results obtained during filtration runs. As a stabilisation process or a lag time between fermentation and filtration can be required, we compared the performance of filtration of a fresh fermentation broth, a frozen (at − 20°C) broth and a sterilised (120°C, 20 min) broth. As filtration fluxes were similar in the three experiments, it can be concluded that the fermentation broths can be preserved by freezing. However, lower fluxes could have been expected for the filtration of the sterilised broth as dead bacteria liberated intracellular proteins which could cause additional fouling. However, the analysis of the proteins (Bradford method) in the fresh and sterilised broths showed that the increase of the protein amount was very small (Table 3). In fact, most of the proteins present in the broths were introduced with yeast extracts and they led to lower fouling than some components of the molasses (Table 2).
3.3. Influence of pore size
Fig. 2. Cross-flow filtration of a lactic acid fermentation broth on different Kerasep membranes. Permeate flux variation with time. Table 4 Membrane resistances to water transfer Membrane
Kerasep 0.8 mm
Kerasep 0.1 mm
Kerasep 300 kDa
Rm (m−1)
6.00×1010
3.27×1011
4.86×1011
According to Matson [10], microfiltration membranes or ultrafiltration membranes with molecular weight cut-off values of at least 100 –300 kDa can be used in cell harvesting. Fig. 2 shows a variation of the permeate flux with time during the filtration of fermentation broths on three Kerasep membranes with average pore size of 0.1 mm, 0.8 mm and a molecular weight cut-off of 300 kDa. These membranes made it possible to clarify thoroughly the broths: no microorganism was detected in the permeate by dry weight analysis and by microscope observation (objectives 40× and 100× ). The resistance of the membranes to water transfer is shown in Table 4. The 0.8 mm membrane, even with the lower intrinsic resistance, led to the lowest permeate flux. This may be due to the blocking of some pores by bacteria. Bacteria were rod shaped and their average size, determined by image analysis, was 19 0.3 mm in diameter and 891 mm long. Thus, the 0.8 mm membrane was not appropriate for this clarification process. The 0.1 mm membrane, which led to the highest permeate flux, was selected for this study.
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3.4. Influence of transmembrane pressure
Fig. 3. Cross-flow filtration of a lactic acid fermentation broth on a 0.1 mm Kerasep membrane. Permeate flux variation with time as a function of transmembrane pressure.
Fig. 3 shows a variation of the permeate flux with time and transmembrane pressure. As soon as the transmembrane pressure was increased, the flux immediately increased and then decreased. Nevertheless the steady-state flux was not reached within the observation times of 150 min. But the difference between the fluxes observed 150 min after the variation of the transmembrane pressure is low: 21% in the 0.5 –2 bar transmembrane pressure range. Results presented in the literature concerning such studies evoke pressure independent steady state permeate flux. For example, during the filtration of a Bacillus subtilis broth, Tanaka et al. [11] observed an almost pressure-independent flux. In the filtration of a Lactobacillus hel6eticus broth [12], the steady-state permeate flux increased with transmembrane pressure only with low values below 0.3 bar. According to the critical pressure equal to 0.35 bar for a cross-flow velocity of 3 m/s (Fig. 7b), we can conclude that our results are in agreement with reference [12]. The steady-state flux is transmembrane pressure independent above 0.35 bar.
3.5. Influence of the cross-flow 6elocity
Fig. 4. Cross-flow filtration of a lactic acid fermentation broth on a 0.1 mm Kerasep membrane. Permeate flux variation with time as a function of cross-flow velocity. (a) Decrease in cross-flow velocity, (b) increase in cross-flow velocity.
Fig. 4 shows an example of permeate flux variation with time and cross-flow velocity. The steadystate fluxes were reached within a time of about one hour. Decreasing the velocity from 4 to 2 m/s led to a reduction of the permeate flux by 27%. These results agree with the usual expectation that increasing cross-flow velocity increases the permeate flow rate, as observed by Shimizu et al. [13] for the filtration of rod shaped Bacillus caldolyticus. However, the increase of cross-flow velocity did not lead to an increase of the flux (Fig. 4b): irreversible fouling occurred. On the other hand, some authors reported a decrease of the permeate flux as cross-flow velocity increased. This can be explained by an increase of the cartridge pressure drop, resulting in higher transmembrane pressures and thereby in a cake layer compaction and a lower permeability (microfiltration of a Bacillus polymyxa broth [14]). Another explanation is that the increase in the specific resistance of the cake
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layer is due to the shear-induced arrangement of the cells parallel to the flow direction, as shown in the microfiltration of L. delbrueckii or B. subtilis broths [11].
constant, the maximum variation is 13% of the mean value. In fact, within the temperature range (25 –70°C) we studied, the ratio of the fluxes at different temperatures is directly linked to that of the permeate viscosity at these temperatures.
3.6. Influence of temperature
3.7. Influence of medium pH
Fig. 5 shows the variation of the permeate flux and of the total resistance to mass transfer with time and temperature. The shape of the permeate flux and temperature plots is almost parallel. The effect of temperature on the filtration rate is thus immediate and the hydraulic resistance is almost
At the end of fermentation, pH was equal to 6.2. The pH of the filtered broth was modified by adding sulphuric acid (95%) or ammonium hydroxide (32%). Steady-states permeate fluxes versus pH are plotted in Fig. 6. Decreasing the pH values below 6.2 decreased the filtration fluxes; whereas the pH increase first improved, and then decreased the filtration rate. The optimum pH range was between 7 and 8. There were large differences between the steady-state fluxes obtained with different pH values. The flux maximal value was twice as large as the minimal value. Thus, pH is a key parameter which has to be taken into account when optimising the clarification process of fermentation broths by microfiltration.
3.8. Influence of permeate flux, identification of critical fluxes Fig. 5. Cross-flow filtration of a lactic acid fermentation broth on a 0.1 mm Kerasep membrane. Permeate flux and total resistance to mass transfer variation with time as a function of temperature.
Fig. 6. Cross-flow filtration of a lactic acid fermentation broth on a 0.1 mm Kerasep membrane. Steady-state permeate flux vs. broth pH.
Fig. 7 shows two examples of filtration runs performed with a stepwise increased or decreased permeate flux. When considering the variation of the transmembrane pressure, two series of permeate flux values were distinguished. At low permeate flux values, transmembrane pressure was almost constant whereas it increased with time with high permeate fluxes. At the highest values, the sharp pressure increase indicated that it was impossible to run filtration under these conditions. The maximum value of the permeate flux at which the transmembrane pressure was stable with time is named ‘critical flux’, it is a function of the cross-flow velocity: for 6= 4 m/s
Jcrit = 50 l/h m2
for 6= 3 m/s
Jcrit = 20 l/h m2
These critical fluxes did not correspond to the strong form of critical flux defined by Field et al.
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4. Conclusions Lactic acid fermentation broths were properly clarified with a 0.1 mm pore diameter membrane and permeate fluxes were high enough. Among the fermentation broth components, the molasses led to the highest permeate flux drop. Nevertheless, we identified critical fluxes that were function of cross-flow velocity. Operating filtration at fluxes below these critical values made it possible to extend filtration times by reducing the formation of the bacterial cell cake at the membrane wall. However, irreversible fouling due to, among others, the adsorption of some of the molasses components always occurred. The broth pH, which is generally determined by the fermentation conditions, was also found to affect the filtration performance. In this case, the pH should be adjusted between the values of 7 and 8 before filtration, as the bacteria are not reused in the batch process.
Acknowledgements Fig. 7. Controlled permeate flux cross-flow filtration of a lactic acid fermentation broth on a 0.1 mm Kerasep membrane. Transmembrane pressure variation with time as a function of permeate flux. (a) 6= 4 m/s, (b) 6 =3 m/s.
[15] or Howell [16] (equivalent to the corresponding clean water flux) because fouling due to, among others adsorption of some molasses components, always occurred. Low permeate flux values made it possible to control only the formation of the bacterial cell cake at the membrane wall. However, the existence of such critical fluxes is very interesting for improving filtration industrial processes as it makes it possible to extend operating times. Such critical fluxes, or more precisely a critical value of the permeate flux ratio on the wall shear stress (J/~w), have already been reported for the microfiltration of a L. hel6eticus broth [12].
The authors would like to acknowledge the Orelis (Saint Maurice de Beynost, France) and Texel (Dange´ Saint Romain, France) companies for their kind gifts of the membranes and of the microorganisms. We are grateful to Drs C. Be´al and E. Latrille (LGMPA-INRA Grignon) for their helpful advice in the improvement of the fermentation process. We are also indebted to G. Rigou (INRA’s translation unit, Jouy-en-Josas) for revising the English version of the manuscript.
References [1] T.B. Vick Roy, in: M. Moo-Young (Ed.), Comprehensive Biotechnology, vol. 3, Pergamon Press, Oxford, 1985, pp. 761– 776. [2] R.L. Evangelista, Z.L. Nikolov, Appl. Biochem. Biotechnol. 57/58 (1996) 471. [3] S. Tejayadi, M. Cheryan, Appl. Microbiol. Biotechnol. 43 (1995) 242. [4] A.S. Kertes, C.J. King, Biotch. Bioeng. 28 (1986) 269. [5] J.S. Kascak, J. Kominek, M. Roehr, in: H.J. Rehm, G. Reed, A. Puhler, P. Stadler (Eds.), Biotechnology,
S. Milcent, H. Carre`re / Separation/Purification Technology 22-23 (2001) 393–401
[6]
[7]
[8] [9] [10]
Product of Primary Metabolism, Ch. 8, vol. 6, VCH, Weinheim, Germany, 1996. M. Bailly, H. Roux de Balmann, P. Aimar, F. Lutin, 2nd European Congress of Chemical Engineering, Montpellier, France, 5 – 7 October, 1999. P. Boyaval, C. Lafforgue-Delorme, G. Goma, in: G. Daufin, F. Rene´, P. Aimar (Eds.), Les Se´parations par Membrane dans les Proce´de´s de l’Industrie Alimentaire, Ch. 13, Tec&Doc Lavoisier, Paris, 1998. Y. Goksungur, U. Guvenc¸, J. Chem. Tech. Biotechnol. 69 (1997) 399. H.E. Spinnler, G. Corrieu, J. Dairy Res. 56 (1989) 755. S.L. Matson, in: R.D. Noble, S.A. Stern (Eds.), Mem-
[11] [12] [13] [14] [15] [16]
.
401
brane Separations Technology, Principles and Applications, Ch. 8, Elsevier, Amsterdam, 1995. T. Tanaka, K. Usui, K. Kouda, K. Nakanishi, J. Chem. Eng. Jpn. 29 (1996) 973. P. Boyaval, C. Lavenant, G. Ge´san, D. Daufin, Biotechnol. Bioeng. 49 (1996) 78. Y. Shimizu, K.I. Shimodera, A. Watanabe, J. Fermentation Bioeng. 76 (1993) 493. N. Nagata, K.J. Herouvis, D.M. Dziewulski, G. Belfort, Biotechnol. Bioeng. 34 (1989) 447. R.W. Field, D. Wu, J.A. Howell, B.B. Gupta, J. Membr. Sci. 100 (1995) 259. J.A. Howell, J. Membr. Sci. 107 (1995) 165.