Growth and lactic acid production by Lactobacillus casei ssp ...

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Growth and lactic acid production by Lactobacillus casei ssp. rhamnosus in batch and membrane bioreactor: influence of yeast extract and Tryptone enrichment A. Olmos-Dichara, F. Ampe, J.-L. Uribelarrea, A. Pareilleux† and G. Goma* Centre de Bioingénierie Gilbert Durand, UMR-CNRS 5504 and L.A. INRA, Institut National des Sciences Appliquées, Complexe Scientifique de Rangueil, 31077 Toulouse cedex 4, France Enrichment of the medium with yeast extract (20 g.l–1) and Tryptone (40 g.l–1) increased the growth of Lactobacillus casei ssp. rhamnosus and its production of lactic acid in both batch and cell-recycle cultures without affecting glucose consumption and the lactic acid production rate.

Introduction Lactic acid has traditionally been used in the food industry as an acidulating and/or preservative agent, and in the chemical industry for cosmetic and textile applications. However, lactic acid fermentation has recently received much attention because of the increasing demand for new materials such as biodegradable and biocompatible polylactic products (Datta et al., 1995). These polymers and co-polymers of L-lactate have potentially large markets in commodity packing, production of prosthetic devices, and controlled delivery of drugs in humans. The most effective way to synthesise L-lactic acid is not through chemical processes but its biosynthesis using fermentation techniques with defined strains (Ye et al., 1996). Indeed, several bacterial strains are able to produce high amounts of L-lactic acid (e.g. L. helveticus, L. casei, L. salivarius and L. delbrueckii) (Hjörleifsdottir et al., 1990; Siebold et al., 1995). However, two major factors limit this biosynthesis and also affect both growth and productivity: the nutrient limiting conditions and the inhibitory effect caused by the lactic acid produced in the culture broth. So far, the study of lactic acid production by lactic acid bacteria has mainly focused on the inhibitory effect of the weak acids on growth (Hongo et al., 1986; © 1997 Chapman & Hall

Loubière et al., 1997) despite the multiple nutritional prerequisites of lactobacilli for growth and production (Ledesma et al., 1977). Little attention has been paid to the effect of complex medium enrichment factors such as corn steep liquor, meat extract, yeast extract and Tryptone. These are often used as growth factors and protein nitrogen sources (Bibal et al., 1989), but their role remains unclear. Only a few preliminary studies have shown that the addition of yeast extract in the culture medium improves growth and lactate production performances of lactobacilli (Ohleyer et al., 1985; Aeschlimann and von Stockar, 1990; Timmer and Kromkamp, 1994), but these studies did not include a detailed analysis of the enrichment effect in a kinetic perspective. Many investigations aimed to improve lactic acid productivity and membrane processes are essential techniques for this purpose (Vick Roy et al., 1983; Boyaval et al., 1987; Bibal et al., 1991). This system can retain the biomass inside the reactor in order to achieve high cell density and an active metabolism, while continuously supplying with fresh medium and removing inhibitory products. The association between a bioreactor and a ultrafiltration unit offers an increase in volumetric productivity as well as a decrease in energy requirements when compared to a conventional fermentation. Biotechnology Letters · Vol 19 · No 8 · 1997

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The current study investigated the effect of the enrichment coefficient (yeast extract + Tryptone) on growth performances and lactic acid production by L. casei ssp. rhamnosus (L. delbrueckii NRRL-B445) in batch and membrane cell-recycle cultures. L. casei was chosen because it both resists to high concentrations of lactic acid, and has multiple nutritional requirements.

the permeate liquid was removed at a desired rate using a peristaltic pump, the excess being recirculated into the fermenter, thus fixing the dilution rate on permeate (D) which was constant over the entire experiment. The supply of fresh medium was provided via a peristaltic pump connected to a level controller, keeping the working volume constant.

Materials and methods Strain and media L. casei ssp. rhamnosus (L. delbrueckii NRRL-B445), a homolactic acid fermentative bacterium was grown on a basal medium consisting of the following composition (in g.l–1): the mineral and carbon source with MgSO4.7H2O, 0.3; (NH4)2SO4, 0.5; KH2PO4, 0.2; K2HPO4, 0.2 and glucose, 50 (unless otherwise mentioned), the organic charge in yeast extract (Biokar, France) and trypsin digest of casein (Biokar, France), the level of which is changing proportionally to a, with a = 1 when yeast extract = 5 g.l–1 and Tryptone = 10 g.l–1.

In total cell recycle experiments, the medium used was supplied with three differents concentrated solutions: (1) glucose 700 g.l–1, (2) yeast extract and Tryptone of casein in a ratio 1:2 at a total amount of 300 g.l–1, and (3) a salt solution twenty times more concentrated than for batch cultures.

The medium for the inoculum preparation was the same as the basal medium except that the glucose concentration was 10 g.l–1. The inoculum was prepared by transferring cells from 10% skimmed milk stock (0.2 ml) stored at –20°C to test tubes (9.5 ml) at various dilutions (medium feed preparation). These tubes were then incubated statically for 18 h at 42°C and transferred to Erlenmeyer flasks of 250 ml (medium, 50 ml) for preculture. After static cultivation for 10 h, the cultures were inoculated in the fermenter at 0.2% (v/v) concentration.

The pH of the cultures was kept constant at 6 by automatic addition of gaseous ammonia, and the temperature was maintained at 42°C. These cultures were performed without bleeding biomass.

Batch cultures Batch culture experiments were performed in 2-l fermenters (Inceltech-SGI, France). The pH was regulated at 6.0 with 2.5 M NH4OH. The fermenter was held at 42°C with constant stirring at 200 rpm, the fermenter was maintained in microaerophilic conditions. Cell-recycle reactor Cultures with cell recycling were undertaken in the same kind of fermenter as batch cultures connected to a cross-flow filtration unit consisting of two mineral membrane modules (Techsep-RP, France; M6 type; surface area of 0.1 m2 nominal cut-off point at 105 Da). The total working volume was 3-l using one filtration module. A variable speed orbital pump (Albin SLP 115, Sweden) was used to withdraw the culture broth from the fermenter in order to provide a sufficient circulation rate to maintain the desired filtration rate (liquid velocity of 4–5 m.s–1 and pressure of 2–3 bar). Part of

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These three solutions were separately supplied to the culture with additional feed of sterile water (0.2 mm pore size) for dilution setting. This approach allowed the preparation of media to be minimised and the ability to set different concentrations of yeast extract and Tryptone thus controlling the a previously described.

Analytical methods For cell dry weight determination, cells were harvested by filtration on 0.2 mm pore size membrane (or by centrifugation in Eppendorf tubes for 5 min at 10 000 g, washed twice with distilled water) and dried to constant dry weight at 60°C under partial vacuum (200 mm Hg). Lactic acid and glucose concentration in the fermentation broth were measured by HPLC using an ion exclusion column (Ion 300, Interaction Chemical, Mountain View, California, USA) with the following conditions: temperature, 50°C; solvent 0.01 N H2SO4; flow rate 0.5 ml.min–1 and a differential refractometer detection. L-Lactic acid concentration was determined by an enzymatic method with an YSI model 2000 analyser (Yellow Spring Instruments, Yellow Springs, Ohio, USA). Calculations The kinetic parameters were estimated after computer treatment of experimental data. For batch cultures, the specific growth rate (m, h–1), the specific rate of glucose consumption (qs, g.g–1.h–1) and the specific rate of lactic acid production (np, g.g–1.h–1) were then calculated by dividing the rates of cell growth (rx), glucose consumption (rs) and lactic acid production (rp) by the estimated

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cell mass concentration (X, g.l–1.dw). For cell-recycle cultures, the values of m, qs, and np, were then calculated by the following equations: m = rx /X, qs = D(Sf – S)/X – (dS/dt)/X and np = D.P/X + (dP/dt)/X, where D is the dilution rate (h–1); Sf, the concentration of glucose in the feed (g.l–1); S, the effluent glucose concentration (g.l–1); and P, the effluent lactic acid concentration (g.l–1) and t is the time. The maximum specific rates (mmax, qsmax and npmax) are the maximal values observed during the culture. Results and discussion Growth of L. casei in batch cultures with various enrichment coefficients (a) L. casei was grown in batch cultures on glucose with an enrichment coefficient (a) ranging from 0.5 (2.5 g.l–1 yeast extract and 5 g.l–1 Tryptone) to 4 (20 g.l–1 yeast extract and 40 g.l–1 Tryptone), and growth parameters were estimated (Table 1). The lactate production yield did not vary significantly with a (Yp/s = 0.96–0.98 g.g–1), the maximum specific rate of glucose consumption (qsmax) and the maximum specific rate of lactic acid production (npmax) were also almost identical for all the cultures. On the other hand, the time of cultivation sharply decreased as a increased, from 50 h with a = 0.5 down to 26 h with a = 4. This decrease could be seen as the result of an increase of both the growth rate (m, from 0.43 h–1 to 0.63 h–1) and the yield of conversion of glucose into biomass (YX/S, from 0.03 g.g–1 to 0.08 g.g–1). The increase in YX/S also provoked a strong increase in the final biomass concentration (Xfinal from 1.4 to 4 g.l–1). Though this increase in Xfinal was already shown (Bibal et al., 1989; Aeschliman and von Stockar, 1990), the

importance of the reaction time was never stressed before, and it is the first time that qsmax and npmax are shown to be independent upon the enrichment coefficient which was demonstrated here to specifically affect the growth. In terms of productivity, increasing the enrichment coefficient from a = 0.5 to 2 lead to an increase in the productivity of 60% and an increase in Xfinal of 140%, whereas a further increase in a from 2 to 4 only gave an increase of 17% in the productivity and of 20% in Xfinal. Therefore, there seem to be a threshold value of a around 2 over which an increase of the enrichment does not significantly improves the overall productivity. The influence of a was further investigated in plotting the growth parameters (m, np, rx and rp) obtained during the batch cultures with various enrichment coefficients versus the advancement of the reaction expressed as P/Pf, P being the lactic acid concentration produced during the culture at the time t, and Pf the final lactic acid concentration in the culture broth (Fig. 1). Results show that µ increased together with a whatever the advancement of the reaction. The biomass production rate (rx) also increased with a, and its maximum value (rxmax) was reached later in the advancement of the reaction for high a than for low a. This indicates that a higher input of yeast extract and Tryptone must have abolished some nutritional limitations. In addition, when a was higher than or equal to 2, growth was maintained until substrate exhaustion, whereas for a lower than 2 growth halted prior to substrate exhaustion, the residual glucose being then entirely converted to lactate.

Table 1 Parameters characteristics of L. casei, growth and lactic acid production for various medium enrichment coefficients in batch cultures Enrichment coefficient of the medium (a)

0.5

1

1.5

2

3

4

Time of cultivation (h)

50

47

36

31.5

30.5

26

Biomass production (anabolism) Biomass final (Xfinal, g.l–1 dw) Maximum growth rate (mmax, h–1) Biomass/glucose yield (YX/S, g.g–1)

1.40 0.41 0.03

1.95 0.46 0.04

2.64 0.48 0.05

3.32 0.51 0.06

3.73 0.58 0.08

4.00 0.63 0.08

Lactic acid production (catabolism) –1

–1

Maximum specific lactic acid productivity (npmax, g.g .h ) Maximum specific glucose consumption (qsmax, g.g–1.h–1) Lactic acid/glucose yield (YP/S, g.g–1)a Lactic acid productivity (g.l–1.h–1) a

2.21 2.29 0.96 0.97

2.27 2.32 0.97 0.98

2.24 2.38 0.96 1.38

2.26 2.36 0.98 1.55

2.26 2.36 0.98 1.52

2.23 2.29 0.98 1.81

L-lactic acid always represented at least 95% of the total lactic acid.


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Figure 1 Influence of the concentration in yeast extract and Tryptone on: the specific growth rate (a); the specific lactate production rate (b); the reaction growth rate (c); and the lactic acid production rate versus the advancement of the reaction (P, lactic acid concentration produced during the culture at the time t; Pf, final lactic acid concentration) during growth of L. casei in batch culture. a = medium enrichment coefficient: h, 0.5; r, 1; n, 1.5; s, 2; j, 3; d, 4.

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On the other hand, the specific rate of lactic acid production (np) did not vary with a whatever the advancement of the reaction. Since rx increased with a, the lactic acid production rate (rp) also increased with a. These results show that increasing a led to a higher fueling of the anabolism without affecting the catabolism, thus increasing the lactic acid production during the idiophase. Interestingly, when a was low, growth halted prior to substrate exhaustion, but this did not affect the specific rate of lactic acid production. This shows that nutritional limitations did not affect the lactic acid production which was then uncoupled to the growth. Indeed, for a = 0.5 a high and constant value of np = 1.2 ± 0.1 g.g–1.h–1 was maintained until the end of the culture (Fig. 1). This suggests that the lactic acid production during the trophophase can be performed with media containing a low charge. Growth of L. casei in cell recycle cultures To further investigate these findings with the objective of a higher scale lactic acid production, we estimated the effect of a during the growth of L. casei in a cellrecycle reactor. This device allowed us to control both the lactic acid inhibition and the nutrient feed, which we performed by increasing the biomass concentration. The dilution rate was set at 0.25 h–1, the glucose concentration in the feed laid between 55–85 g.l–1 and the glucose was added so that this substrate was not limiting. The concentration of yeast extract and Tryptone varied by simply adjusting the incoming media, the concentration of these elements were directly proportional to the value of a. After establishing the pseudo steady-state with a = 1, the conditions were changed by decreasing a from 1 to 0.5. Each pseudo steady-state was maintained during at least ten residence times (40 h). We chose condition when the biomass remained constant and no growth was observed. Decreasing a from 1 to 0.5 did not alter the production of lactic acid as only a small decrease in np was observed (Table 2). This slight decrease was probably the result of cellular death as already shown by Cachon and Diviès (1993). This was here corroborated by the decrease in the slight percentage of glucose utilisation (Table 2). However, due to accuracy of measurement we do not consider this difference significant. Growth in the cell-recycle reactor led to a biomass concentration (X ≈ 77 g.l–1) nineteen times higher than

Table 2 Parameters characteristics of L. casei, growth and lactic acid production for various medium enrichment coefficients in cell recycle cultures, pseudo steady-state during a perfusion cycle (D = 0.25 h–1; m ≈ 0; balanced performed during 40 h) Enrichment coefficient of the medium (a) Biomass (g.l–1)a Lactic acid (g.l–1)a,b Glucose utilisation (%)a Specific lactic acid productivity (np, g.g–1.h–1)a Specific glucose consumption (qs, g.g–1.h–1)a Lactic acid/glucose yield (YP/S, g.g–1)a Lactic acid productivity (g.l–1.h–1)a

0.5

1

76.8 ± 0.05 57.2 ± 0.6 85 ± 0.3

76.8 ± 0.1 57.8 ± 0.2 86 ± 0.4

0.18 ± 0.004 0.19 ± 0.003 0.19 ± 0.001 0.19 ± 0.002 0.95 ± 0.01

0.96 ± 0.01

14.2 ± 0.2

14.4 ± 0.1

a

Values given are the mean for all the period of each pseudo steady-state ± the standard deviation. b L-lactic acid always represented 95% of the total acid lactic.

in batch cultures. The productivity also increased eight times while the yield YP/S was constant and equal to that found for batch cultures. It should be noted that in the beginning of the perfused culture, the maximum specific growth rate (mmax = 0.16 h–1) and lactic acid production (npmax = 1.1 g.g–1.h–1) were found to be much lower than those previously observed in batch cultures, probably as the result of mechanical stresses generated in the system by pumping. Conclusion Altogether, these results clearly show that a high production of lactic acid can be maintained while keeping a low enrichment of the medium. On this concept, a two-step culture (such as a two-level cascade reactor) would be an optimised process with high biomass concentration: during the first step, a would be kept high to produce the cell biomass, and in the second one a could be strongly decreased without affecting the productivity of lactic acid, though decreasing the production costs. Acknowledgements The authors wish to thank Dr. V. Guillou for stimulating discussions. A. Olmos-Dichara would like to thank the “Consejo Nacional de Ciencia y Tecnología” (CONACyT) from México and the “Société Française d’Exportation des Ressources Educatives” (SFERE) from France, for their financial support. Biotechnology Letters · Vol 19 · No 8 · 1997

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References Aeschlimann, A. and von Stockar, U. (1990). Appl. Microbiol. Biotechnol. 32: 398–402. Bibal, B., Vayssier, Y., Tournou, M. and Pareilleux, A. (1989). Appl. Microbiol. Biotechnol. 30: 630–635. Bibal, B., Vayssier, Y., Goma, G. and Pareilleux, A. (1991). Biotechnol. Bioeng. 37: 746–754. Boyaval, P., Corre, C. and Terre, S (1987). Biotechnol. Lett. 9: 207–212. Cachon, R. and Diviès, C. (1993). Appl. Microbiol. Biotechnol. 40: 28–33. Datta, R., Tsai, S-P., Bonsignore, P., Moon, S-H. and Frank, R. J (1995). FEMS Microbiol. Rev. 16: 221–231. Hjörleifsdottir, S., Seevaratnam, S., Holst, O. and Mattiasson, B. (1990). Current Microbiol. 20: 287–292. Hongo, M., Nomura, Y. and Iwahara, M. (1986). Appl. Env. Microbiol. 52: 314–319.

Ledesma, O.V. Ruiz-Holgado de, P. A., Oliver, G., De Giori, S.G., Raibaud, P. and Galpin, V.J. (1977). J. Appl. Bacteriol. 42: 123–133. Loubière, P., Cocaign-Bousquet, M., Matos, J., Goma, G. and Lindley, N.D. (1997). J. Appl. Bacteriol. 81: 1–6. Ohleyer, E., Blanch, H.W. and Wilke, C.R. (1985). Appl. Biochem. Biotechnol. 11: 317–332. Siebold, M., Frieling, V.P., Joppien, R., Rindfleisch, D., Schügerl, K. and Röper, H. (1995). Process Biochem. 30: 81–95. Timmer, J.M.K. and Kromkamp, J. (1994). FEMS Microbiol. Rev. 14: 29–38. Vick Roy, T.B., Mandel, D.K., Dea, D.K., Blanch, H.W. and Wilke, C.R. (1983). Biotechnol. Lett. 5: 665–670. Ye, K., Jin, S. and Shimizu, K. (1996). J. Ferm. Bioeng. 81: 240–246.

Received 25 April 1997; Revisions requested 22 May 1997; Revisions received 2 June 1997; Accepted 5 June 1997

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