Alternative media for lactic acid production by lactobacillus delbrueckii NRRL b 445

0960–3085/03/$23.50+0.00 # Institution of Chemical Engineers www.ingentaselect.com = titles = 09603085.htm Trans IChemE, Vol 81, Part C, September 2003 ALTERNATIVE MEDIA FOR LACTIC ACID PRODUCTION BY LACTOBACILLUS DELBRUECKII NRRL B-445 S. J. TE ´ LLEZ-LUIS 1,2 , A. B. MOLDES 2 , M. VA ´ ZQUEZ 1 ,3 and J. L. ALONSO 2 1 Department of Food Science and Technology, UAM Reynosa-Aztla´n, Universidad Autonoma de Tamaulipas, Me ´ xico 2 Department of Chemical Engineering, University of Vigo (Facultad de Ourense), Vigo, Spain 3 A ´ rea de Tecnolog‡´a de los Alimentos, Escuela Polite´cnica Superior, Departamento de Qu‡´mica Anal‡´tica, Universidad de Santiago de Compostela, Lugo, Spain L actic acid bacteria are generally recognized as nutritionally fastidious. The complexity of the media increases the cost of lactic acid production. In this study a low-cost nutrient medium based on corn steep liq uor (CSL) was developed for lactic acid pro- duction by Lactobacillus delbrueckii NRRL B-445. Starting from a medium containing 90 g l ¡ 1 glucose and 20 g l ¡ 1 CSL as a sole nutrient source, 70.7 g l ¡ 1 lactic acid was obtained with an economic ef ciency of 98 g lactic acid per „ nutrient. Other media, made with CSL and each individual component (yeast extract, peptone, sodiu m acetate, sodium citrate, K 2 HPO 4 , MgSO 4 ¢ 7H 2 O, MnSO 4 ¢ H 2 O or FeSO 4 ¢ 7H 2 O) of a general (Mercier) lactobacilli medium were also assayed. The highest economical ef ciency (134 g lactic acid per „ nutrient) was obtained supplementing 10 g l ¡ 1 CSL with 0.05 g l ¡ 1 FeSO 4 ¢ 7H 2 O. Additionally, lactic acid production and glucose consumption were mathematically modelled and the regression parameters obtained were correlated with CSL concentration by linear or exponential equations. Keywords: Lactobacillus delbrueckii; lactic acid, corn steep liquor, n utrients study; mathematical modelling. INTRODUCTION Many applications in dairy, beverage, confectionery, meat and poultry industries have been found for lactic acid (2-hydroxy propionic acid) and its derivates. Lactic acid is employed in food industry as acid ulant,  avour and preser- vative due to its mild taste that does not hide the weaker aromatic  avours of some foods. Additionally, it has a technological application during cheese and yoghurt produc- tion, producing the coagulation of the casein fraction. An other important appli cation of this compound is the production of polylactic acid (PLA)-based degradable plas- tics (Chahal, 1991; Ozen and Ozilgen, 1992). Lactic acid can be obtained by chemical synthesis from petroleum-based products or by microbial fermentation. Many lactobaci lli strains a nd some fungus like Rhizopus oryzae can bioconvert glucose and other sugars to lactic acid (Zhou et al. , 1999; Hofvendahl and Hahn-Hagerdal, 2000). Owing to its asymmetric c arbon, lactic acid can occur in two optically active enantiomers, L and D (Vick-Roy, 1985), but an important advantage of microbial fermentation over chemical synthesis is that it is possible to produce exclu- sively one of the isomers. Lactic acid bacteria u se sugars via different pathways resulting in homo-, hetero- o r mixed acid fermentations. Homofermentation gives only lactic ac id as the end product of glucose metabolism by the Embden–Meyerhof–Parnas pathway. In heterofermentation, equimolar amounts of lactic acid, carbon dioxide and ethanol or acetate are formed from glucose via the phosphoke tolase pathway (Chahal, 1991). Several factors that affect lactic acid production by micro- organisms are medium composition (carbohydrate source, sugar concentration and growth factors), temperature, presence of oxygen, pH and product concentration (Burgos-Rubio et al. , 2000). Lactobacillus delbrueckii NRRL B-445, also named as Lactobacillus rhamnosus ATCC 10863 (Hofvendahl and Hahn-Hagerdal, 2000), is a homofermentative lactic acid b acterium that produces mainly L-lactic acid. In spite of the advantages, fermentations must be cost competitive with chemical synthesis. Fermentation medium can represent almost 30% of the cost for a microbial fermentation (Miller and Churchill, 1986). Lactic acid bacteria have limited capacity to synthesize B-vitamins and amino acids (Hofvendahl and Hahn-Hagerdal, 2000). Yeast extract is used as the main source of nitrogen and vitamins for lactic acid production by microorganisms, but it is too expensive for larg e-scale fermentations. Complex media commonly employed for growth of lactic acid bacteria are not economically attractive due to their high amount of expensive nutrients such as yeast extract, peptone and salts (Mercier et al. , 1992). Various nitrogen sources were tested for lactic acid production by bacteria but none of these gave lactic acid concent rations as high as that obtained with yeast extract (Nancib et al. , 2001; Te´llez- Luis et al. , 2003). However, new low-cost media for lactic acid fermentation are desirable in order to decrease the production cost. 250 Corn steep liquor (CSL) is a low-cost nutritional medium employed successfully in the production of ethan ol by Zymomonas mobilis (Kadam and Newman, 1997; Silveira et al. , 2001), succinic acid by Anaerobiospirillum succini- ciproducens (Lee et al. , 2000) or arabinanase by Fusarium oxysporum Cheilas et al. , 2000). It could replace some of the expensive nutrients in the complex medium employed to grow L. delbrueckii . The aim of t his study was to develop a low-cost nutrient medium based on CSL for lactic acid production by L. delbrueckii NRRL B-445. Additionally, lactic acid produc- tion and glucose consumption were modelled and the regres- sion parameters obtained were correlated with CSL concentration. MATERIALS AND METHODS Strains and Culture Conditions L. delbrueckii NRRL B-445 was obtain ed from the United States Department of Agriculture Northern National Research Laboratory in Peoria, IL. The strain was grown on plates using the complete media proposed by Mercier et al. (1992), which contains 20 g l ¡ 1 glucose, 5 g l ¡ 1 yeast extract, 10 g l ¡ 1 peptone, 5 g l ¡ 1 sodium acetate, 2 g l ¡ 1 sodium citrate, 2 g l ¡ 1 K 2 HPO 4 , 0.58 g l ¡ 1 MgSO 4 ¢ 7H 2 O, 0.12 g l ¡ 1 MnSO 4 ¢ H 2 O, 0.05 g l ¡ 1 FeSO 4 ¢ 7H 2 O and 10 g l ¡ 1 agar at 37 ¯ C for 24 h. Inocula were prepared by washing cells from plates with 5 ml sterile water. Biomass in the inocula was measured by optical density at 600 nm and adjusted to equivalent values by dilution with water to obtain 6 g l ¡ 1 dry cells. Inocula were 5 ml. The experiments were carried out in 250 ml Erlenmeyer  asks with a  nal volume of 100 ml usi ng different media. The content of nitrogen in the yeast extract, peptone and CSL used was 11.9, 12 and 13% in dry basis, respectively. The content of water in the yeast extract, peptone and CSL used was 3.5, 4 and 50%, respectively. Calcium carbonate (10 g) was added previously to inocu- lation. After inoculation, fermentations were carried out in orbital shakers at 41.5 ¯ C and 200 rpm for 96–98 h. The pH was kept constan t around 6 due to the lactic acid formed was neutralized by the present of calcium carbonate. Samples (2 ml) were taken at random time in tervals and centrifuged at 16,000 g for 3 min. The supernatants were used immedi- ately for various analyses. Analytical Methods Glucose, lactic acid and ac etic acid were determined by high performance liquid chromatography (HPLC) using a Transgenomic ION-300 column and an isocratic elution with a  ow rate of 0.4 ml min ¡ 1 . The mobile phase was 0.0025 M H 2 SO 4 . The oven temperature was 65 ¯ C and a refractive index (RI) detector was used. Statistical Analysis All experimental data were obtained in triplicate and mean values are g iven. Linear and non-linear regression analyses of experimental data were performed usi ng commercial software (Microsoft Excel 2000, Microsoft Corporation, Redmond, WA, USA). RESULTS AND DISCUSSION The cost of nutrients is an important aspect in the fermentation of glucose to lactic acid by L. delbrueckii . General lactobacilli media such as Mercier medium and MRS medium are very complex with many expensive nutrients. Table 1 shows the components of the Mercier medium as well as the cost of each nutrient. The price of CSL is also included in the same table. As it can be noted, the price of CSL is two to three times lower than the price of yeast extract and peptone, respectively. Comparative Study Between Mercier Medium and CSL-based Media Media containing different concentrations of CSL (1, 3, 5, 10 or 20 g l ¡ 1 ) were tested in the fermentation of 90 g l ¡ 1 glucose to lactic acid. For comparative purposes a batch fermentation using the Mercier medium with 90 g l ¡ 1 glucose was also performed. Figure 1 shows the results of the batch experiments for lactic acid and glucose concentrations. The highest lactic acid concentration was obtained using the Mercier medium (76.2 g l ¡ 1 ). However, a similar pattern was shown by the fermentation with 20 g l ¡ 1 CSL, obtaining 70.7 g l ¡ 1 lactic acid at the end of the fermentation. Using lower concentrations of CSL, lower lactic acid concentrations and volumetric productivities were obtained. These facts suggest that CSL at concentration lower than 20 g l ¡ 1 did not supply the require d nutrients for the metabolism of L. delbrueckii . Additionally, acetic acid was quanti ed and negligible concentrations were obtained (data not shown). This was important because it demonstrated that L. delbrueckii maintains the homofermentative pathway in the presence of CSL. In the experiment using the Mercier medium, the glucose concentration was completely consumed at the end of the fermentation (Figure 1b). However, a  nal glucose concen- tration of 12.0 g l ¡ 1 was observed in the experiments carried out with medium con taining 20 g l ¡ 1 CSL. Using lower concentrations of CSL, higher concentrations of residual glucose were obtained. This suggested that CSL is limited in some nutrients. Table 2 shows numerical values of lactic acid concentra- tion, product yield ( Y p = s ), product ef ciency ( E p = s ) and economic ef ciency ( E p = „ ) after 98 h of fermentation. Product yield was de ned as grams lactic acid produced per gram glucose consumed, product ef ciency as g rams lactic acid produced per gram initial glucose and economic Table 1. Prices of nu trients used in experiments. Nutrient Cost ( „ = kg) Corn steep liquor 36.06 Yeast extract 76.74 Peptone 112.27 Sodium acetate 13.94 Sodium citrate 20.73 K 2 HPO 4 30.29 MgSO 4 10.58 MnSO 4 15.03 FeSO 4 11.54 ALTERNATIVE MEDIA FOR LACTIC ACID PRODUCTION 251 Trans IChemE, Vol 81, Part C, September 2003 ef ciency as grams lactic acid produced per cost unit of nutrients ( „ ). As it can be observed, E p = s decreased with the decrease of CSL concentration. However, Y p = s was main- tained around 0.9 g g ¡ 1 using the Mercier medium or media containing 20 or 10 g l ¡ 1 CSL. It i s interesting to select the cheapest media that allow the highest lactic acid concentra tion to be obtained. The E p = „ is an adequate parameter to compare media from an econom- ical poin t of view. The parameter E p = „ showed that it was more pro t able to use a medium with 20 g l ¡ 1 CSL than the Mercier medium because using 20 g l ¡ 1 CSL, 98 g of lactic acid were produced per euro of n utrients while only 50 g lactic acid were obtained per euro when the Mercier medium was employed. Mathematical Modelling of Fermentation with CSL as a Sole Nutritional Source Lactic acid production and glucose consumption were mathematically modelled and the regression parameters obtained were correlated with CSL concentration by linear or exponential equati ons. A mathematical model was adopted from another study to describe the fermentative production of lactic acid (Mercier et al. , 1992): d P d t ˆ P r P 1 ¡ P P m  ´ (1) where t is time, P is lactic acid concentration, P m is maximum concentration of lactic acid, and P r is the ratio between the initial volumetric rate of produ ct formation ( r p ) and the initial product concentration P 0 . Equation (1) can be directly solved to give the following expression: P ˆ P 0 P m e P r t P m ¡ P 0 ‡ P 0 e P r t (2) From the series of experimental data for lactic acid concentration during fermentation, the model parameters P 0 , P m and P r can b e calculated for each fermentation medium by non-linear regression using the least-squares method. Table 3 shows the kinetic and statistical parameters. Figure 1a shows the experimental and predicted data for these batches. The coef cient r 2 showed a good agreement between experimental and predicted data. The value of the F -test probability showed that the model for 1 g l ¡ 1 CSL medium is the least accurate due to the low value of lactic acid concentration obtained for this medium. The models predict maximum lactic acid concentrations of 74.9 g l ¡ 1 for the Mercier medium, 66.6 g l ¡ 1 for 20 g CSL l ¡ 1 medium and 25.9 g l ¡ 1 for 10 g CSL l ¡ 1 medium at 98 h. The regression parameters obtained for each experiment were Figure 1. Experimental and calculated dependence of lactic acid and glucose concentrations on the fermentation time correspon ding to fermen- tations of 90 g l ¡ 1 glucose with different concentrations of corn steep liquor and the Mercier medium. Table 2. Results for the lactic acid pro duction by Lactobacillus delbrueckii using different concentrations of CSL and the Mercier medium. All fermentations were with 90 g l ¡ 1 glucose. Medium Lactic acid concentration (g l ¡ 1 ) Y p = s (g g ¡ 1 ) E p = s (g g ¡ 1 ) E p = „ (g „ ¡ 1 ) Mercier medium 76.20 0.85 0.85 50 20 g l ¡ 1 CSL 70.73 0.91 0.79 98 10 g l ¡ 1 CSL 27.13 0.90 0.30 75 5 g l ¡ 1 CSL 15.59 0.66 0.17 86 3 g l ¡ 1 CSL 8.55 0.42 0.10 79 1 g l ¡ 1 CSL 2.57 0.20 0.03 71 Table 3. Results obtained by regression analysis of lactic acid p roduction and glucose consumption by Lactobacillus delbrueckii u sing different media (all media include 90 g l ¡ 1 glucose). Lactic acid production Glucose consumption Medium P 0 (g l ¡ 1 ) P m (g l ¡ 1 ) P r (h ¡ 1 ) r 2 F-test of probability Y p = s (g g ¡ 1 ) r 2 F-test of probability Mercier medium 2.81 74.97 0.181 0.9982 0.9759 0.91 0.9966 0.9931 20 g l ¡ 1 CSL 4.82 66.66 0.091 0.9783 0.9041 0.91 0.9935 0.9933 10 g l ¡ 1 CSL 1.69 25.91 0.137 0.9851 0.9311 0.86 0.9834 0.9767 5 g l ¡ 1 CSL 0.99 15.38 0.166 0.9941 0.9559 0.63 0.9883 0.9782 3 g l ¡ 1 CSL 0.67 8.88 0.199 0.9892 0.9432 0.46 0.9808 0.9648 1 g l ¡ 1 CSL 0.28 2.73 0.412 0.9856 0.8505 0.21 0.9536 0.9775 Trans IChemE, Vol 81, Part C, September 2003 252 TE ´ LLEZ-LUIS et al. correlated with the CSL concentration by mean of empirical equations. P 0 , P m and P r were related to the CSL concentra- tion given by equations (3 )–(5), respectively: P 0 ˆ 0:2371 C csl ¡ 0:1555 (3) P m ˆ 3:3189 C csl ¡ 1:9735 (4) P r ˆ 0:3788 ¢ C ¡ 0:4733 csl (5) The coef cient r 2 (0.9710) for P 0 , (0.9858) for P m and (0.9749) for P r con rmed that the empirical equations  t the data well. By combining equations (3)–(5) with the model of equation (2), it is possible to predict the lactic acid concen- tration at any time for CSL concentrations and time in the range studied (0–98 h and 1–20 g l ¡ 1 CSL). Figure 2 shows how the generalized model predicts the dependence of lactic acid concentration on different CSL concentra tions and time using the model p arameters. This kind of surface response allows the selection o f different conditions in order to achieve the same results. The consumptionof glucose by L. delb rueckii can be given by the following equation (obtained from the Y p = s de nition): S ˆ S 0 ¡ 1 Y p = s ( P ¡ P 0 ) (6) where Y p = s , P and P 0 were de ned above, S is the glucose concentration (g l ¡ 1 ) and S 0 is the initial glucose concentra- tion. Using the series of experimental data concernin g glucose concentration = time and the regression parameters of equation (2), the model parameter Y s = p can b e calculated for each fermentation medium by non-linear regression using the least-squares method. Table 3 lists the numerical values of Y p = s and statistical parameters obtained for the glucose consumption and Figure 1b shows the experimental and predicted data for these fermentations. The parameter Y p = s varied with C csl according to the following equation: 1 Y p = s ˆ 4:163 ¢ C 0:5072 csl (7) The statistical parameter r 2 for the empirical equation (7) was signi cant (0.9645). Combining equation (5) with equation (6), a generalized model for predicting glucose consumption in CSL media was also developed. Figure 3 shows the prediction of the generalized model for the dependence of glucose concentration with the CSL concen- tration and time. The model predicts that more than 45 g l ¡ 1 glucose remaine d in the medium when less than 10 g l ¡ 1 CSL is used. Both models would be very useful for op timization. Fermentation of Supplemented CSL Media In order to increase the lactic acid production and the economic ef ciency, experiments were conducted using 10 g l ¡ 1 CSL supplemented with one component of the Figure 2. Prediction of the generalized model for the dependence of lactic acid concentration on the corn steep liquor concentration and time. Figure 3. Prediction of the generalized model for the dependence of glucose concentration on the corn steep liquo r concentration and time. Table 4. Results for the lactic acid production by Lactobacillus delbrueckii using CSL supplemented with other nutrients from the Mercier medium. Medium Lactic acid concentration (g l ¡ 1 ) Y p = s (g g ¡ 1 ) E p = s (g g ¡ 1 ) E p = „ (g lactic acid per „ nutrients) 10 g l ¡ 1 CSL 27.13 0.90 0.30 75 10 g l ¡ 1 CSL ‡ 10 g l ¡ 1 peptone 76.71 0.85 0.85 52 10 g l ¡ 1 CSL ‡ 5 g l ¡ 1 yeast extract 77.64 0.89 0.86 104 10 g l ¡ 1 CSL ‡ 2 g l ¡ 1 citrate 41.30 0.72 0.46 103 10 g l ¡ 1 CSL ‡ 5 g l ¡ 1 acetate 40.04 0.77 0.44 111 10 g l ¡ 1 CSL ‡ 2 g l ¡ 1 K 2 HPO 4 29.10 0.66 0.32 69 10 g l ¡ 1 CSL ‡ 0.58g l ¡ 1 MgSO 4 25.49 0.84 0.27 67 10 g l ¡ 1 CSL ‡ 0.12g l ¡ 1 MnSO 4 37.35 0.94 0.41 103 10 g l ¡ 1 CSL ‡ 0.05g l ¡ 1 FeSO 4 48.53 0.85 0.54 134 Trans IChemE, Vol 81, Part C, September 2003 ALTERNATIVE MEDIA FOR LACTIC ACID PRODUCTION 253 Mercier medium at a time, in the same concentrations. Table 4 lists the medium used and results for lactic acid concentration obtained and Y p = s , E p = s and E p = „ calculated at 98 h of fermentation for media of supplemented CSL. Figure 4 shows the experimental and calculated results from the fermentation of 10 g l ¡ 1 CSL alone as control an d 10 g l ¡ 1 CSL supplemented with peptone or yeast extract (the main nutritional components of the Mercier medium). Both exhibited a signi cant effect. The highes t lactic acid concentration (77.64 g l ¡ 1 ) was obtained by supplementing 10 g l ¡ 1 CSL with 5 g l ¡ 1 yeast extract. Similar lactic acid concentration (76.71 g l ¡ 1 ) was also obtained by supple- menting with 10 g l ¡ 1 peptone. These values compare very well with those achieved with th e Mercier medium and the 20 g l ¡ 1 CSL medium. The E p = s was also higher in the above two cases. Although the glucose consumed was different, the Y p = s was similar or slight lower than that without supplementation. The values of Y p = s compare well with those reported using other microorganisms like Rhizopus oryzae (Zhou et al. , 1999). The E p = „ was decreased by 30.66% using the medium supplemented with peptone and increased by 38.66% when using the medium supplemented with yeast extract (Table 4). The E p = „ values showed that it is most economically interesting to supplement CSL with yeast extract than with peptone. The importan ce of yeast extract in the preculture media is stressed (Amrane and Prigent, 1994). They proposed that the main contributors of yeast extract are the purine and pyridine bases and B group Figure 4. Experimental and calculated dependence of lactic acid and glucose concentrations on the fermentation time correspon ding to fermen- tations of 90 g l ¡ 1 glucose with different concentrations of corn steep liquor alone and supplemented with peptone or yeast extract. Figure 5. Experimental and calculated dependence of lactic acid and glucose concentration on the fermentation time corresponding to fermenta- tions of 90 g l ¡ 1 glucose with different concentrations of corn steep liquor supplemented with citrate, acetate or phosphate. Figure 6. Experimental and calculated dependence of lactic acid and glucose concentration on the fermentation time corresponding to fermenta- tions of 90 g l ¡ 1 glucose with different concentrations of corn steep liquor supplemented with MgSO 4 , MnSO 4 or FeSO 4 . Trans IChemE, Vol 81, Part C, September 2003 254 TE ´ LLEZ-LUIS et al. vitamins. The importance of yeast extract to Lactobacilli has been reported (Hujanem and Linko, 1996). Figure 5 shows experimental results for the fermentat ion of 10 g l ¡ 1 CSL supplemented with the carboxylic salts and mineral acids (sodium citrate, sodium acetate and sodium phosphate) of the Mercier medium. Citrate, acetate and phos- phate decreased signi cantly the value of Y p = s but the E p = s was increased after supplementing with citrate or acetate. Using 10 g l ¡ 1 CSL alone, lactic acid concentration was 27.13 g l ¡ 1 . This value slightly increased with supplementa- tion with acetate or citrate but enhancement was not observed with phosphate.The involvement of citrate and acetate in the metabolism cycles could be the explanation.The supplemen- tation of CSL with citrate and acetate enhanced the economy of the lactic acid fermentation. E p = „ was 111 g lactic acid per „ nutrient for the fermentation of the 10 g l ¡ 1 CSL medium supplemented with 5 g l ¡ 1 acetate. The E p = „ of med ia with citrate was the same as that for yeast extract (Table 4). Figure 6 shows the experimental results from the fermen- tation of 10 g l ¡ 1 CSL supplemented with MgSO 4 , MnSO 4 and FeSO 4 , the mineral sources of the Mercier medium. The lactic acid concentration obtained by supplementing CSL with MgSO 4 remained the same as that of medium without supplementation at 98 h . It is reported that magnesium is a key element in lactic acid fermentation (Thomas and Ingledew, 1990). In our study, addition of MgSO 4 had no effect on la ctic acid production. This must have occurred because CSL contains 1.5% Mg 2 ‡ on a dry basis (Zabriskie et al. , 1980). Using MgSO 4 as a component of the CSL medium decreased the E p = „ of the process. Better results were obtained by supplementing with MnSO 4 or FeSO 4 (Table 4). Although the highest lactic acid concentration was obtained by supplementing with yeast extract, the E p = „ showed that the better supplement is 0.05 g l ¡ 1 FeSO 4 because 134 g lactic acid per „ of nutrients was obtained. This value is 75% higher than the E p = „ obtained with 10 g l ¡ 1 CSL, 36% higher than to 20 g l ¡ 1 CSL medium and 168% higher than the E p = „ value when the Mercier medium was used. Mathematical Modelling of Fermentation with CSL Supplemented with Nutritional Source The experimental l actic acid production and glucose concentration d ata were examined using equation (2) and equation (6). The kinetic parameters of P 0 , P m and P r were calculated for each fermentation medium by non-linear regression. The results are shown in Table 5 together with the statistical p arameters. The coef cient r 2 showed that all the equations obtained were well  tted and Fig ures 4–6 con rm the good agreement between experimental and predicted data. The value of F -test probability also showed that the model was accurate. The values of P 0 obtained are higher than the values reported by others (Para jo´ et al. , 1996). This was because the CSL contains a low concentra- tion of lactic acid (Hull et al. , 1996). Table 5 also shows the parameter Y p = s and statistical parameters for the consumption of glucose. The coef cient showed a good agreement between experimental and predicted data. Figures 4–6 also display comparison between experimental and predicted data. The values of Y p = s are in agreement with those reported in the literature (Parajo´ et al. , 1996). CONCLUSIONS Alternative media based on CSL were evaluated in order to improve the eco nomic ef ciency of the lactic acid production by Lactobacillus delbrueckii NRRL B-445. CSL is a cheaper nutrient source than other complex media like that proposed by Mercier. In this work, it was demonstrated that a medium containing 1 0 g l ¡ 1 corn steep liquor is more economically ef cient than the Mercier medium but it is not a balanced nutritional medium for Lactobacillus delbrueckii . It can be improved by adding other supplements such as yeast extract or mineral salts. A medium containing CSL (10 g l ¡ 1 ) with 0.05 g l ¡ 1 FeSO 4 is an economically ef cient medium for lactic acid production by Lactobacillus delbrueckii NRRL B-445. REFERENCES Amrane, A. and Prigent, Y., 1994, Mathematical model for lactic acid production from lactose in batch culture: model development and simula- tion, J Chem Technol Biotechnol, 60: 241–246. Burgos-Rubio, C.N., Okos, M.R. and Wankat, P.C., 2000, Kinetic studies of the conversion of differen t substrates to lactic acid using lactobacillus bulgaricus, Biotech nol Prog, 16: 305–314. Chahal, S.P., 1991, Lactic acid, in Ullmann’s Encyclopedia of Industrial Chemistry (VCH, Berlin, Germany), Vol A15, pp 97–105. Table 5. Results obtained by regression analysis of lactic acid production and glucose consumption by Lactobacillus delbrueckii using CSL supplemented with other components of Mercier medium (all media include 90 g l ¡ 1 glucose). Lactic acid production Glucose consumption Medium a P 0 (g l ¡ 1 ) P m (g l ¡ 1 ) P r (h ¡ 1 ) r 2 F-test of probability Y p = s (g g ¡ 1 ) r 2 F-test of probability CSL 1.51 26.09 0.141 0.9967 0.9606 0.80 0.9987 0.9602 CSL ‡ peptone 3.80 72.64 0.150 0.9898 0.9309 0.81 0.9858 0.9956 CSL ‡ yeast extract 3.46 73.95 0.169 0.9890 0.9350 0.84 0.9942 0.9578 CSL ‡ citrate 3.72 45.59 0.048 0.9859 0.9298 0.62 0.9978 0.9129 CSL ‡ acetate 4.47 42.89 0.048 0.9720 0.9081 0.76 0.9930 0.9573 CSL ‡ K 2 HPO 4 2.02 27.89 0.111 0.9977 0.9377 0.71 0.9926 0.9137 CSL ‡ MgSO 4 2.47 22.83 0.078 0.9859 0.9291 0.77 0.9952 0.9492 CSL ‡ MnSO 4 4.11 38.14 0.051 0.9773 0.9036 0.67 0.9956 0.9675 CSL ‡ FeSO 4 4.78 60.38 0.041 0.9771 0.9232 0.74 0.9925 0.9176 a The CSL concentration was 10 g l ¡ 1 in all the experiments and the concentrations of the other nutrients were the same as used in Mercier medium. 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Te´llez-Luis, S.J., Moldes, A.B., Alonso, J.L. and Va ´ zquez, M., 2003, Optimization of lactic acid productio n by Lactobacillus delbrueckii through response surface methodology, J Food Sci, 37: 267–274. Thomas, K.C. and Ingledew, W.N., 1990, Fuel ethanol production: effects of free amino nitrogen on fermentation of very-high-gravity wheat mashes, Appl Environ Micro biol, 56: 2046–2050. Vick-Roy, T.B., 1985, Comprehensive Bio technology, Moo Young, M. (ed.) (Pergamon Press, Oxford, UK). Zabriskie, D.W., Armiger, W.B., Phillips, D.H. and Albano, P.A., 1980, Traders’ Guide to Fermentation Med ia Formulation (Traders Protein, Memphis, TN, USA). Zhou, Y., Dom‡ ´ nguez, J.M., Cao, N.J., Gong, C.S. and Tsao, G.T., 1999, Optimization of L-lactic acid production from glucose by Rhizopus oryzae ATCC 52 311, Appl Biochem Biotechnol, 77–79: 401–407. ACKNOWLEDGEMENTS The Authors are grateful to Xunta de Galicia for the  nancial support of this work (Project XUGA PGIDT00PXI38301PR). A grant from the PROMEP program of the Secretar‡ ´ a de Educacio´n Pu ´ blica (Me´xico) to author Te´llez-Luis is gratefully acknowledged. ADDRESS Correspondence concerning this paper should be addressed to Dr M. Va´zquez, A ´ rea de Tecnolog‡´a de los Alimentos, Escuela Polite´cnica Superior, Departamento de Qu‡ ´ mica Anal‡ ´ tica, Universidad de Santiago de Compostela, Campus de Lug o, 27002 Lugo, Spain. E-mail: vazquezm@lugo.usc.es The manuscript was received 7 May 2002 and accepted for publication after revision 30 April 2003. Trans IChemE, Vol 81, Part C, September 2003 256 TE ´ LLEZ-LUIS et al. . economically ef cient medium for lactic acid production by Lactobacillus delbrueckii NRRL B-445. REFERENCES Amrane, A. and Prigent, Y., 1994, Mathematical model for lactic acid production from lactose. to grow L. delbrueckii . The aim of t his study was to develop a low-cost nutrient medium based on CSL for lactic acid production by L. delbrueckii NRRL B-445. Additionally, lactic acid produc- tion. obtained by regression analysis of lactic acid p roduction and glucose consumption by Lactobacillus delbrueckii u sing different media (all media include 90 g l ¡ 1 glucose). Lactic acid production