Você está na página 1de 6

A media design program for lactic acid production coupled with extraction by electrodialysis

KiBeom Lee
a

a,b,*

Department of Environmental Science and Engineering, Gwangju Institute of Science and Technology (GIST), Gwangju, South Korea b The Wistar Institute, 3601 Spruce Street, Philadelphia, PA 19104-4268, USA Received 21 November 2003; received in revised form 12 November 2004; accepted 18 November 2004 Available online 20 January 2005

Abstract The aim of this study was to investigate industrial media for lactic acid fermentation to reduce the cost of nitrogen sources. Corn steep liquor (CSL) was successfully used at 5% (v/v) in batch fermentations. Use of soluble CSL improved the productivity approximately 20% with an advantage of clearer fermentation broth. Yeast extract (YE)-complemented CSL media further increased the productivity. It was found that 3.1 g L1 yeast extract and 5% CSL could be an eective substitute for 15 g L1 yeast extract in 10% glucose medium. Spent brewery yeast was also used as a sole nitrogen source equivalent to 5% CSL. Lactic acid was recovered by electrodialysis from the cell free broth. Depleted cell free broth supplemented with 5 g L1 of yeast extract performed reasonably in batch cultures. Reuse of the fermentation broth may reduce the cost of raw materials as well as minimize the fermentation wastes. 2005 Elsevier Ltd. All rights reserved.
Keywords: Lactic acid; Nitrogen sources; Broth reuse; Industrial media

1. Introduction Lactic acid has a wide range of applications in the food, pharmaceutical and cosmetics industries (Datta et al., 1995; Hujanen and Linko, 1996). In recent years, new applications, such as degradable plastics and coatings made from poly (lactic) acid, have the potential to greatly expand the market for lactic acid (Cheng et al., 1991; Goncalves et al., 1992). Up to date, approximately 50% of lactic acid has been produced by microbial fermentation, while the remainder is manufactured by chemical synthesis (VickRoy, 1985). Because products obtained by biological origin are preferred by food industry and consumers, microbial fermentation must be cost competitive with chemical synthesis. Lactic acid
Address: The Wistar Institute, 3601 Spruce Street, Philadelphia, PA 19104-4268, USA. Tel.: +1 215 898 0662; fax: +1 215 898 0664. E-mail address: klee@wistar.upenn.edu 0960-8524/$ - see front matter 2005 Elsevier Ltd. All rights reserved. doi:10.1016/j.biortech.2004.11.010
*

production complex requires a basal medium which results in increased production cost. The eciency and economics of the lactic acid fermentation is still a problem from several points of view and medium composition plays a vital role in the improvement of lactic acid production (Aeschlimann and von Stockar, 1990; Arasaratnan et al., 1996; Cheng et al., 1991; Heriban et al., 1993; Hujanen and Linko, 1996; Monteagudo et al., 1993). Current research eorts are focused on looking for eective nutritional sources and new fermentation techniques. It has been suggested that for lactic acid production, economical and industrial media be investigated to reduce related costs. Corn steep liquor (CSL) is a by-product of the corn wet-milling industry. Currently, CSL is evaporated to a 50% solids syrup and marketed primarily as an animal feed supplement in the cattle industry. CSL is a cost eective medium for fermentations and contains vitamins, minerals and carbohydrates (Formanek, 1998;

1506

K. Lee / Bioresource Technology 96 (2005) 15051510

Parekh et al., 1999). It is also used as a nutrient source for production of ethanol by yeast (Amartey and Jeries, 1994; Kadam and Newman, 1997; Sreenath and Jeries, 1996) and Escherichia coli (Lawford and Rousseau, 1996). However, CSL exhibits many disadvantages over yeast extract, such as increased fermentation time and relative diculty of product separation because of strong impurities in the broth. Korea has several large beer breweries and there is an abundant waste yeast products generated from this industry. Spent brewery yeast is currently used as an ingredient for animal feed. If spent brewery yeast can be eciently used as a raw material for the production of value-added products with commercial importance, then a considerable reduction in fermentation costs would be possible (Carvalheiro et al., 2004). Microbial fermentation requires the use of an ecient and economic downstream process to recover lactic acid from the fermentation broth. Depleted cell free broth is a by-product of lactic acid recovery from fermentation broth using microltration and electrodialysis. It consists of water, glucose, and small amounts of other nutrients. The disposal of depleted cell free broth presents an environmental problem, leading to research into depleted cell free broth utilization. Consequently, there is a need to examine depleted cell free broth utilization while still resuing the fermentation broth. The objective of this work was to study the production of lactic acid by a mixture of ve lactobacillus strains (LBM 5), in IK1 media supplemented with yeast extract and dierent nitrogen sources. The inuence of combined eects of yeast extract and CSL concentrations on lactic acid production was tested. Further investigation was the conversion of spent brewing yeast wastes to lactic acid without the use of a supplementing nitrogen source. Depleted cell free broth was used as a potential reuse of the fermentation broth supplemented with glucose and yeast extract, and nutrient salts.

on MRS-agar (Merck, NJ, USA) medium and subcultured fortnightly. 2.2. Preparation of inoculum The inoculum was developed in modied MRS medium (deMan et al., 1960) in which starch had been substituted by glucose. The medium composition per liter was as follows (Lee et al., 2001): (a) Bacto Protease Peptone 10 g, Bacto Beef Extract 10 g, Tween 80 1 g, K2HPO4 2 g, CH3COONa 3H2O 5 g, MgSO4 7H2O 0.1 g, MnSO4 H2O 0.05 g, ammonium citrate 2 g; and (b) glucose 20 g. Composition (a) and (b) were autoclaved separatively and aseptically mixed together before starting the cultivation. Seed culture was prepared by growing a single colony in a 25 mL of the previously described nutrient broth at 42 C overnight with 120 rpm agitation. The ask culture medium was incubated with 10% (v/v) of the seed culture. Cultures were incubated in 500 mL Erlenmeyer asks containing 100 mL of the medium above (120 rpm, pH 6.3, 42 C). 2.3. Fermentations in bioreactor All fermentations were performed under anaerobic conditions in a 3.7 L stirred tank fermenter (Bioengineering, Uppsala, Sweden). Temperature and agitation rate were controlled at 42 C and 120 rpm min1, respectively. The pH was maintained constant at 5.5 by automatic addition of 10 N NaOH solution. The total time of fermentation was approximately 4870 h. All experiments were performed at least in triplicate. The fermentation medium (IK1) was inoculated with 10% (v/v) of the ask culture. The medium composition per liter was as follows: (a) Bacto Yeast Extract 15 g, K2HPO4 0.5 g, KH2PO4 0.5 g, CH3COONa 3H2O 1 g, MgSO4 7H2O 0.5 g, MnSO4 H2O 0.05 g, ammonium citrate 1 g, FeSO4 7H2O 0.03 g; and (b) glucose 100120 g. Compositions (a) and (b) were autoclaved separatively, followed by aseptically mixing them together before starting the cultivation. Yeast extract of dierent amounts was added to IK1 medium to bring the nal concentration to 0, 2.5, 3.1, 4.2, 8.3, 15 g L1. To IK1 medium, CSL, various yeast extract and CSL, waste brewing yeast extract, and soluble CSL were added. Soluble CSL was washed thoroughly with distilled water and centrifuged at 4000 rpm (Marathon 10K, Fisher Scientic, Pittsburgh, PAUSA) for 30 min and the clear supernatant was collected. At the end of the fermentation periods, the lactic acid was recovered by electrodialysis (ED) from the cell free broth. Depleted cell free broth supplemented with glucose (100 g L1), organic nitrogen source (an initial yeast extract of 1/3), and nutrient salts performed in batch culture. The medium was sterilized at 121 C for 15 min.

2. Methods 2.1. Microorganism A mixture of ve lactobacillus strains (LBM 5) was used in this study. These strains were identied as L. delbrueckii sup. lactis (ATCC 12315), L. casei (NRRLB1445), L. delbrueckii (NRRL-B445), L. helveticus (NRRL-B1937), and L. casei (NRRL-B1922). The LBM 5 was obtained from the Argonne National Laboratory (Argonne, IL, USA). Strain LBM 5 is extensively known for its ability to produce high amounts of lactic acid in the culture (Tsai et al., 1995). This strain was stored in MRS broth medium (Merck, NJ, USA) with 10% glycerol at 80 C. The bacteria were maintained

K. Lee / Bioresource Technology 96 (2005) 15051510

1507

2.4. Lactic acid recovery from fermentation broth using electrodialysis The electrodialysis unit was supplied by Tokuyama Corp (TS-2-10), Japan. The ED stack was operated with ve cell pairs of cation and anion exchange Neosepta membranes (Tokuyama Soda, Tokuyama, Japan; Lee et al., 1998). The initial concentration of lactate in the fermentation broth used as a feeding solution was 80 100 g L1. Permeate and feed solutions were calculated with a ow rate of 1.681.72 L min1. The acid compartment for lactic acid recovery was lled with either 0.1% lactic acid or 0.1% NaOH, with 0.1% NaOH being used as the initial base compartment solution. In this study, electrodialysis was operated for 100 min with current of 812 A. Finally, depleted cell free broth that was obtained after electrodialysis, was used as a fermentation broth supplemented with glucose, yeast extract and nutrient salts. 2.5. Analysis Bacterial growth was monitored by spectrophotometric measurement at 560 nm. The concentration of glucose was measured enzymatically with a glucose assay kit (Sigma, USA). Lactic acid was analyzed by liquid chromatography (HPLC Waters, Milford, USA) equipped with a dierential refractive index detector (Waters, Milford, USA). The column used was an Aminex HPX-87H (Bio-Rad Co., USA) operated at 50 C, and the ow rate was 0.6 mL min1. The protein content was estimated by the bicinchoninic acid method (Smith et al., 1985). 2.6. Reproducibility All the experiments were independently carried out three times. The data are reported as the average values and standard deviations were obtained from these experiments.
Glucose consumption (g L-1)

140

Yeast extract 15 gL-1 CSL 3% CSL 5% CSL 7.5%

140

100

100

80

80

60

60

40

40

20

20

0 0 10 20 30 40 50 60

0 70

Time (hr)

Fig. 1. Glucose utilization and lactic acid production in batch fermentation with yeast extract 15 g L1 and various CSL concentrations. Mean SE values from triplicates are shown.

low lactic acid production were observed. This low yield in lactic acid production may be explained by the fact that lactic acid bacteria growth was limited by nitrogen limitation because CSL was decient (Charalampopoulos et al., 2002). It was shown that the lactic acid production was dierent at levels of CSL 3% and 5%. The yield in lactic acid production related to glucose consumption increased with an increase in CSL supplementation up to 5%, above this gave no further increase in this yield. The rate of glucose consumption was 1.18 g L1 h1 at CSL 3% as compared with 1.52 g L1 h1 at CSL 5%. Although there was an increase in the CSL up to 7.5%, the rate of consumption of glucose remained constant at 1.56 g L1 h1. Higher levels of CSL may have been inhibitory to the culture. This could be due to nutrient source limitation or the growth inhibition of cells by impurities remaining in CSL during lactic acid fermentation (Underwood et al., 2004). 3.2. Eect of CSL supplemented with yeast extract on lactic acid production

3. Results and discussion 3.1. Eect of corn steep liquor on lactic acid production Three dierent concentrations of CSL (3%, 5%, and 7.5%) were tested in comparison with a standard media containing yeast extract 15 g L1. As shown in Fig. 1, the glucose consumption rate increased rapidly and thus high level of lactic acid was produced in the media containing yeast extract. Glucose used at a concentration of 120 g L1 in media was almost consumed by the cells in 24 h and completely utilized in 40 h. CSL was added to the fermentation media at 3%, 5%, and 7% and fermentation was allowed without supplementing with any other nutrients. Incomplete glucose utilization and In a previous work, the eect of dierent nitrogen sources on the glucose consumption and the lactic acid production was shown. Because the expensive yeast extract used for lactic acid production can not be used in high volume in fermentor, use of CSL as a source of additional nitrogen was examined. Although CSL is high in protein and is an optimal source of micro-nutrients including vitamins and metal salts (Formanek, 1998), it suers from some drawbacks. For example, impurities must be removed from the product stream in order to obtain high-purity lactic acid, and this could add to the purication cost. It is therefore desirable to use as little supplement as possible, while still obtaining high lactic acid yield within a reasonable fermentation

Lactic acid production (g L-1)

120

120

1508

K. Lee / Bioresource Technology 96 (2005) 15051510


-1 Yeast extract 2.5 g L + CSL 3% (1:6) -1 Yeast extract 3.1 g L + CSL 5% (1:8) -1 Yeast extract 8.3 g L + CSL 5% (1:3) -1 Yeast extract 4.2 g L + CSL 5% (1:3)

140

140

Glucose cons umption (g L -1 )

120

120

140

140

100

100

Glucose consumption (g L-1)

120

120

Lactic acid production (g L-1)

80

80

100

100

60

60

80

80

40

40

60

60

20

20

40

40

0 0 8 16 24 32 40 48

20

20

Time (hr)
0 0 8 16 24 32 40 48 0

Time (hr)

Fig. 2. Glucose utilization and lactic acid production in fermentation using yeast extract supplemented CSL media [ratio of yeast extract to CSL (w/w)]. Mean SE values from triplicates are shown.

Fig. 3. Glucose utilization and lactic acid production in batch fermentation using soluble CSL 5%. Mean SE values from triplicates are shown.

time. At optimization of CSL 5% level, the yeast extract supplementation is an attractive approach since it can provide a positive eect for an optimal fermentation. When CSL 5% was used in combination with YE levels ranging from 2.5 g L1 to 8.33 g L1 (Fig. 2), the results indicated that the glucose consumption and lactic acid production rates with YE 8.33 g L1 at CSL 5% (YE-CSL ratios: 1:3) were similar to those obtained with YE 3.1 g L1 at CSL 5% (YE-CSL ratios: 1:8), conrming that a considerable benecial eect of YE was not seen in lactic acid production yield with the addition of high level YE. With YE 2.5 g L1 and lower CSL concentration (3%), equivalent to YE-CSL ratios (1:6), despite better results were found than at a only CSL 5% (Fig. 1), lower glucose consumption and lactic acid production rates were measured than other media containing dierent CSL-YE ratios. In the current study, the highest yield in lactic acid production was reached in media with YE 3.1 g L1 at CSL 5% (YE-CSL ratios: 1:8). This value is comparable to that achieved with CSL 5%, showing that the time taken for fermentation decreased from 68 h (CSL 5%) to 48 h (YE 3.1 g L1CSL 5%). Cornelius et al. (1996) found that a supplementation of YE and CSL ratios (1:3) was enough for a high-performance lactic acid production by Lactobacillus plantarum ATCC 8014. However, in this work, better lactic acid production yield was observed for higher CSL-YE ratio (1:8). Therefore, using YE 3.1 g L1 with CSL 5% in a 1:8 ratio would reduce the cost of production. 3.3. Comparison of fermentation between CSL and soluble CSL A potential disadvantage of CSL supplementation is the concentration of impurities remaining after fermen-

tation which must be removed in order to produce high purity lactic acid. Removal of the suspended CSL particles results in even higher lactic acid production and yields. There have been reports of successful attempts to produce ethanol by using soluble CSL (Lawford and Rousseau, 1997). In the current work, the suspended CSL particles were removed by centrifugation and a supplementation of soluble CSL improved the ability of ethanol production up to 1.33-fold. On the basis of these observations, CSL was centrifuged and the supernatant was collected for fermentation. As seen in Fig. 3, when compared with CSL 5% (1.60 g L1 h1), the soluble CSL displayed an increased lactic acid productivity of approximately 24% (1.98 g L1 h1). The results conrm that soluble CSL is an eective and economic supplement for lactic acid bacteria medium while increasing lactic acid production and decreasing separation costs. 3.4. Lactic acid fermentation using brewing yeast wastes Brewing yeast wastes is a by-product of beer fermentation. The disposal of excess brewing yeast wastes can be detrimental to environmental quality, leading to research into brewing yeast wastes utilization. One such possibility is the production of lactic acid from brewing yeast wastes by fermentation with lactic acid bacteria. To determine total protein concentration, 15 g of brewing yeast wastes were reduced to powder using a hammer mill (Falling Number AB, Sweden) tted with a sieve of 0.5 mm aperture size, and powdered brewing yeast wastes were washed with 1 L of deionized water and then protein content was measured by the bicinchoninic acid method (Smith et al., 1985). In comparing the pure yeast extract (14 g L1) to brewing yeast wastes (12.5 g L1), they were nearly equal in terms of the total protein concentration. The time course of 68 h fermen-

Lactic aci d production (g L -1 )

K. Lee / Bioresource Technology 96 (2005) 15051510


140 140
Glucose concentration of FM Glucose concentration of RM Lactic acid concentration of FM Lactic acid concentration of RM Protein concentration of RM Protein concentration of FM

1509

Glucose consumption (g L -1)

Lactic acid production (g L -1)

120

120

Glucose and lactic acid concentration (g L-1)

120

32 30

100

100

80

80

100 28 80 26 60 24 22 40 20 20 18 0 0 10 20 30 40 50 16

60

60

40

40

20

20

0 0 10 20 30 40 50 60

0 70

Time (hr)

Fig. 4. Glucose consumption and lactic acid production with waste brewing yeast. Mean SE values from triplicates are shown.

Time (hr)

tation and the glucose consumption and lactic acid production rate with brewing yeast wastes were 1.40 g L1 h1 and 1.55 g L1 h1 (Fig. 4). By comparison with batch culture on CSL 5% medium, the glucose consumption was lower than that in CSL 5% medium (1.52 g L1 h1). Nevertheless, brewing yeast wastes yielded lactic acid similar to the CSL 5% medium (1.60 g L1 h1). Even though the protein concentration in brewing yeast wastes is high, lactic acid productivity by brewing yeast wastes was very low compared to pure yeast extract. This might be due to a limitation of growth resulting from a decrease in growth factors because of various organic sources and salts (Phadwal and Singh, 2003; Tamara et al., 2002). Yet, the possibility of an alternative nitrogen source from brewing yeast wastes could also be considered when an impurity must be eectively removed from the brewing yeast wastes. 3.5. Eect of reused fermentation broth on lactic acid production The pure media contained 100 g of glucose, 15 g of yeast extract, and 2930 g of initial protein concentration (g L1). Using the pure media, 48 h culture resulted in complete glucose depletion and protein concentration decreased to 16.7 g L1. Prior to recovery of lactic acid from fermentation broth, microorganisms, particulate matter and higher molecular weight proteins were removed using a microltration membrane. From 4.2 L fermentation broth, the lactic acid rich fermentation broth treated in a microltration membrane was 3.8 L. It was found that more than 97% of lactic acid was recovered from 3 L fermentation broth by electrodialysis and nal fermentation broth wastes were approximately 1.9 L. The concentration of protein in the fermentation broth wastes was 1819 g L1. A test was performed to assess which levels of yeast extract supplement added to depleted cell free broth were potentially

Fig. 5. Glucose, protein utilization and lactic acid production in batch fermentation with reuse and fresh medium (RM: reused medium, FM: fresh medium). Mean SE values from triplicates are shown.

important to inuence growth rate (l). When the strain was cultivated using a 200 mL shake-ask for 40 h on a pure medium with 15 g L1 YE, growth rate was 0.166 h1. By adding the depleted cell free broth with 0, 2, 5, 10, and 15 g L1 YE, growth rates varied. The highest growth rate was obtained with 5 g L1 YE (0.161 h1). The growth rates for 0, 2, and 10 g L1 YE were 0.092, 0.149, 0.158 h1, whereas lower value of 0.147 h1 was estimated for 15 g L1 YE. Therefore, the optimum supplementation of YE to the depleted cell free broth was 5 g L1. The fermentation results were compared in the pure and reused medium (Fig. 5). The level of initial protein concentration in reused media was 27 g L1, which was lower than that of nal protein concentration in pure media (30 g L1). However, the level of nal protein concentration in reused media was 18.8 g L1, which was higher than that of nal protein concentration in pure media (16.7 g L1). Therefore, the reuse of the fermentation broth supplemented with yeast extract resulted in rather low protein consumption. When the lactic acid production was compared for media with reused fermentation and pure broth, a similar result was observed. With the reused fermentation media, 2.18 g L1 was obtained, whereas 2.22 g L1 was obtained with the pure media. These observations showed that high lactic acid productivity could be obtained using reused fermentation broth with supplementation of a minimal level of nutrients.

4. Conclusions Based on this analysis, it was apparent that high lactic acid productivity could be obtained under culture condition utilizing 5% CSL. Incorporation of soluble CSL resulted in 1.24-fold higher production of lactic

Protein concentration (g L-1)

1510

K. Lee / Bioresource Technology 96 (2005) 15051510 Heriban, V., Stikey, V., Sturdik, E., Matus, P., 1993. Nutrition and broth alterations in the lactic acid fermentation. Acta Biotechnol. 35 (3), 283288. Hujanen, M., Linko, Y.Y., 1996. Eect of temperature and various nitrogen sources of L(+)-lactic acid production by Lactobacillus casei. Appl. Microbiol. Biotechnol. 45, 307313. Kadam, K.L., Newman, M.M., 1997. Development of a low-cost fermentation medium for ethanol production biomass. Appl. Microbiol. Biotechnol. 47, 625629. Lawford, H.G., Rousseau, J.D., 1996. Studies on nutrient requirements and cost-eective supplements for ethanol production by recombinant E. coli. Appl. Biochem. Biotechnol. 5758, 307326. Lawford, H.G., Rousseau, J.D., 1997. Corn steep liquor as a costeective nutrition adjunct in high performance Zymomonas ethanol fermentations. Appl. Biochem. Biotechnol. 6365, 287 304. Lee, E.G., Moon, S.H., Chang, Y.K., Yoo, I.K., Chang, H.N., 1998. Lactic acid recovery using two-stage electrodialysis and its modeling. J. Membr. Sci. 145, 5366. Lee, K.B., Lee, J.W., Kim, Y.H., Moon, S.H., Park, Y.H., 2001. Unique properties of four lactobacilli in amino acid production and symbiotic mixed culture for lactic acid biosynthesis. Curr. Microb. 43, 383390. Monteagudo, J.M., Rincon, J., Rodriguez, L., Fuertes, J., Moya, A., 1993. Determination of the best nutrient medium for the production of L -lactic acid from best molasses a statistical approach. Acta Biotechnol. 13, 103110. Parekh, M., Formanek, J., Blascheck, H.P., 1999. Pilot-scale production of butanol by Clostridium beijerinckii BA101 using a low-cost corn steep water based fermentation medium. Appl. Microbiol. Biotechnol. 51, 152157. Phadwal, K., Singh, P.K., 2003. Eect of nutrient depletion on bcarotene and glycerol accumulation in two strains of Dunaliella sp. Bioresource Technol. 90, 5558. Smith, P.K., Krohn, R., Hermanson, E.K., 1985. Measurement of protein using bicinchoninic acid. Anal. Biochem. 150, 7685. Sreenath, H.K., Jeries, T.W., 1996. Eect of corn steep liquor on fermentation of mixed sugars by Candida shehatae FPL-702. Appl. Biochem. Biotechnol. 5758, 551561. Tamara, H., Alexandra, S., Margot, B., Andrea, V., Uwe, V., Erhard, B., 2002. High-salinity-induced iron limitation in Bacillus subtilis. J. Bacteriol. 184, 706717. Tsai, S.P., Moon, S.H., Coleman, R., 1995. US Patent 5,464,760., November 7. VickRoy, T.B., 1985. Lactic acid. In: Blanch, H.W., Drew, S., Wang, D.I.C. (Eds.), Comprehensive Biotechnology, vol. 3. Pergamon Press, Oxford, pp. 761776. Underwood, S.A., Buszko, M.L., Shanmugam, K.T., Ingram, L.O., 2004. Lack of protective osmolytes limits nal cell density and volumetric productivity of ethanologenic, Escherichia coli k011 during xylose fermentation. Appl. Environ. Microbiol. 70 (5), 27342740.

acid in ve mixed lactobacillus strain, LBM 5. Considering CSL supplemented with yeast extract, the optimal YE: CSL ratio of medium should be 1:8. The fermentation yield at brewing yeast wastes was similar to that at CSL 5%. To decrease the wastes treatment cost by reusing remaining nutrients, when an initial yeast extract of one third in pure media was supplemented to fermentation broth, a satisfactory level of lactic acid production could be obtained. References
Aeschlimann, A., von Stockar, V., 1990. The eect of yeast extract supplementation on the production of lactic acid from whey permeable by Lactobacillus helveticus. Appl. Microbiol. Biotechnol. 32, 398402. Amartey, S., Jeries, T.W., 1994. Comparison of corn steep liquor with other nutrients in the fermentation of D -xylose by Pichia stipitis CBS 6054. Biotechnol. Lett. 16, 211214. Arasaratnan, V., Senthuran, A., Balasubaramaniam, K., 1996. Supplementation of whey with glucose and dierent nitrogen sources for lactic acid production by Lactobacillus delbrueckii. Enzyme Microb. Technol. 19, 482486. Carvalheiro, F., Duarte, L.C., Medeiros, R., Girio, F.M., 2004. Optimization of brewerys Spent grain dilute-acid hydrolysis for production of pentose-rich culture media. Appl. Biochem. Biotechnol. 113116, 10591072. Charalampopoulos, D., Pandiella, S.S., Webb, C., 2002. Growth studies of potentially probiotic lactic acid bacteria in cereal-based substrates. J. Appl. Microbiol. 92 (5), 851859. Cheng, P., Muller, R.E., Jaeger, S., Bajpai, R., Iannoti, E.L., 1991. Lactic acid production from enzyme-thinned corn starch using Lactobacillus amylovorus. J. Ind. Microbiol. 7, 2734. Cornelius, C., Erpicum, Th., Jacques, Ph., Thonart, Ph., 1996. Comparison of fermentation industrial components such as corn steep and yeast extract for lactic bacteria production. Med. Fac. Landbouww. Univ. Gent. 61/4a, 14611463. Datta, R., Tsai, S.P., Bonsignore, P., Moon, S.H., Frank, J.R., 1995. Technological and economic potential of poly(lactic acid) and lactic acid derivatives. FEMS. Microbiol. Rev. 16, 221231. deMan, J.C., Rogosa, M., Sharpe, M.E., 1960. A medium for the cultivation of lactobacilli. J. Appl. Bacteriol. 23, 30135. Formanek, J.A., 1998. Genetic manipulation and characterization of solvent-producing clostridia. Ph.D. Thesis. University of Illinois, IL, USA. Goncalves, L.M.D., Barreto, M.T.O., Xavier, A.M.B., Carrondo, M.J.T., Klein, J., 1992. Inert supports for lactic acid fermentation a technological assessment. Appl. Microbiol. Biotechnol. 38, 305 311.