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

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

# Institution of Chemical Engineers www.ingentaselect.com=titles=09603085.htm Trans IChemE, Vol 81, Part C, September 2003

ALTERNATIVE MEDIA FOR LACTIC ACID PRODUCTION

S J TE´LLEZ-LUIS 1,2 , A B MOLDES 2 , M VA´ZQUEZ 1,3 and J L ALONSO 2

1Department of Food Science and Technology, UAM Reynosa-Aztla´n, Universidad Autonoma de Tamaulipas, Me´xico

2Department of Chemical Engineering, University of Vigo (Facultad de Ourense), Vigo, Spain

3A´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 liquor (CSL) was developed for lactic acid

pro-duction by Lactobacillus delbrueckii NRRL B-445 Starting from a medium containing

90 g l¡1glucose and 20 g l¡1CSL as a sole nutrient source, 70.7 g l¡1lactic acid was obtained

with an economic efŽ ciency of 98 g lactic acid per „ nutrient Other media, made with CSL

and each individual component (yeast extract, peptone, sodium acetate, sodium citrate,

K2HPO4, MgSO4¢7H2O, MnSO4¢H2O or FeSO4¢7H2O) of a general (Mercier) lactobacilli

medium were also assayed The highest economical efŽ ciency (134 g lactic acid per „ nutrient)

was obtained supplementing 10 g l¡1CSL with 0.05 g l¡1FeSO4¢7H2O 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, nutrients 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 acidulant,  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 application 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 lactobacilli strains and 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 carbon, lactic acid can occur in two

optically active enantiomers,LandD(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 use sugars via different pathways

resulting in homo-, hetero- or mixed acid fermentations

Homofermentation gives only lactic acid 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 phosphoketolase 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 bacterium that produces mainlyL-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 large-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 concentrations 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

Corn steep liquor (CSL) is a low-cost nutritional medium

employed successfully in the production of ethanol 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 this 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 consumpproduc-tion 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 obtained 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 K2HPO4, 0.58 g l¡1 MgSO4¢7H2O,

0.12 g l¡1MnSO4¢H2O, 0.05 g l¡1FeSO4¢7H2O 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¡1dry cells Inocula were 5 ml The experiments

were carried out in 250 ml Erlenmeyer  asks with a Ž nal

volume of 100 ml using 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 constant around 6 due to the lactic acid formed was

neutralized by the present of calcium carbonate Samples

(2 ml) were taken at random time intervals and centrifuged

at 16,000g for 3 min The supernatants were used

immedi-ately for various analyses

Analytical Methods Glucose, lactic acid and acetic 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 H2SO4 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 given Linear and non-linear regression

analyses of experimental data were performed using

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¡1CSL, 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¡1did not supply the required nutrients for the metabolism of

L delbrueckii Additionally, acetic acid was quantiŽ 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 Ž nal glucose concen-tration of 12.0 g l¡1was observed in the experiments carried out with medium containing 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 (Yp=s), product efŽ ciency (Ep=s) and

economic efŽ ciency (Ep=„) after 98 h of fermentation Product yield was deŽ ned as grams lactic acid produced per gram glucose consumed, product efŽ ciency as grams lactic acid produced per gram initial glucose and economic

Table 1.Prices of nutrients used in experiments.

efŽ ciency as grams lactic acid produced per cost unit of

nutrients („) As it can be observed, Ep=sdecreased with the

decrease of CSL concentration However, Yp=s was

main-tained around 0.9 g g¡1using the Mercier medium or media

containing 20 or 10 g l¡1CSL

It is interesting to select the cheapest media that allow the

highest lactic acid concentration to be obtained The Ep=„ is

an adequate parameter to compare media from an

econom-ical point of view The parameter Ep=„ showed that it was

more proŽ table to use a medium with 20 g l¡1CSL than the

Mercier medium because using 20 g l¡1CSL, 98 g of lactic

acid were produced per euro of nutrients 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 equations A mathematical model was adopted from another study to describe the fermentative

production of lactic acid (Mercier et al., 1992):

dP

dt ˆ PrP 1 ¡P P

m

³ ´

(1)

where t is time, P is lactic acid concentration, Pm is

maximum concentration of lactic acid, and Pr is the ratio

between the initial volumetric rate of product formation (rp)

and the initial product concentration P0 Equation (1) can be directly solved to give the following expression:

P ˆ P0PmePrt

From the series of experimental data for lactic acid concentration during fermentation, the model parameters

P0, Pm and Pr can be 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 coefŽ cient r2showed 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¡1for the Mercier medium, 66.6 g l¡1for 20 g CSL

l¡1medium and 25.9 g l¡1for 10 g CSL l¡1medium 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 corresponding 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 production 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 ) (g gYp=s¡1 ) (g gEp=s¡1 ) (g „Ep=„¡1 )

Table 3 Results obtained by regression analysis of lactic acid production and glucose consumption by Lactobacillus delbrueckii using different media (all

media include 90 g l ¡1 glucose).

probability

correlated with the CSL concentration by mean of empirical

equations P0, Pmand Prwere related to the CSL

concentra-tion given by equaconcentra-tions (3)–(5), respectively:

P0ˆ 0:2371Ccsl¡ 0:1555 (3)

Pmˆ 3:3189Ccsl¡ 1:9735 (4)

Prˆ 0:3788 ¢ C¡0:4733

The coefŽ cient r2(0.9710) for P0, (0.9858) for Pmand

(0.9749) for PrconŽ rmed that the empirical equations Ž 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 concenconcen-trations and time in the

range studied (0–98 h and 1–20 g l¡1CSL) Figure 2 shows

how the generalized model predicts the dependence of lactic

acid concentration on different CSL concentrations and time

using the model parameters This kind of surface response

allows the selection of different conditions in order to

achieve the same results

The consumptionof glucose by L delbrueckiican be given

by the following equation (obtained from the Yp=sdeŽ nition):

S ˆ S0¡Y1

p=s(P ¡ P0) (6)

where Yp=s, P and P0were deŽ ned above, S is the glucose

concentration (g l¡1) and S0is the initial glucose

concentra-tion Using the series of experimental data concerning

glucose concentration=time and the regression parameters

of equation (2), the model parameter Ys=pcan be calculated for each fermentation medium by non-linear regression using the least-squares method Table 3 lists the numerical

values of Yp=s and statistical parameters obtained for the glucose consumption and Figure 1b shows the experimental and predicted data for these fermentations The parameter

Yp=svaried with Ccslaccording to the following equation: 1

Yp=sˆ 4:163 ¢ Ccsl0:5072 (7)

The statistical parameter r2for the empirical equation (7) was signiŽ 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 remained in the medium when less than 10 g l¡1CSL

is used Both models would be very useful for optimization

Fermentation of Supplemented CSL Media

In order to increase the lactic acid production and the economic efŽ 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 liquor 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 ) Yp=s (g g ¡1 ) Ep=s (g g ¡1 )

Ep=„ (g lactic acid per „ nutrients)

Mercier medium at a time, in the same concentrations.

Table 4 lists the medium used and results for lactic acid

concentration obtained and Yp=s, Ep=sand Ep=„ 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¡1CSL alone as control and

10 g l¡1 CSL supplemented with peptone or yeast extract

(the main nutritional components of the Mercier medium)

Both exhibited a signiŽ cant effect The highest lactic acid

concentration (77.64 g l¡1) was obtained by supplementing

10 g l¡1 CSL with 5 g l¡1yeast extract Similar lactic acid

concentration (76.71 g l¡1) was also obtained by

supple-menting with 10 g l¡1peptone These values compare very

well with those achieved with the Mercier medium and the

20 g l¡1CSL medium The Ep=swas also higher in the above

two cases Although the glucose consumed was different,

the Yp=s was similar or slight lower than that without

supplementation The values of Yp=s compare well with

those reported using other microorganisms like Rhizopus

oryzae (Zhou et al., 1999) The Ep=„ 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 Ep=„ values showed that it

is most economically interesting to supplement CSL with

yeast extract than with peptone The importance 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 corresponding 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 MgSO4, MnSO4or FeSO4.

vitamins The importance of yeast extract to Lactobacilli has

been reported (Hujanem and Linko, 1996)

Figure 5 shows experimental results for the fermentation

of 10 g l¡1CSL 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 signiŽ cantly the value of Yp=sbut the Ep=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 Ep=„ was 111 g lactic acid per

„ nutrient for the fermentation of the 10 g l¡1CSL medium

supplemented with 5 g l¡1acetate The Ep=„ of media 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¡1CSL supplemented with MgSO4, MnSO4

and FeSO4, the mineral sources of the Mercier medium The

lactic acid concentration obtained by supplementing CSL

with MgSO4remained 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 MgSO4had no

effect on lactic acid production This must have occurred

because CSL contains 1.5% Mg2‡on a dry basis (Zabriskie

et al., 1980) Using MgSO4 as a component of the CSL

medium decreased the Ep=„ of the process

Better results were obtained by supplementing with

MnSO4 or FeSO4 (Table 4) Although the highest lactic

acid concentration was obtained by supplementing with

yeast extract, the Ep=„ showed that the better supplement

is 0.05 g l¡1 FeSO4 because 134 g lactic acid per „ of

nutrients was obtained This value is 75% higher than the

Ep=„ obtained with 10 g l¡1 CSL, 36% higher than to

20 g l¡1 CSL medium and 168% higher than the Ep=„

value when the Mercier medium was used

Mathematical Modelling of Fermentation with CSL

Supplemented with Nutritional Source

The experimental lactic acid production and glucose

concentration data were examined using equation (2) and

equation (6) The kinetic parameters of P0, Pmand Prwere calculated for each fermentation medium by non-linear regression The results are shown in Table 5 together with

the statistical parameters The coefŽ cient r2showed that all the equations obtained were well Ž tted and Figures 4–6 conŽ 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 P0obtained are

higher than the values reported by others (Parajo´ 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 Yp=s and statistical parameters for the consumption of glucose The coefŽ cient showed a good agreement between experimental and predicted data Figures 4–6 also display comparison between experimental and predicted data The values of

Yp=sare 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 economic efŽ 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 10 g l¡1corn steep liquor is more economically efŽ 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¡1FeSO4is an economically efŽ 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 different substrates to lactic acid using lactobacillus bulgaricus , Biotechnol 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).

probability

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.

Cheilas, T., Stoupis, T., Christakopoulos, P., Katapodis, P., Mamma, D.,

Hatzinikolaou, D.G., Kekos, D and Macris, B.J., 2000, Hemicellulolytic

activity of Fusarium oxysporum grown on sugar beet pulp Production

of extracellular arabinase, Proc Biochem, 35: 557–561.

Hofvendahl, K and Hahn-Hagerdal, B., 2000, Factors affecting the

fermen-tative lactic acid production from renewable resources, Enzyme Microb

Technol, 26: 87–107.

Hujanen, M and Linko, Y.Y., 1996, Effect of temperature and various

nitrogen sources on L ‡ lactic acid production by Lactobacillus casei,

Appl Microbiol Biotechnol, 45: 307–313.

Hull, S.R., Peters, E., Cox, C., Montgomery, R., 1996, Composition of

corn steep water during experimental steeping, J Agric Food Chem, 44:

3521–3527.

Kadam, K.L and Newman, M.N., 1997, Development of a low-cost

fermentation medium for ethanol production from biomass, Appl

Microbiol Biotechnol, 47: 625–629.

Lee, P.C., Lee, W.G., Lee, S.Y., Chang, H.N and Chang, Y.K., 2000,

Fermentative production of succinic acid from glucose and corn steep

liquor by Anaerobiospirillum succiniciproducens, Biotechnol Bioprocess

Eng, 5: 379–381.

Mercier, P., Yerushalmi, L., Rouleau, D and Dochain, D., 1992, Kinetics

of lactic acid fermentation on glucose and corn by Lactobacillus

amylophilus , J Chem Tech Biotechnol, 55: 111–121.

Miller, T.L and Churchill, B.W., 1986, Substrates for large-scale

fermenta-tions, in Manual of Industrial Microbiology and Biotechnology,

Demain, A.L and Solomon, L.A (eds) (American Society for

Micro-biology, Washington, DC, USA).

Nancib, N., Nancib, A., Boudjelal, A., Benslimane, C., Blanchard, F.

and Boudrant, J., 2001, The effect of supplementation by

different nitrogen sources on the production of lactic acid from

data juice by Lactobacillus casei subsp rhamnosus, Biores Technol,

78(2): 149–153.

Ozen, S and Ozilgen, M., 1992, Effects of substrate concentration on

growth and lactic acid production by mixed cultures of Lactobacillus

bulgaricus and Streptococcus thermophillus, J Chem Technol Biotechnol,

54: 57–61.

Parajo´, J.C., Alonso, J.L and Santos, V., 1996, Lactic acid from wood, Proc

Biochem, 31: 271–280.

Silveira, M.N., Wisbeck, E., Hoch, I and Jonas, R., 2001, Production of glucose-fructose oxidoreductase and ethanol by Zimomonas mobilis ATCC29191 in medium containing corn steep liquour as a source of

vitamins, Appl Microbiol Biotechnol, 55: 442–445.

Te´llez-Luis, S.J., Moldes, A.B., Alonso, J.L and Va´zquez, M., 2003,

Optimization of lactic acid production 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 Microbiol, 56: 2046–2050.

Vick-Roy, T.B., 1985, Comprehensive Biotechnology, 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 Media 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 52311, Appl Biochem Biotechnol, 77–79: 401–407.

ACKNOWLEDGEMENTS

The Authors are grateful to Xunta de Galicia for the Ž 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 Lugo, 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.