ELSEVIER

Lactic acid production from deproteinized whey

by mixed cultures of free and coimmobilized
Lactobacillus casei and Lactococcus Lactis cells
using fed batch culture
T. Roukas and P. Kotzekidou
Department of Food Science and Technology, Aristotle University of Thessaloniki, Thessaloniki, Greece
The production of lactic acid from deproteinized whey by mixed cultures of free and coimmobilized Lactobacillus casei and
Lactococcus lactis cells in batch and fed batch culture was investigated. Fed batch culture proved to be a better fermentation
system for the production of lactic acid than batch culture. The maximum lactic acid concentration (46 g/h) in fed batch
culture was obtained with both free cells mixture and coimmobilized cells at a substrate concentration of 100 g 1-l and a
feeding rate of 250 ml/h. In repeated fed batch culture, coimmobilized L. casei and L. lactis cells gave a higher overall lactic
acid concentration compared with the free cells mixture. The coimmobilized L. casei and L. lactis cells in Ca-alginate beads
retained their ability to produce lactic acid for 20 days. 0 1998 Elsevier Science Inc.
Keywords: Lactic acid: deproteinized whey; mixed cultures: fed batch culture

Introduction
Among the many carbohydrate materials used for the production of lactic acid, cheese whey lactose deserves special
consideration. Cheese whey is a clean, wholesome, abundant food-grade material and a potential environmental pollutant. It is
the product separated from milk during cheesemaking and consists of water, lactose (4-5%) proteins, vitamins, and mineral salts.
Lactic acid is commercially produced by fermentation of corn sugars, molasses, and whey with homofermentative lactic acid
bacteria. It is used in the pharmaceutical, chemical, and food industries primarily as an acidulant and preservative.’
The production of lactic acid from whey permeate by free and immobilized lactic acid bacteria cells using batch and
continuous culture has been reported. (cite 2-8) Complex mixed cultures have also been used in several fermentation processes,
e.g., the production of ethanol, aspartic acid, and succinic acid from glucose; however, mixed cultures have not been used yet on
an industrial scale for the production of chemicals, because it is difficult to establish optimum culture conditions for both strains
for parameters such as nutrient supply, temperature, oxygen demand, pH, etc. Only a few reports concern the production of lactic
acid from starch, nonfat dry milk, and whey permeate by mixed cultures in batch fermentation. (cite 9-10)
Fed batch culture is a batch culture which is fed continuously or sequentially with substrate without the removal of fermentation
broth. It is widely used for the production of microbial biomass, ethanol, organic acids, antibiotics, vita- mins, enzymes, and
other compounds. When a portion of the fermentation broth is withdrawn at intervals and the residual part of the culture is used
as an inoculum for the next fed batch culture, the system is operated as a repeated fed batch culture. In addition to increased
productivity, a repeated fed batch culture has the advantage of not requiring new inocula for each consecutive fed batch and the
contamination of the medium is also lower than in the continuous culture; thus, repeated fed batch culture is considered one of
the most useful systems for the economic lactic acid production. The production of lactic acid from deproteinized whey by mixed
cultures using fed batch and repeated fed batch culture has not been investigated.
The aim of this investigation was to examine the potential of fed batch and repeated fed batch culture as fermentation systems for
lactic acid production from deproteinized whey by mixed cultures of free and coimmobilized Lactobacillus casei and
Lactococcus lactis cells.

Materials and methods

Microorganisms and culture conditions
The organisms used in the experiments were L. casei SU No 22 obtained from Applied Microbiology Division, University of
Strathclyde, Glasgow, UK and L. lactis WS 1042 obtained from Institut fur Mikrobiologie, Technische Universitat Munchen,
Freising, Germany. They were propagated in MRS broth (Merck, Darmstadt, Germany, Cat. No. 10661) and Ml7 broth (Merck,
Cat. No. 15029) at 30°C for 24-48 h. The inoculum was obtained from cultures in the logarithmic phase of growth cultivated in
200 ml MRS broth or Ml7 medium. The pH of the broth was adjusted to 5.5 with 1 N NaOH.

Immobilization of cells
MRS broth (200 ml) and an equal volume of Ml7 broth containing 1.4 X 10´10 cells/ml of L. casei or 1.2 X 10´10 cells/ml of L.
lactis were dilluted with 200 ml sterile-distilled water. The mixed culture of both strains was mixed with 800 ml of a 5% sterile
alginic acid sodium salt solution (Sigma, St. Louis, MO, A-2033). The mixture was extruded drop by drop with a peristaltic
pump into a sterile 2% CaCl2 solution at room temperature while stirring it continuously. The beads (2-3 mm diameter) were
hardened in CaCl2 solution for 2 h. The particles were washed with sterile physiological saline solution to remove excess calcium
ions and unentrapped cells.

Treatment of whey
Cheese whey was obtained from a local feta cheese plant. It contained 5% (w/v) lactose and had a pH of 5.0. Protein precipitation
was induced by heating the whey at 90°C for 20 min. Precipitated proteins were removed by centrifugation at 4,000 g for 15 min.
The supernatant (5% lactose) was concentrated at 50°C under vacuum to obtain 7.5, 10, and 15% lactose. The pH of the solution
was adjusted to 6.0 with 5 N NaOH and the medium was sterilized at 121°C for 20 min. After cooling, the whey was
supplemented with 0.4% yeast extract, 0.02% peptone from casein, 0.04% MgSO, 7H2O, and 0.007% MnSO, 4H2O (the
solutions of nutrients were sterilized separately). Samples of whey prepared in this way (production medium) were used for the
production of lactic acid by free and coimmobilized L. casei and L. lactis cells.

Fermentation conditions
Batch culture. The fermentation was performed in a 9L stirred tank fermentor with a working volume of 6 L. The fermentor was
built in our department. It consisted of a glass vessel with stainless steel endplates and three equally spaced vertical baffles.
Agitation was provided by a six-flat-blade impeller (diameter 6 cm) located 4 cm above the bottom of the vessel. The fermentor
was sterilized at 121 ºC for 15 min. After cooling, 6L of production medium containing 50 g/L lactose was added into the
fermentor. The medium was inoculated with 200 ml inoculum of L. casei or L. lactis to give a final concentration of 4.6 X 10´8
cells/ml or 4.0 X 10´8 cells/ml of each culture, respectively. In the case of the mixed culture, the medium was inoculated with
400 ml inoculum.
The fermentor was incubated at 32°C in a thermostatized chamber. The impeller speed was 300 rpm. The pH of the medium was
maintained at 6.0 +/- 0.3 by addition of 5 N NaOH.
Fed batch culture. Mixed cultures (400 ml) of L. casei and L. lactis or 1 kg of Ca-alginate beads with coimmobilized cells of the
microorganisms (2.5 x10^9 cells/g beads of L. casei and 2.2 x l0^9 cells/g beads of L. lactis) were mixed with 3 L of production
medium (50 g/L lactose) and the mixture was added into the fermentor. The fermentation was performed in two phases. In the
first phase, the lactic acid bacteria were grown in batch culture for 12 h. In the second phase, the production medium containing
75, 100, and 150 g/L lactose was continuously added into the reactor from its bottom with a peristaltic pump (Watson-Marlow
503 S, Smith and Nephew, UK) at a constant feeding rate of 250 ml/h (12 h fermentor filling-up time) to a total volume of 6 L.
The fermentation conditions were the same as described in batch culture. In the case of immobilized cells, mild agitation was
used to prevent the dissolution of the Ca-alginate beads.
Repeated fed batch culture. Production medium containing 100 g/L lactose was used as substrate for the production of lactic
acid using repeated fed batch cultures. After 24 h of fermentation (batch and fed batch culture), 3 L of fermentation broth was
withdrawn from the fermentor and an equal volume of production medium (100 g/L lactose) was added into the fermentor at a
constant feeding rate of 250 ml/h up to a total volume of 6 L. The above fed batch fermentations were repeated ten times. In the
case of immobilized cells, the total fermentation broth (6 L) was removed, the gel particles were washed twice with sterile-
distilled water, and resuspended in 3 L of production medium containing 50 g/L lactose. After 12 h of fermentation, fresh
medium (100 g/L lactose) was added into the fermentor with a constant feeding rate of 250 ml/L until reaching a total volume of
6 L. The above fed batch fermentations were repeated 20 times.

Analytical techniques
In free-cell fermentation, the number of living cells was determined by plate counting. L. casei was cultivated on MRS agar, L.
lads on Ml7 agar, and mixed cultures on both MRS and Ml7 agar at 30°C for 48 h. In the case of coimmobilized cells, the
concentration of living cells entrapped in Ca-alginate beads was determined by dissolving six beads in 10 ml of 0.3 M sodium
citrate solution (adjusted to pH 5.0 with 1 M citric acid) for 20 min with continuous stirring. The number of living cells liberated
from the gels was determined as described above.
The fermentation broth was centrifuged at 10,000 g for 20 min and the supernatant was used for the determination of lactic acid
and residual lactose (in the case of immobilized cells the fermentation broth was not centrifuged). Total lactic acid and residual
lactose concentration were determined as described by Lawrence (cite 12) and Dubois et al (cite 13) respectively.
The lactic acid productivity (R) was calculated using the equation: R = P/t where P is the lactic acid concentration (g/L) and t is
the total fermentation time (h). Lactic acid yield and lactose utilization were expressed as g lactic acid (100 g) ^-1 lactose utilized
and g lactose utilized (100 g) ^-1 initial lactose, respectively.

Results
Batch culture with free cells
The production of lactic acid from deproteinized whey (50 g/L lactose) by free cells of L. casei or L. lactis and mixed culture of
the above strains in batch culture is shown in
Figure 1. L. casei, L. lactis, and the mixed culture differed considerably in their capacity to produce lactic acid. In all culture
systems, the concentration of lactic acid increased with the increase in fermentation time. Lactic acid concentration increased
rapidly during the first 18 h of fermentation and kept increasing at a slower rate to reach a maximum after 24 h of incubation.
The biomass concentration followed a pattern similar to lactic acid concentration with maximum viable cell number observed at
the same time as the maximum concentration of lactic acid was observed. The maximum concentration of viable cells in
fermentation by L. casei, L. la&, and the mixed culture were 1.5 x 10^11, 1.0 x 10^11”, and 3.4 x 10^10 ufc/ml, respectively,
after 24 h of fermentation (data not shown).
As expected, the concentration of residual lactose de- creased during the fermentation. This coincides with an increase in lactic
acid production (Figure I). The concentration of residual lactose fell rapidly during the first 18 h of fermentation after which it
decreased slowly. The highest concentration of residual lactose was obtained in fermentation with L. lactis while the lowest
concentration was found in fermentation with mixed cultures of L. casei and L. lactis. When the maximum concentration of lactic
acid was achieved, 40, 33.3, and 48.4% of the lactose consumed was converted to lactic acid in medium fermented by L. casei, L.
lactis, and the mixed culture, respectively. In this case, the total amount of lactose utilized was 80, 63, and 93% in fermentations
by L. casei, L. lactis, and the mixed culture, respectively. The above results showed that the mixed culture system gave better
results than single cultures regarding lactic acid concentration and sugar utilization. For this reason, mixed cultures of L. casei
and L. lactis were used for all subsequent studies.

Fed batch culture with free cells

The production of lactic acid was performed by feeding substrate containing 75, 100, and 150 g/L lactose as shown in Figure 2.
The lactic acid concentration increased slightly with the increase in lactose concentration up to 100 g/L but remained constant
beyond this value. The highest concentration of lactic acid (46 g/L) was obtained at lactose concentrations of 100 and 150 g/L
after 24 h of incubation. In culture grown at 75 g/L lactose, the maximum lactic acid concentration was reduced to 42 g/L. The
number of viable cells (cfu/ml) increased slightly during fermentation while a marked increase in the total number of viable cells
was obtained (data not shown).
The concentration of residual lactose decreased in batch fermentation while it was increased during fed batch culture (except at
75 g/L feeding substrate). By increasing the lactose concentration from 75 to 150 g/L , a significant rise in the residual lactose
concentration was obtained. At the highest lactic acid concentrations in the media, the lactic acid yields were 73% from 75 g,
77% from 100 g, and 66% from 150 g of the feeding substrate concentration while the lactose utilization decreased (92% from 75
g, 80% from 100 g, and 70% from 150 g of the feeding substrate concentration).

Effect of feeding rate with free cells

The production medium containing 100 g/L initial lactose was pumped into the fermentor at a feeding rate of 250, 334, 500, and
1,000 ml/h up to a total volume of 6 L (fermentor filling-up time 12, 9, 6, and 3 h, respectively). As shown in Figure 3, the lactic
acid concentration decreased as the feeding rate was increased from 250 to 1,000 ml/h. The highest value of lactic acid
concentration (46 g/L) was achieved at the lowest feeding rate (250 ml/h). The lactic acid productivity and the lactic acid yield
remained practically constant with an increase in feeding rate from 250 to 334 ml/h, but decreased at higher feeding rates (500-
1,000 ml/h). Maximum lactic acid yield (77%) was obtained at F = 250 ml/h). The above results showed that the optimum
feeding rate for the lactic acid concentration, productivity, and yield was 250 ml/h.

Batch versus fed batch culture

From the results already described in Figures 1 and 3 (batch mixed culture 24 h, fed batch culture 24 h, Sf = 100 g/L, F = 250
ml/h), various kinetic parameters describing the performance of the two fermentation systems are presented in Table 1. In the
case of the fed batch culture, the lactic acid productivity was twice higher than the batch productivity. The fed batch culture also
supported a higher lactic acid concentration, lactic acid yield, specific lactic acid production rate, and specific sugar uptake rate
compared with batch culture. On the other hand, the batch culture gave higher values of biomass concentration, biomass yield,
specific biomass production rate, and fermentation efficiency. Based on the above observations, fed batch culture proved to be a
better fermentation system for the production of lactic acid from deproteinized whey than batch culture; thus, fed batch culture
was selected over batch culture for all subsequent studies.

Fed batch culture with coimmobilized cells

The production of lactic acid from deproteinized whey containing 75, 100, and 150 g/L lactose by coimmobilized L. casei and L.
lactis cells using fed batch culture is shown in Figure 4. AS shown there, the lactic acid concentration reached its maximum value
(47 g/L) at a lactose concentration of 100 g/L and remained constant beyond this value. At the maximum lactic acid
concentration. the lactic acid yield and the lactose utilization were 76.5%, 85.5%, 72.3% and 89%, 73%, and 65% from 75,100,
and 150 g/L of the feeding substrate concentration, respectively.

Repeated fed batch culture with free and coimmobilized cells

The production of lactic acid from deproteinized whey by free and coimmobilized L. casei and L. lactis cells using repeated fed
batch culture is shown in Figure 5. In the free cells system, the overall lactic acid concentration and the number of viable cells
decreased significantly after 5 days of fermentation. On the other hand, the coimmobilized L. casei and L. lactis cells in Ca-
alginate gel retained their activity to produce lactic acid for 20 days, then the cells began to show signs of aging, since there was a
decrease in biomass and lactic acid concentration.

Discussion
In a batch culture system with free cells of L. casei and L. lactis, the highest concentration of lactic acid (22.5 g/L) was obtained
in the mixed culture system while in single culture fermentations of L. casei or L. lactis, the maximum concentration of lactic
acid was 16 and 10.5 g/L, respectively. Guoqiang et al (cite 14) and Audet et al (cite 7) reported that a high concentration of
lactic acid (25 and 9 g/L) was obtained when free cells of L. casei and L. lactis were grown in a chemically defined medium and
whey permeate, respectively, in batch culture. Ozen and Ozilgen (cite 10) found that mixed cultures of Lactobacillus bulgaricus
and Streptococcus thermophilus produced 16 g 1-l lactic acid when 20% (w/v) nonfat dry milk was fermented in conical flasks.
There are some possible reasons for these differences including the strain of organism used, the chemical composition of the
substrate, the fermentation system, and generally, the conditions under which the fermentation takes place.
The mixed culture system gave better results than single cultures regarding lactic acid concentration, lactic acid yield, and sugar
utilization. Mixed culture fermentations offer a number of advantages over fermentations using a single culture such as higher
product yield and better utilization of the substrate, and they enable the utilization of cheap and impure substances. (cite 15)
During lactic acid production in a fed batch culture system with free cells by feeding substrate containing increased lactose
concentrations, a significant increase in the residual lactose concentration was obtained. The de- creased lactose utilization with
higher concentrations was probably due to osmotic effects. It has been reported that, above a critical substrate concentration, the
reduced water activity combined with plasmolysis causes a decrease in the rate of fermentation and sugar utilization.16 The
lactose concentration of the feeding substrate had a significant effect on the kinetic parameters of fermentation using mixed
cultures of L. casei and L. lactis. The optimum lactose concentration of the feeding substrate for the maximum final lactic acid
concentration and lactic acid yield was found to be 100 g/L.
The maximum production of lactic acid (47 g/L) from deproteinized whey by fed batch culture with coimmobilized L. casei and
L. lactis cells was obtained at a lactose concentration of 100 g/L. Tuli et al (cite 5) and Audet et (cite 7) found that maximal lactic
acid concentrations of 33 and 8 g/L were obtained when immobilized L. casei or L. lactis cells in agar and k-carrageenan,
respectively, were grown in whey permeate in batch culture. In a previous work, a maximum lactic acid concentration (41 g/L)
was obtained when coimmobilized L. casei and L. lactis cells in Ca- alginate beads were grown in deproteinized whey in static
culture after 48 h of fermentation. Kurosawa (cite 9) reported that a maximum lactic acid concentration (25 g/L) was obtained
when coimmobilized Aspergillus awamori and L. lactis cells in Ca-alginate beads were grown in starch in batch culture. These
differences were due to the strain applied, the nature of the substrate, the immobilization matrix, the fermentation system, and the
conditions employed during fermentation. The optimum lactose concentration for lactic acid production coinciding with a high
lactic acid yield and sugar utilization was found to be 100 g/L. Generally, the coimmobilized cell system gave the same lactic
acid concentration compared to the free cell system under the same fermentation conditions. The lactic acid yield was higher in
the case of coimmobilized cells compared to free cells. On the other hand, free cells gave a higher sugar utilization.
In repeated fed batch culture, the coimmobilized L. casei and L. lactis cells in Ca-alginate gel retained the activity to produce
lactic acid for 20 days versus 5 days for free cells. Demirci and Pometto (cite 17), who studied the production of lactic acid from
glucose by immobilized L. casei cells in biofilm reactor with plastic-composite supports using repeated batch culture, have
shown that the cells retained their ability to produce lactic acid for 72 days. Hang (cite 18) reported that Rhizopus oryzae cells
immobilized in Ca-alginate beads produced lactic acid from glucose for 17 days without loss of their activity using repeated
batch fermentation. This ability of immobilized cells to produce lactic acid for a long time has not been explained yet. This may
be due to the protection of cells by the immobilization matrix. Rychtera et al (cite 19) reported that immobilized cells can retain
enzyme activities for a long time due to the different composition of cells (proteins, lipids, RNA, DNA, and inorganic sub-
stances) compared with free cells.

Conclusions
Our results showed several important aspects of lactic acid production from deproteinized whey by free and coimmobilized L.
casei and L. lactis cells. The mixed culture system gave better results regarding lactic acid production compared to free L. casei
or L. Lactis cells. Fed batch culture was a better fermentation system than batch culture. Under the same fermentation conditions,
both free and coimmobilized L. casei and L. lactis cells gave the same maximum lactic acid concentration in fed batch culture. In
repeated fed batch culture, coimmobilized L. casei and L. Lactis cells in Ca-alginate beads retained their activity to produce lactic
acid for a long time. The deproteinized whey was an attractive medium for the production of lactic acid by free and
coimmobilized L. casei and L. lactis cells using fed batch culture.

References
1.
Roukas, T. and Kotzekidou, P. Production of lactic acid from deproteinized whey by coimmobilized Lactobacillus casei and
Lactococcus lactis cells. Enzyme Microb. Technol. I99 1, 13, 33-38 2. Chiarini, L., Mara. L., and Tabacchioni, S. Influence of
growth
supplements on lactic acid production in whey ultrafiltrate by Lactobacillus helveticus. Appl. Microbial. Biotechnol. 1992. 36,
461-464 3. Gltje, G. and Gottschalk. G. Limitation of growth and lactic acid production in batch and continuous cultures of
Luctobacillus hel- veticus. Appl. Microbial. Biotechnol. 1991, 34, 446-449 4. Tejayadi, S. and Cheryan, M. Lactic acid from
cheese whey
permeate. Productivity and economics of a continuous membrane bioreactor. Appl. Microbial. Biotechnol. 1995. 43, 241-238 5.
Tuli, A., Sethi, R. P.. Khanna, P. K., Marwaha. S. S., and Kennedy. J. F. Lactic acid production from whey permeate by
immobilized Lactobacillus casei. Enzyme Microb. Technol. 1985, 7. 164- I68 6.
Roukas. T., Kotzekidou, P., and Alichanidis, E. Production of lactic acid from whey by free and immobilized cells of
.!,actoba~$lfu,~ plantawn. Abstract of Eighth Tnt. Biotechnol. Symp. Paris. France Lafayette, Paris, 1988. 219 7.
Audet, P., Paquin, C., and Lacroix, C. Sugar utilization and acid production by free and entrapped cells of Streptococcus
,saliwrius subsp. thermophilus. Lactobucillus delbrueckii subsp. bulgaricus, and Lacrococcus lactis subsp. lactis in a whey
permeate medium. Appl. Environ. Microbial. 1989, 55, 185-I 89 8.
Norton, S.. Lacroix, C.. and Vuillemard, J. C. Kinetic study of continuous whey permeate fermentation by immobilized Lactoba-
cillus helveticus for lactic acid production. Enzyme Microb. Tech- nol. 1994, 16, 457-466 9
Kurosawa, H.. lshikawa, H., and Tanaka, A. L-lactic acid production from starch by coimmobilized mixed culture system of
Aspergillus awamori and Streptococcus lactis. Biotechnol. Bioeng. 1988. 31, 183-187 10.
bzen, S. and Gzilgen, M. Effects of substrate concentration on growth and lactic acid production by mixed cultures of Lutobacil-
lus bulgaricus and Streptococcus thermophilus. J. Chem. Tech. Biotechnol. 1992, 54, 57-61 11.
Stanbury. P. F. and Whitaker, A. Principles of Fermentation Technology. Pergamon, Oxford, 1984 12.
Lawrence, A. J. Determination of lactic acid in cream. Aust. J. Dairy Technol. 1975. 30, 14-15 13.
Dubois. M., Gilles, K. A., Hamilton. J. K., Rebers, P. A., and Smith. F. Calorimetric method for determination of sugars and
related substances. Anal. Chem. 1956, 28, 350-356 14.
Guoqiang, D., Kaul. R., and Mattiasson, B. Evaluation of alginate- immobilized Luctobacillus casei for lactate production. Appl.
Mi- crobiol. Biotechnoi. 1991, 36, 309-3 14 15.
Hesseltine. C. W. Mixed-culture fermentations: An introduction to oriental food fermentations. In: Mixed Culfures in
Biotechnology (Zeikus. J. G. and Johuson, E. A., Eds.). McGraw-Hill, New York, 1991, l-16 16.
Roukas, T.. Lazarides, H., and Kotzekidou, P. Ethanol production from deproteinized whey by Saccharomyces cerevisiae cells
en- trapped in different immobilization matrices. Milchwissenschaft 1991, 46,438-441 17.
Demirci, A. and Pometto. A. L. Repeated-batch fermentation in biofilm reactors with plastic-composite supports for lactic acid
production. Appl. Microbial. Biotechnol. 1995, 43, 585-589 18.
Hang, Y. D., ihmramci, H., and Woodams, E. E. Production of L(+)-lactic acid by Rhizopus oryzae immobilized in calcium alg-
inate gels. Biotechnol. L&t. 1989. 11, 119-120 19.
Rychtera, M.. Basarova, G., and lvanova, V. Behavior and proper- ties of released and in calcium alginate gel immobilized cells
of Saccharomyces cerevisiae in continuous culture. In: 4th European Congress on Biotechnology Vol. 2 (Neijssel, 0. M., van der
Meer. R. R., and Luvben, K., Eds.). Elsevier Science Publishers, Amster- dam. 1987. 107-109
204 Enzyme Microb. Technol., 1998, vol. 22, February 15