LACTIC ACID PRODUCTION USING IMMOBILIZED LACTOBACILLUS

4.1. INTRODUCTION

Self immobilization of microorganisms as films, floes and pellets is a widespread

phenomenon in nature. The intentional use of immobilized cells in bioprocesses,

however, is a relatively recent development. The classical fermentation suffers from

various constraints such as low cell density, nutritional limitations and batch mode of

operation with high-down times. It has been recognized that microbial cell density is of

prime importance to attain higher volumetric productivity. The major limitation in the

development of continuous fermentation process has been the wash out of cells from

the bioreactor. The cell immobilization technology can circumvent these problems and

it has been considered a useful method for the long-term utilization of biocatalysts in

fermentation (Nampoothiri & Pandey 1998). Lactic acid production in batch mode

requires a high concentration of cells in the reactor, which can be achieved by cell

immobilization.

Biochemical processing with immobilized microbial cells offers a number of

unique advantages over traditional fermentation processes with free cells, such as

relative ea se o f ceil mass separation from the bulk liquid for possible re-use, prevention

of washout, reduced risk of contamination and operational stability. Furthermore, using

the entrapment technique, a dense cell culture can be established leading to improved

productivity (Kourkoutas et al. 2004). This has triggered a surge of research activity in

this exciting and rapidly growing field. The biotransformational and biosynthetic abilities

of immobilized growing microbial cells for the production of diverse valuable products

like antibiotics, organic acids, enzymes and alcohols have been well demonstrated.

Proper selection of immobilization techniques and supporting materials is needed to

minimize the disadvantages of immobilization (Ramakrishna & Prakasham 1999). One

65
of the most suitable methods for cell immobilization is entrapment in calcium alginate,

because this technique is simple and cheap. Sodium alginate is a readily available non­

toxic biological material and is therefore suitable as an immobilization matrix for bio­

molecules and microorganisms (Mattiasson et al. 1984). Beads of calcium alginate

prepared under mild conditions have been used extensively for microencapsulating and

entrapping cells (Jamuna & Ramakrishna 1992).

The most widely used immobilization method for lactic acid production is gel

entrapment (Roy et al. 1987; Dong et al. 1991; Wang et al. 1995; Y o o et al. 1996).

Entrapment o f lactic acid-producing bacteria in gel matrices can protect them from

bacteriophages, contaminating bacteria and physico-chemical stresses (Garbayo et al.

2004).

4.2. MATERIALS AND METHODS

4.2.1. Microorganism and whole cell Immobilization
Lactobacillus delbrueckii NCIM 2025 and Lactobacillus easel NCIMB 3254

cultures were used for the immobilization.

For entrapment o f cells, 25 mL of inoculum was centrifuged at 5870 g for 10

min at 4 °C. Cells were washed, re-suspended in 5 mL physiological saline, and mixed

thoroughly with 20 mL o f sterile 2,5% (w/v) sodium alginate solution. The mixture was

dropped into a sterile solution o f 0.6 M CaCl2 using a syringe. Beads were cured in the

presence of 0.5 M C aC bfor 4 h at 4 °C. After washing in physiological saline they were

used to inoculate 100 mL of cassava bagasse hydroiysate-based production medium.

The cultures were immobilized individually and also as mixed culture in 1:1

ratio and the efficacy o f each of the immobilized system to produce lactic acid were

evaluated. Various process parameters for whole cell immobilization were optimized

such as, (1) concentration of alginate (1-3.5%) (2) CaCl2 (0.1-1 M) (3) initial cell

concentration (0.05-0.4 g) (4) curing time (4-24 h) (5) bead size (2-8 mm) (6) agitation
(static and shaked with an rpm of 80) and (7) incubation time (24 h interval up to 144 h)

for maximum production.

Cell leakage during the course of fermentation is measured by plating a serially

diluted aliquot o f the culture medium in MRS agar plate and counting the CPU after 24

h o f Incubation and expressed as CFU/mL. The biomass within the beads is measured

by dissolving the beads in sodium phosphate buffer (pH 6.5) and plating an aliquot in

MRS medium for counting the CPU.

The reusability o f beads was investigated by transferring the immobilized whole

cell beads to fresh medium at the end of each fermentation cycle after washing with

sterile CaCb curing solution. All optimization experiments were conducted in 250 mL

Erlenmeyer flasks in duplicate and the value indicated Is the average with standard

error.

4.2.2. Packed column bioreactor

A glass column bioreactor (26 x 2 cm, bed volume was 69 cm^) was packed

with L delbrueckii, immobilized in calcium alginate beads under the conditions

optimized for batch experiments. A diagrammatic representation of the experimental

set up is shown in Pig. 4.1. The cassava bagasse starch hydrolysate-based medium (2

L) In the reservoir was continuously pumped at the rate of 2 mL/min through column

bloreactor and the medium coming through the outlet of the reactor was redirected to

the reservoir. The pH o f the system was maintained by CaCOa supplemented in the

medium. The consumption of reducing sugar and lactic acid production were monitored

at regular intervals of 24 h. The cycle was continued until the residual sugar in the

medium dropped to a trace amount, after which one-fourth of the fermented medium

was replaced with fresh medium so as to maintain the concentration of reducing sugar

and the cycle w as resumed.

A

Bypass valve for

sampling out and
recirculation

Fig. 4.1. Packed c o lu m n btoreactor. (a) S che m atic diagram o f the c o lu m n b io re a cto r, and
(b) E xpe rim e ntal s e tu p o f the bio re a cto r. Arrow marks show the circulation o f medium
(A; resen/oir, B; pump, C; packed column, D: bypass valve for sampling out and recirculation and
E: sampling port)

4. 3. RESULTS AND DISCUSSION

4.3.1. Optimization of immobilization parameters
Concentration and type of alginate used for Immobilization determines the

properties o f beads as alginate may contain different proportions of mannuronic acid

and guluronic acid (Zhang et al. 2000). Depending upon the organism used and the

product of interest, the alginate concentration required for immobilization may differ (Lu

& Chen 1988; Nasin et al. 1989; Nampoothiri & Pandey 1998). In the present study, the
alginate concentration required for immobilization o f L. delbrueckii was 2.5%. Upon

addition to the cell and sodium alginate mixture, Ca^* brings about instant interfacial

polymerisation of alginate with precipitation of calcium alginate. Calcium ions can

diffuse through the alginate and induce gradual gelation of interior part of the beads

(Zhang et al. 2000). Alginate beads are unstable in the presence of other metal Ions

like Wlg^ or (Lu & Chen 1988; Nampoothiri & Pandey 1998). Hence, care was taken

not to supplement these metal ions in the production medium. The behaviour of

entrapped cells depends on the nature of biological material in beads (Garbayo et al.

2004). Optimum conditions were defined on the basis of maximum lactic acid

production and minimum cell leakage in the fermentation medium.

Immobilized L delbrueckii showed better production (29.3 g/L) than L. casei

(20.18 g/L) or the mixed culture ((23.23 g/L) in the initial screening and hence L

delbrueckii was selected for the further experiments.

Although the large scale production of calcium alginate gel beads without any

sophisticated equipments is easy, there are a number of immobilization parameters

that need to be optimized. In order to find out the optimum concentration of sodium

alginate for bead formation, five different concentrations of sodium alginate (1, 1.5, 2,

2.5, 3 and 3.5%, w/v) were tested (cell concentration: 0.2 g, CaCl2 concentration; 0.2 M,

bead size 2mm, fermentation time 120 h). If the concentration o f sodium alginate is

higher, the possibility of cell leakage was lower. As the concentration of sodium

alginate was increased from 1 to 2.5%, the cell leakage was found to decrease and

correspondingly the lactic acid yield increased (Table 4.1). Increasing the sodium

alginate concentration beyond this level decreased the lactic acid, though cell leakage

was low. This was due to decrease in the transport of nutrients from the medium across

the beads.
 Table 4.1. Effect of sodium alginate concentration on cell leakage and lactic acid yield
Sodium alginate Lactic acid production Cell leakage (x 10”
concentration (%, w/v) (g/L)at96h CFU/ mL) at 14 h
1 19.49 14.5
1.5 19.84 4.48
2 23.67 4.20
2.5 29.29 1.80
3 20.11 1.30
3.5 19.57 1.00

With the intention o f generating beads with minimum cell leakage and

maximum lactic acid fomiation, the concentration o f CaC k in the curing medium w as

varied (0.1, 0.2, 0.3, 0.5, 0.75 and 1 M), sodium alginate concentration: 2.5%, w/v,

initial cell mass: 0.2 g, bead size 2mm, fermentation time 120 h. A s in the ca se o f

sodium alginate, the cell leakage decreased and lactic acid yield increased with

increase in C aC b concentration up to 0.5 M (Fig. 4.2). Still higher concentrations o f

CaCl 2 resulted in decreased lactic acid yield.

Calciumchbride(M)
Fig. 4.2: Effect of CaCb concentration on cell leakage (■) and lactic acid production
(□) by immobilized L. delbrueckii
 The effect of initial cell concentration on cell leakage and the corresponding

lactic acid yield was investigated by varying the initial concentration of cells (0.05, 0.1,

0.2, 0.3 and 0.4 g, on the basis of wet weight) used for infimobilization (sodium alginate

concentration: 2.5%, w/v, CaCIa concentration: 0.5 M, t>ead size 2mm, fermentation

time 120 h). The results (Table 4.2) indicated that lactic acid yield increased with

increase in initial biomass and 0.3 g wet biomass in 25 mL alginate solution was the

optimum for highest lactic acid yield, beyond which there was no increase in lactic acid

production with increase in initial biomass. However, cell leakage was directly

proportional to initial biomass and hence, initial cell concentration above 0.4 g was not

tested.

Table 4.2. O ptim ization o f in itia l cell co n cen tra tion fo r the im m o bilizatio n o f L. delbrueckii

Initia l ceil conc. La ctic acid p ro du ction Ceii leakage (x 10°

(g) (g/L) a t 96 h CFU/mL) a t 14 h
0.05 12.34 0.11
0.1 18.95 0.126
0.2 29.2 0.149
0.3 33.42 0.165
0.4 33.8 0.21

The effect of bead size and curing time (with a conditions of sodium alginate

concentration: 2.5%, cell mass: 0.3 g, CaCl2: 0.5 M, initial reducing sugar: 42 g/L) on

final yield of lactic acid is illustrated in Table 4.3. The experiments showed that 2 mm

beads were suitable for highest lactic acid yield and was selected for further studies.

Beads were cured at 4 °C for 4, 12 and 24 h and it was obsen/ed that curing for 4 h

was good enough to impart rigidity to beads for maximum lactic acid yield and minimum

cell leakage when compared to long time curing (Table 4.4). To study the effect of

aeration and agitation, fermentation was carried out with immobilized beads under mild

agitation (80 rpm) as well as under static conditions. Results revealed that static

cultures gave better yield (35.9 g/L lactic acid) than agitated ones (8.5 g/L lactic acid).
 Table 4.3. Influence o f th e size o f im m obilized beads on la ctic a cid p ro d u ctio n
by L. delbrueckii

Bead diam eter La ctic acid Cell leakage (x 1o"

(mm) (g/L) CFU/mL)

2 37.12 0.162
4 36.12 0.165
6 35.10 0.162
8 30.62 0.163

Table 4.4. Influence o f c u rin g tim e on la ctic a cid p ro d u ctio n by im m o bilized L. delbrueckii

C u ring tim e La ctic acid Cell leakage (x 10”

(h) (gfl-) CFU/mL)

4 37.1 0.164
12 36.96 0.162
24 36.33 0.161

Alginate beads with immobilized L delbrueckii were prepared under optimized

conditions and fermentation was carried out for 144 h. The time course o f lactic acid

production by immobilized L. delbrueckii is shown in Fig. 4.3. Using L. delbrueckii,

maximum conversion of reducing sugar to lactic acid was at 120 h with a yield of 0.93 g

lactic acid/g reducing sugar.

X ■uS
bO .o Fig. 4.3. Time course o f lactic
o
acid production (■) by
X
D
bn immobilized L. delbrueckii,
U reducing sugar consumption
(□ ) and cell growth (A ) at
different hours of
fermentation
u
O

Incubation tn e (h)
 To check the reusability of beads, repeated batch fermentation was carried out

for 120 h in flasks by transferring the beads to fi-esh medium after each cycle. The yield

was almost constant up to G®' consecutive batches (38.9, 38.5, 38.2, 37.9, 36.9 and

36.6 g/L lactic acid) after which it declined and resulted in less than 30 g/L in 9*^ cycle.

4.3.2. Packed column bioreactor

Reusability o f immobilized beads with steady production rates was shown to be

possible up to six repeated batches. Based on this, a column bioreactor (Fig. 4.1) was

designed and was used for continuous fermentation for three weeks. Productivity in the

packed column bioreactor was higher than in batch fermentation. Higher productivity o f

immobilized cells in column bioreactors in comparison to those in batch fermentation in

flasks was also reported earlier (Nomura 1987). Generally in continuous fermentation,

utilization of reducing sugar will be low and hence the residual sugar in fermented

medium will be very high, which will affect purification of lactic acid from femnented

broth. So in the present study the medium was recirculated to reservoir and again

pumped to column continuously to make it a modified continuous fennentation. There

was a fluctuation in productivity from 0.63 In the initial h to 0.33 in the final h; it may be

due to the variation in nutrient levels of the medium. As the yield decreased with

respect to the time increased so fennentation time in the last cycles were higher than

the initial one.

The cassava bagasse hydrolysate-based medium in reservoir was

continuously pumped to the column through a pump and the effluent from the column

was redirected to the reservoir after passing through a bypass valve through which

sample was collected for estimation. The redirection of effluent back to the reservoir for

recirculation through the column made the system more efficient in that the conversion

of reducing sugar to lactic acid was very high. Once the residual sugar concentration in
the effluent became low, fermented broth in reservoir was replaced with fresh medium

as described in materials and methods.

In the first cycle, the maximum production of lactic acid (75.6 g) resulted after

120 h. For subsequent cycles, based on the sugar consumption rate, replacement of

fermented medium with fresh medium was done at every 36 h inten/al for 9 days (6

replacements), 48 h Interval for 8 days (4 replacements) and the yield obtained was

0.75 - 0.95 g lactic acid/g reducing sugar with a production rate o f 0.33 - 0.63 g/L/h for

21 d.

In conclusion, immobilization of the cells is attractive in terms of reusability of

cells for repeated batches or continuous fermentation with high cell mass and less

contamination problems. The present study enabled to set various immobilization

parameters for L. delbrueckii for L-lactic acid production in batch and repeated batch or

continuous mode for maximum conversion of the reducing sugar of the hydrolysate of

cassava bagasse to lactic acid yield. As reported earlier (Champagne 1994), cell

immobilization technology for lactic acid production is well recognized due to high cell

masses achieved, good protection from oxygen in growth medium, and few

contamination problems.