Letters in Applied Microbiology ISSN 0266-8254

ORIGINAL ARTICLE

Lactic acid production by Lactobacillus delbrueckii in a dual

reactor system using packed bed biofilm reactor
V. Rangaswamy and S.V. Ramakrishna
Industrial Biotechnology Group, Reliance Life Sciences Pvt. Ltd, Navi Mumbai, India

Keywords
dual reactor, lactic acid fermentation,
Lactobacillus delbrueckii, molasses, packed
bed bioreactor, polyurethane foam.
Correspondence
V. Rangaswamy, Industrial Biotechnology
Group, Reliance Life Sciences Pvt. Ltd,
Dhirubhai Ambani Life Sciences Centre,
R-282, TTC area of MIDC, Thane–Belapur
Road, Rabale, Navi Mumbai 400 701, India.
E-mail: vidhya_rangaswamy@relbio.com
2007 ⁄ 0066: received 12 January 2007,
revised 1 March 2008 and accepted 3 March
2008
doi:10.1111/j.1472-765X.2008.02362.x
Abstract
Aims: An integrated dual reactor system for continuous production of lactic
acid by Lactobacillus delbrueckii using biofilms developed on reticulated poly-
urethane foam (PUF) is demonstrated.
Methods and Results: Lactobacillus delbrueckii was immobilized on PUF,
packed in a bioreactor and used in lactic acid fermentation. The rate of lactic
acid production was significantly high with a volumetric productivity of
5 g l)1 h)1 over extended period of time. When coupled to a bioreactor, the
system could be operated as dual reactor for over 1000 h continuously without
augmentation of inoculum and no compromise on productivity.
Conclusions: Polyurethane foams offer an excellent support for biofilm
formation.
Significance and Impact of the Study: The system was very robust and could
be operated for prolonged period at a volumetric productivity of 4–6 g l)1 h)1.

Introduction Materials and methods

Majority of the reports on lactic acid fermentation focus
on improving the volumetric productivity, a measure of
process intensification. Adsorption of microbial cells on
inert supports (Goncalves et al. 1992) is one such
approach wherein lactic acid bacteria biofilms are devel-
oped by adsorption on to supports such as sintered glass,
ceramic materials, polyurethane foams (PUFs) or poly-
propylene composite chips (Guioquiang et al. 1992; Yan
et al. 1999; Krishnan et al. 2001; John et al. 2007). These
biofilm reactors have several advantages including mild
treatment conditions, autoclavability and low cost.
In this report, we have developed biofilm of Lactobacil-
lus delbrueckii on commercially available synthetic carrier,
PUF for lactic acid fermentation. This study presents data
on the utility of this system in various modes of fermen-
tation. Besides, a novel dual reactor system has also been
developed which is an integrated system comprising of a
stirred tank bioreactor coupled with packed bed biofilm
column that can be operated in a continuous mode.
Bacterial strains, media and growth conditions
Lactobacillus delbrueckii NCIM 2365 was cultivated
without agitation at 42C in MRS medium (De Man
et al. 1960). The concentration and source of sugar
used in the medium has been indicated at appropriate
places.
Reticulated PUF sheets were procured locally. Cubes of
reticulated PUF (1 cm · 1 cm · 1 cm) were washed with
deionized water and sterilized at 121C for 20 min before
use.
Sugarcane juice, molasses and bagasse were procured
locally. The initial sugar concentration of the sugarcane
juice varied between 8% and 10% whereas molasses had a
sugar concentration between 40% and 45%. Bagasse was
treated with 1% sulfuric acid (w ⁄ v) at a ratio of 1 : 10
and autoclaved at 121C for 1 h. The supernatant was
squeezed out and neutralized prior to using in the fer-
mentation. The final sugar concentration in the medium

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Journal compilation ª 2008 The Society for Applied Microbiology, Letters in Applied Microbiology 46 (2008) 661–666 661
Lactic acid production using a dual reactor system
was adjusted to 2% irrespective of the source unless men-
tioned otherwise.
Analytical procedures
Reducing sugar was estimated by dinitrosalicylic acid
(DNSA) method (Miller 1959). Wherever sucrose, sugar-
cane juice or molasses was used as source of sugar, the
samples were acid hydrolysed with 6 mol l)1 HCl at 95C
for 5 min, neutralized and then used in the DNSA assay.
Lactic acid was estimated either colorimetrically (Taylor
1966) or by HPLC on an organic acid column (Phenome-
nex Rezex (Phenomenex, Inc., Torrance, CA) ROA
column, 210 nm, mobile phase: 0Æ05 mol l)1 H2SO4 at
0Æ6 ml min)1) (Kiran and Divakar 2001).
Development of biofilm
The washed cells from a overnight grown L. delbrueckii
culture suspended in 100 ml of sterile MRS medium was
contacted with 2 g of sterile reticulated PUF. After incu-
bation at 42C for 12 h on a rotary shaker, the PUF cubes
were washed with sterile distilled water to remove the un-
adsorbed cells. Fresh MRS medium (100 ml) was added
to the PUF cubes and incubated at 42C for 24 h to allow
the cells to proliferate inside the PUF matrix. This proce-
dure was repeated three times to ensure proper biofilm
formation within the PUF matrix. The cells adhere to the
PUF and proliferate (Fig. 1).
Figure 1 A SEM micrograph showing Lactobacillus delbrueckii cells
lodged inside the pores of the PUF. A single pore of the PUF was
magnified at 800· to show the presence of cells (arrow).
662 Journal compilation ª 2008 The Society for Applied Microbiology, Letters in Applied Microbiology 46 (2008) 661–666
V. Rangaswamy and S.V. Ramakrishna
Batch experiments
All batch experiments were carried out in 250 ml Erlen-
meyer flasks containing 2 g of cell-loaded PUF cubes
(unless indicated otherwise) in 100 ml of MRS medium
and incubated at 42C. Samples were drawn every 4 h
and analysed for lactic acid and residual sugar in the
medium. To study the effect of cell loading, different
quantities of cell-loaded PUF (2, 5 and 7Æ5 g) was added
to the sterilized medium. At higher concentrations of
PUF, practically no free liquid is left in the flask and to
circumvent these hurdles, all further experiments were
carried out at 2% cell-loaded PUF and the fermentation
time was typically 8–10 h.
Effect of various carbon sources on lactic acid produc-
tion was investigated by replacing the glucose in MRS
medium with 2% (w ⁄ v) of either sucrose, molasses or sug-
arcane juice. By temperature optimization, it was found
that incubating at 42C was most suitable and hence all
experiments were carried out at this temperature. To eval-
uate the potential of cell-loaded PUF for long-term opera-
tion, repeated batch experiments were carried out. After
24 h of batch operation, the fermented medium was
drained and fresh sterilized media was added aseptically
and fermentation was continued for next 24 h. The proce-
dure was repeated every 24 h; lactic acid produced and
residual sugar was estimated. Data collected from five
independent experiments are represented in Table 1.
Development of biofilm reactor
The packed bed biofilm reactor (PBBR) consisted of a
jacketed glass column of 5Æ5 cm (o.d.) · 4Æ8 cm (i.d.) and
60 cm length with provision for feed entry at the bottom
and outlet at the top. The glass reactor with 16 g of PUF
cubes was sterilized at 121C for 20 min. The cell attach-
ment was accomplished by circulating 1000 ml of over-
night culture of L. delbrueckii grown in MRS medium
through a peristaltic pump. After 12 h of circulation, the
Table 1 Effect of various carbon sources on lactic acid production in
batch experiments
Lactic acid % Sugar conversion
Sugar (g l)1) to LA in 8 h
Bagasse hydrolysate 10Æ8 50Æ2 ± 4Æ2
Sucrose 20 99 ± 0Æ9
Sugarcane juice 14Æ8 75Æ5 ± 2Æ1
Glucose 15Æ7 79 ± 0Æ5
Molasses 15 75Æ3 ± 3Æ6
The concentration was maintained at 2% for all the sugar sources.
The lactic acid values are given for a typical experiment. % Sugar con-
version data is from a set of five different experiments.
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V. Rangaswamy and S.V. Ramakrishna Lactic acid production using a dual reactor system

media was replaced with fresh sterilized medium and cir- Among various carbon sources including glucose,
culated for another 24 h to facilitate the adsorbed cells to sucrose, molasses, sugarcane juice and bagasse hydrolysate
grow and proliferate inside PUF matrix. The temperature tested for lactic acid fermentation, sucrose was the pre-
inside the PBBR was maintained at 42C by circulating ferred source of sugar than glucose giving 20% higher
water from a 42C water bath through the jacket of the yield of lactic acid (Table 1). Sugarcane juice, molasses
column continuously. After two replacements of fresh and bagasse hydrolysates resulted in slightly lower amount
sterilized medium for 48 h, the reactor was ready for use. of lactic acid as compared to pure carbon sources.
The feed containing 2% carbon source was introduced
from the bottom and the outflow was collected from top.
Lactic acid fermentation in dual reactor
The residence time was adjusted at 3 h by controlling the
flow of the feed at 100 ml h)1. Data were collected from When the PBBR with L. delbrueckii loaded PUF was oper-
at least 100 samples. ated alone, the pH dropped to 4Æ0 within 3 h due to pro-
duction of 12–14 g l)1 of lactic acid. No further increase
in the yield of lactic acid was observed. Therefore, the
Experiments in dual reactor system
packed bed reactor was coupled with the STR, which has
Since pH adjustment is of utmost importance for continu- a pH control thereby giving us a dual reactor system.
ous lactic acid fermentation, the PBBR (volume 800 ml) When the dual reactor system was operated in batch
was coupled to a 2Æ2 l fermentor (Model Biostat B, M ⁄ s B mode, 45–60 g l)1 of lactic acid was produced in 24 h.
Braun Biotech International, Melsungen, Germany). The The percent conversion of sugars to lactic acid was in the
outlet from the biofilm reactor was connected to the range of 90–95% and the volumetric productivity of the
fermentor inlet and the media overflow from the fermen- system was 2–2Æ5 g l)1 h)1. This is about four times
tor was circulated through the reactor by means of peri- higher than the productivity of 0Æ65 g l)1 h)1 obtained
staltic pump. The reactor was connected to the fermentor using free cell system carried out independent of the
as an outer loop. Sterilized medium (1000 ml) using PBBR (Fig. 3a).
molasses as carbon source (sugar concentration adjusted
to 5–10%) was circulated through the biofilm reactor con-
Continuous fermentation using the packed bed
tinuously. After few hours, the leached out cells from the
bioreactor
reactor proliferated in the fermentor resulting in initiation
of dual reactor system. Since pH was accurately monitored The dual reactor system was operated in continuous
and controlled (by the addition of 26% NH4OH), the mode at various flow rates including 200, 250 and
fermentation proceeded until all the sugars were converted
to lactic acid. Experiments were carried out at pH 6Æ0 and
42C. Samples were drawn every 4 h and residual sugar
and lactic acid was estimated. The reactor was operated at
a flow rate of 250 ml h)1 with a dilution rate of
0Æ0833 h)1. The productivity was calculated on the basis
of lactic acid produced at the operating flow rate and the
volume of the stirred tank (2Æ2 l) and dual reactor system
(3 l). Reproducibility of long-term use of the dual reactor
system was tested at least three times.

Results
Lactic acid fermentation in a dual reactor system consti-
tuting a packed bed PUF reactor immobilized with L. del-
brueckii and a stirred tank reactor (STR) is the focus of
the current investigation.
In flask level fermentation using cell-loaded PUF cubes,
Figure 2 A schematic representation of the dual reactor system
complete sugar conversion took place in 12 h. Increasing
where the PBBR is connected to the fermentor. The feed passes
the cell loading by using 7Æ5% PUF cubes significantly through the PBBR and flows into the fermentor through the outlet
reduced the time for complete conversion of lactic acid to at top. The pH drop due to the lactic acid produced in the PBBR is
just 2 h (data not shown) indicating the potential of such rapidly adjusted to pH 6Æ0 by liquid ammonia. The overflow from the
a support. fermentor is recycled into the PBBR.

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Journal compilation ª 2008 The Society for Applied Microbiology, Letters in Applied Microbiology 46 (2008) 661–666 663
Lactic acid production using a dual reactor system V. Rangaswamy and S.V. Ramakrishna

Table 2 Volumetric productivity of continuous lactic acid fermenta- (a)

tion using the dual reactor system

Flow rate Residence Volumetric productivity

(ml h)1) time (h) (g l)1h)1)

200 15 4
250 12 5
300 10 4Æ5

The carbon source used was molasses and initial sugar concentration
was 10%.

300 ml h)1. The highest volumetric productivity of

5 g l)1 h)1 could be achieved at 250 ml h)1 flow rate (b)
(Table 2). The feasibility of long-term operation of dual
reactor system was tested by carrying out the fermenta-
tion for 1000 h without interruption or augmentation
of inoculum at a flow rate of 250 ml h)1. A constant
volumetric productivity of 5 g l)1 h)1 was obtained
(Fig. 3b).

Discussion
It is a well-known fact that the product inhibition during
lactic acid fermentation is a major bottleneck in obtaining
higher yields. Immobilization of cells has been widely
used to achieve high productivity of lactic acid fermenta-
tions since it is possible to enhance the cell density and
facilitate continuous fermentations without cell wash-out.
Lactic acid fermentation using L. delbrueckii NCIM
2365 immobilized on an inert-support viz. PUF to
achieve high productivity was evaluated. Lactobacillus del-
brueckii NCIM 2365 derived Uc-3 mutant has been widely
used in several studies dealing with utilization of sugars
from various biomass including sugarcane bagasse (Adsul
et al. 2007), molasses (Dumbrepatil et al. 2008), fructose
from cane sugar (Patil et al. 2006) for production of lac-
tic acid in a free cell fermentation. Highest productivity
value of 4Æ15 g l)1 h)1 was achieved for lactic acid pro-
duction from molasses (Dumbrepatil et al. 2008). There
is only one report in which L. delbrueckii NCIM 2365
immobilized on functionalized alginate matrix (Rao et al.
2007) was used for fermentation and was shown to pro-
duce selectively l(+)lactic acid.
In the current study, sucrose was the preferred source
of sugar than glucose giving 20% higher yield of lactic
acid (Table 1). Sugarcane juice, molasses and bagasse
hydrolysates gave lower yield of lactic acid probably due
to presence of some inhibitory by-products in these
sources. Long-term efficacy of biofilm in repeated batch
experiments indicated that the biofilms could be main-
tained very effectively with the conversion of sugars to
lactic acid predominantly between 85% and 95% for over
Figure 3 (a) Comparison of volumetric productivities of fermentation
in stirred tank alone ( ) and in the dual reactor system ( ). The initial
sucrose concentration in both systems were 10% and the fermenta-
tion was carried out at 42C and pH 6Æ0. (b) Long-term sustainability
of the dual reactor mode of lactic acid fermentation. Continuous LA
fermentation was carried out at 250 ml h)1 flow rate, 42C, pH 6Æ0
and 10% sucrose.
100 batches. Also, no augmentation of inoculum was
required during the entire period of operation.
In a PBBR with L. delbrueckii loaded PUF, the fermen-
tation could not be sustained beyond 3 h due to rapid
drop in pH to 4Æ0 and development of a pH gradient
across the length of the column. The yield of lactic acid
hovered around 12–14 g l)1 irrespective of the sugar con-
centration. Therefore, it necessitated the integration of
the reactor to a pH stat or to another fermentor vessel
where pH can be adjusted resulting in a dual reactor
system.
Dual reactor system
The dual reactor system (Fig. 2) basically operates by
coupling the PBBR to an STR where the free cells released
from the biofilm will proliferate and ferment the sugar to
lactic acid in the STR. When the system was operated on
batch mode, the cell release from the PBBR inoculates the
STR and fermentation in the dual reactor by free and
immobilized cells in separate vessels is initiated. The bio-
mass generated in STR indicates the potential of such

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664 Journal compilation ª 2008 The Society for Applied Microbiology, Letters in Applied Microbiology 46 (2008) 661–666
V. Rangaswamy and S.V. Ramakrishna
scheme where the viable count was as high as
4Æ5 · 1011 CFU ml)1 in both the PBBR and fermentor
samples (data not shown).
The dual reactor system gave higher productivity than
a packed bed reactor or a free cell fermentation. This is
in sharp contrast to the report where free cells produced
more lactic acid as compared to cells entrapped in j-car-
rageenan (Audet et al. 1989). The operational stability of
a biocatalyst is an important consideration for bioreactors
that is evaluated by its half-life, the time when it loses
50% of its activity. The current dual reactor system was
very robust as proven by uninterrupted fermentation for
1000 h without augmentation of inoculum. A constant
volumetric productivity of 5 g l)1 h)1 was obtained at a
flow rate of 250 ml h)1 (Fig. 3b).
Recently, lactic acid fermentation using PUF as a carrier
has been reported by another group (John et al. 2007).
However, the process involves solid state fermentation
using Lactobacillus casei using cassava bagasse starch.
Other matrices reported in the literature such as the
chitosan-based polypropylene (Krishnan et al. 2001), poly-
ethylenemine-treated ceramic (Guioquiang et al. 1992),
calcium alginate (Roukas and Kotzekidou 1996), j-carra-
geenan (Buyukgungor 1992) are less effective with either
low operational life or low productivity or low yields of
lactic acid. The polypropylene matrix-based biocatalyst
could be recycled only 11 times although the yield and
productivity were 97% and 7Æ66 g l)1 h)1. The j-carra-
geenan beads could be recycled effectively four times with
100% conversion and a low productivity of 0Æ83 g l)1 h)1.
Coimmobilized L. casei and Lactococcus lactis in calcium
alginate beads in continuous packed bed column reactor
had an operational life of 24 days with a productivity of
2Æ5 g l)1 h)1. However, the yield of lactic acid was only
55% (Roukas and Kotzekidou 1996). Senthuran et al.
(1997) investigated lactic acid production by immobilized
L. casei cells in a recycle PBCR and observed that the bio-
catalyst could be recycled only 12 times with a yield of
93% with a productivity of 5 g l)1 h)1. Effective recycling
of 11 times with a yield of 82% was obtained when L. del-
brueckii immobilized in calcium alginate in a continuous
PBCR was carried out (Stenroos et al. 1982). It is thus
evident that, among all these systems, the PUF based dual
reactor system is by far the most superior and robust with
respect to its long-term usage and productivity.
Thus, the present report successfully demonstrates the
utility of a cheap, re-usable, inert, autoclavable support
with high porosity for biofilm-based fermentation which
has been cited by John et al. (2007). The system is advan-
tageous as loss of biomass from the support is constantly
replenished by fresh cells from the fermentor. Concomi-
tantly, the PBBR continuously inoculates the fermentor
thereby alleviating any need for intermitted inoculation.
ª 2008 The Authors
Journal compilation ª 2008 The Society for Applied Microbiology, Letters in Applied Microbiology 46 (2008) 661–666
Lactic acid production using a dual reactor system
Acknowledgements
The authors gratefully acknowledge the encouragement
and support of Reliance Life Sciences Pvt. Ltd, in carrying
out the research work.
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