1. SHORT COMMUNICATION
Effective conversion of industrial starch waste to L -Lactic acid
by Lactococcus lactis in a dialysis sac bioreactor
Seema Bhanwar & Arashdeep Singh & Abhijit Ganguli
Received: 15 August 2013 /Accepted: 29 October 2013
# Springer-Verlag Berlin Heidelberg and the University of Milan 2013
Abstract We describe here a simple technological process
based on the direct fermentation of potato starch waste
(PSW), an inexpensive agro-processing industrial waste, by a
potential probiotic strain, Lactococcus lactis subsp. lactis, for
enhancing L-lactic acid production. To maximize bioconver-
sion and increase cell stability, we designed and tested a novel
dialysis sac-based bioreactor. Shake flask fermentation (SFF)
and fed batch fermentation in the dialysis sac bioreactor were
compared for L-lactic acid production efficiency. The results
showed that the starch (20 g/L) in the PSW-containing medium
was completely consumed within 24 h in the dialysis sac
bioreactor, compared with 48 h in the SFF. The maximum
lactic acid concentration (18.9 g/L) and lactic acid productivity
(0.79 g/L·h) obtained was 1.2- and 2.4-fold higher in the
bioreactor than by SFF, respectively. Simultaneous saccharifi-
cation and fermentation was effected at pH 5.5 and 30 °C. L.
lactis cells were viable for up to four cycles in the fed batch
fermentation compared to only one cycle in the SFF.
Keywords Dialysis sac bioreactor . Lactococcus lactis .
Fermentation . L-Lactic acid . Potato starch waste
Many food and agro-based industries throughout the world
produce large volumes of waste which are often discharged
into the environment, possibly with harmful ecological effects.
Effective utilization, recycling and reprocessing of these wastes
would therefore be beneficial to the environment (Abdel and
Sonomoto 2010) and potentially economically advantageous.
For example, potato processing plants release an appreciable
amount of starch into wastewater streams which could be
utilized as a cheap substrate by microorganisms to produce
economically valuable organic acids, such as lactic acid, acetic
acid, among others. The wide application of lactic acid (2-
hydroxypropionic acid, CH3CHOHCOOH) in the food, cos-
metic, pharmaceutical and chemical industries (Dumbrepatil
et al. 2008; Gegios et al. 2010; Walton et al. 2010; Wanga
et al. 2010; Zhao et al. 2010 ) has significantly increased
interest in lactic acid production on a large scale (Palaniraj
and Nagarajan 2012). Although lactic acid can be
manufactured either via chemical synthesis or by a biological
approach, the former is associated with several major disad-
vantages, including environmental issues and the depletion of
petrochemical resources. Consequently, in recent years, focus
has been directed towards the development of economically
effective and sustainable biological/biotechnological ap-
proaches to produce lactic acid on an industrial scale (Wee
et al. 2006).
Microorganisms in general play an important role in waste
utilization through their ability to degrade/convert organic
waste, such as wastes from various food-processing indus-
tries, and lactic acid bacteria (LAB) can produce lactic acid
using such wastes as substrates (Datta et al. 1995). Historical-
ly, refined sucrose was the most commonly used substrate for
the industrial production of lactic acid. More recently, corn
refiners have started to manufacture lactic acid from corn
starch, which is produced as a by-product from the wet milling
of corn: the starch is liquefied and saccharified by enzymes to
glucose, which is then fermented to lactic acid (Shen and Xia
2006). However, the industrial application of an microorgan-
ism which could produce lactic acid directly from starch-
containing wastes would obviate the need for saccharification
and/or liquefaction, resulting in large savings in production
costs. As a first step in this direction, various LAB, including
Lactococcus lactis ssp. lactis ATCC 19435, L. lactis and
Lactobacillus delbrueckii, L. casei NRRL B-441 and L.
amylovorus ATCC 33620, have been shown to have the
S. Bhanwar :A. Singh :A. Ganguli (*)
Department of Biotechnology and Environmental Sciences, Thapar
University, Patiala, PB 147004, India
e-mail: aganguli@thapar.edu
Ann Microbiol
DOI 10.1007/s13213-013-0754-2

2. ability to produce lactic acid directly from starchy substrates
(John et al. 2009).
The aim of our study was to develop a process in which L-
lactic acid can be produced from a cheap agro-processing
industry waste, namely, potato starch waste, by direct fermen-
tation. We also attempted to increase lactic acid production by
setting up a novel dialysis sac-based bioreactor to facilitate the
recycling and reuse of the L. lactis cells for an extended
period.
Lactococcus lactis subsp. lactis used in this study is a lactic
acid-producing strain isolated from pickled yam (Bhanwar
et al. 2013). Three types of media were used in this study
(1) de Man–Rogosa–Sharpe (MRS medium; Sigma, St. Louis,
MO), (2) modified MRS medium (containing potato starch
instead of D-glucose), (3) potato starch waste (PSW; a kind
gift from PepsiCo., Patiala, India; stored at 4 °C until used).
The strain was maintained on MRS agar plates at 4 °C and
sub-cultured weekly. All other chemicals and reagents used
were of highest grade available commercially.
To test the feasibility of improving lactic acid production
by free cells, various experiments were conducted in MRS
medium, modified MRS medium and PSW by inoculating
approximately 108
CFU/mL seed culture into flasks contain-
ing 100 mL medium. The flasks were incubated with shaking
at 120 rpm, 30 °C for 96 h. The concentration of carbon source
in the media was varied (0.5, 1, 2 %). PSW was diluted to
achieve the same final concentration of starch as the carbon
source in the other two media. Aliquots (5 mL) of each culture
were withdrawn at 8-h intervals and centrifuged at 10,000 rpm
for 10 min; residual starch and lactic acid were estimated in
the supernatant. In a parallel experiment, the pellet was
washed and resuspended in 0.85 % saline before absorbance
was read at 600 nm to obtain the cell density.
To facilitate the recycling and reuse of the L. lactis cells,
we used a novel bioreactor equipped with a dialysis mem-
brane (Ganguli and Tripathi 2002) for improved lactic acid
production (Fig. 1). Briefly, dialysis tubing (Cellulose; Sigma-
Aldrich) was thoroughly washed with sterile triple-distilled
water and clipped at one end to produce a sac. The wet weight
of log phase L. lactis cells (10 mg) corresponding to 5.5×
108
CFU/mL was packed into the dialysis sac. Each sac was
then tied at the upper end with clips and submerged complete-
ly in a vessel containing one of the culture media and incu-
bated with stirring at 30 °C for 96 h. Aliquots (5 mL) of
medium were withdrawn at various intervals and used to
estimate residual starch and lactic acid.
During fermentation, the pH was maintained at 5.5 by
adding 2 mol/L NaOH or 1 mol/L HCl, and the temperature
Fig. 1 Experimental setup for the
study of starch utilization in fed
batch fermentation by cells of
Lactococcus lactis in the potato
starch waste (PSW) medium. The
volume in the vessel was
maintained at 500 mL and the
temperature was monitored using
a temperature sensor
Ann Microbiol

3. was kept at 30 °C, which had been determined earlier to be the
optimal culture temperature (data not shown). Liquid samples
(5 mL) were taken at predetermined time intervals and centri-
fuged at 10,000 rpm for 10 min. The supernatants were
collected and properly diluted for high-performance liquid
chromatography (HPLC) analysis. Cell growth was monitored
by measuring the optical density at 600 nm using a UV-
spectrophotometer.
Due to starch depletion from the media over time, during the
course of the experiment we withdrew medium from the
dialysis bioreactor and replaced it with fresh medium. The
allowed us to check the reusability of the dialysis sac-
entrapped cells for lactic acid production. The viability and
stability of the cells entrapped in the dialysis sac was checked
by diluting them in phosphate buffered saline (pH 7.2) and
plating them on MRS agar plates under sterile conditions.
Lactic acid production was estimated as described below.
Lactic acid was analyzed on an HPLC system ( 1200
Series; Agilent Technologies, Santa Clara, CA) coupled with
an UV–VIS detector and an HPLC column (Acclaim Organic
Fig. 2 Kinetic study of L-lactic
acid production in shake flask
fermentation (SFF) using
modified de Man–Rogosa–
Sharpe (MRS) medium (a), MRS
medium (b) and PSW-containing
medium (c)
Ann Microbiol

4. acid Column; 5 μm, 4×250 mm; Thermo Scientific, Waltham,
MA). The mobile phase was a sodium sulfate (100 mM)
solution (pH 2.65 adjusted with MSA), and an isocratic elu-
tion was used at a flow rate of 0.6 mL/min. The detection of
lactic acid was set at λ=210 nm (Naveena et al. 2005). The
calculation of lactic acid content was based on the peak area
registered at the specific retention time for lactic acid and by
taking the regression curve factor into account. Residual
starch was detected as described by Giraud et al. (1993).
All analyses were done in triplicate, and the statistical
comparison of data was performed by analysis of variance to
reveal significant differences for each parameter among spe-
cies. The correlation coefficient (R2
) and P value were used to
show correlations and their significance. A probability value
of P< 0.05 was adopted as the criterion for a significant
difference.
Lactic acid production was initially quantified in shake
flasks (control) under previously optimized conditions of tem-
perature (30 °C) and pH (5.5) (unpublished data). Lactic acid
productivity increased with increasing cell number which led
in turn to a decreasing starch concentration (Zhou et al. 1999).
There was a gradual increase in lactic acid production with
increasing initial starch concentration from 0.5 to 2 %, but
there was no significant increase at 3 % starch concentration
(data not provided). One possible explanation for this findings
is decreased free availability. It is possibly that at lower
concentrations (0.5–2 % starch), the starch was readily avail-
able to L. lactis cells, but as the concentration increased to
>2 %, the availability of free starch increased to such a level
that it did not dissolve completely in water to become avail-
able to the cells, thereby decreasing the production of lactic
acid.
Fig. 3 Kinetics of lactic acid
production and starch degradation
in PSW-containing medium in
SFF (a) and fed batch
fermentation (b) in a dialysis sac
bioreactor
Ann Microbiol

5. The maximal lactic acid concentration in MRS medium
(19.5 g/L) (Fig. 2a) was 1.11-fold higher than that in modified
MRS medium (15.6 g/L) (Fig. 2b) and 1.59-fold higher than
that in PSW-containing medium(12.25 g/L) (Fig. 2c). These
results indicate that the glucose in the MRS media is a more
readily available carbon source for the production of lactic
acid (Cock and de Stouvenel 2006) than starch granules.
When L. lactis cells were directly used in the shake flask
fermentation (SFF) containing either MRS broth or modified
MRS broth or PSW, the cells remained viable for 2 days.
Complete starch degradation was not achieved due to the loss
in cell viability caused by the acidic environment that developed
in the culture flasks. The production of lactic acid did not
increase significantly even after starch supplementation
(Fig. 3a). Therefore, to overcome these problems and facilitate
recycling and reuse of the cells, we tested the media and L.
lactis cells in a two-in-one dialysis sac-based bioreactor (Fig. 1).
Starch/glucose (20 g/L) was completely consumed within
24 h in the dialysis sac bioreactor (Fig. 3b) in comparison to
>48 h by free cells in the control shake flasks. Lactic acid
productivity (0.79 g/L·h) and yield (0.94 g/g) in the dialysis
bioreactor were 2.4- and 1.2-fold higher, respectively, than
those (0.33 g/L·h and 0.78 g/g) in the control flasks. The final
lactic acid concentration (18.9 g/L) after 24 h of culture in the
dialysis bioreactor with 20 g/L starch as substrate was 1.2-fold
higher than that in the control (15.6 g/L) after 48 h. When the
initial glucose/starch concentration was increased to 30 g/L
(data not shown), glucose/starch was not completely mixed
within the medium and became unavailable to the cells, there-
by decreasing lactic acid productivity and yield (0.55 g/L·h
and 0.51 g/g, respectively).
To test the reusability of the cells, after each batch of
fermentation, the broth was withdrawn and an equal volume
of fresh medium was added to the bioreactor. Figure 3a shows
that lactic acid productivity and yield dropped in the shake
flasks after the first cycle of fermentation and remained almost
unchanged after the second cycle of fermentation. The re-
duced lactic acid productivity and yield might be ascribed
to the acidic environment (pH 2.5–3.0) of the fermenta-
tion broth which negatively affected the activity of the
cells whose maximum activity is at pH 5.5–6.2. Lactic
acid productivity (0.79 g/L·h) and yield (0.94 g/g) were higher
in the dialysis bioreactor than in the control and increased for
up to four cycles of fermentation (Fig. 3b), following which the
productivity remained unchanged. The reusability of cells im-
plies that the dialysis bioreactor could be an efficient alternative
for the treatment of PSW.
There have been several reports on the production of lactic
acid using electrodialysis (Min-tian et al. 2005; Huang et al.
2007), but to our knowledge there has been no report on the use
of a dialysis sac bioreactor for lactic acid production. More
specifially, we believe that our study is the first to examine the
recovery of lactic acid from an industrial waste using probiotic
bacteria in the setting of a dialysis sac bioreactor. One additional
interesting finding is that the cells contained in the dialysis sac
and the enzymes produced can be recovered and may be used
for industrial use.
The dialysis sac-based bioreactor used in our study was
effective in improving lactic acid production from PSW.
Higher levels of lactic acid productivity, yield and starch
degradation were achieved in the dialysis sac bioreactor than
in the SFF (control). Based on our results, we suggest that the
dialysis sac bioreactor represents an easier setup and is more
cost effective than electrodialysis, which has been used for
years to produce lactic acid. This is the first report of such a
reactor being adapted for direct starch conversion to L-Lactic
acid production. Rigorous large-scale studies are warranted
before this technology can be used in industrial-scale waste-
water treatment plants. However, the technology may lead to a
new process for the treatment of starch-enriched agro-waste
and the production of a non-toxic by-product (lactic acid)
using indigenous LAB isolated from pickled yam. We have
demonstrated that our novel dialysis bioreactor not only in-
creases lactic acid productivity but that it is also cost effective
due to the use of a relatively inexpensive substrate.
Acknowledgments The authors thank the University Grants Commis-
sion for financial support in the form of a fellowship (RGNF) to one of the
authors (Ms. Seema Bhanwar) and the Director, Thapar University, for
providing the infrastructure for the work.
Conflict of interest None.
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