Optimization of lactic acid production from cheap raw material: sugar cane molasses

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Pak. J. Bot., 44(1): 333-338, 2012.
OPTIMIZATION OF LACTIC ACID PRODUCTION FROM CHEAP RAW
MATERIAL: SUGARCANE MOLASSES

UMAR FAROOQ1*, FAQIR MUHAMMAD ANJUM2, TAHIR ZAHOOR2, SAJJAD-UR-RAHMAN3,
MUHAMMD ATIF RANDHAWA2, ANWAAR AHMED4 AND KASHIF AKRAM1

1Institute of Food Science and Nutrition, University of Sargodha, Sargodha.
2National Institute of Food Science and Technology, University of Agriculture, Faisalabad.
3Department of Veterinary Microbiology, University of Agriculture, Faisalabad.
4Department of Food Science and Technology, University of Arid Agriculture, Rawalpindi, Pakistan.

Abstract

Biotechnological process is advantageous over chemical one as we can utilize cheap raw materials as carbon source
such as agro-industrial byproducts and can produce the pure lactic acid in a very economic way. Sugar cane molasses is
available in many countries as byproduct of sucrose production, which can be used as substrate for lactic acid fermentation.
Process variables were optimized for the production of lactic acid by Lactobacillus delbrueckii using food wastes as
substrates. The productivity was found to be affected by fermentation time, temperature and the level of substrate. The
maximum lactic acid production was achieved after 7 days of fermentation in media containing 18% substrate level with a
mean value of 7.76±0.08 g/100 ml (77.6 g/l) at 42ºC. The maximum recovery of lactic acid with respect to initial total sugar
contents of the media (9.91±0.20 g/100 ml) was 78.30%.

Introduction

Lactic acid is one of the functional and valuable
compounds utilized in food, pharmaceutical and chemical
industries. It is industrially produced either by chemical
synthesis or by microbial fermentation. A biological
method has advantage that an optically pure lactic acid
can be obtained by choosing a strain of lactic acid
bacteria, whereas chemical synthesis always results in a
racemic mixture of lactic acid (Ryu et al., 2003).
Lactic acid bacteria are traditionally fastidious
microorganisms and have complex nutrient requirements
(Fitzpatrick & OKeeffe, 2001). Refined sugars (glucose
or sucrose) have been more frequently used to produce
lactic acid (Hofvendahl & Hahn, 1997). However, these
are economically not feasible due to high cost of pure
sugars whereas the product (lactic acid) is relatively
cheap. The production of lactic acid through fermentation
technology in industry is mainly dependent on cost of raw
material to be used. Therefore, it is mandatory to have a
raw material for industrial production of lactic acid with
several characteristics such as low cost, minimum level of
contaminants, rapid fermentation rate, high lactic acid
production yields, little or no by-product formation and
year-round availability (Ryu et al., 2003).
Food industrial wastes, high in moisture and rich in
carbon source have been considered as attractive nutrient
source for industrial lactic acid production. In Pakistan,
thousand tons of agro-based industrial wastes are
generated every year (John et al., 2009). The production
of sugar cane molasses only has been reported to be 1.3 to
2.1 million tones during 1994 to 2008 (Anon., 2009),
which is a cheaper substrate as a source of sugars to be
utilized in a fermentation process subsequently for lactic
acid production (Rashid & Altaf, 2008).
The molasses is a syrupy material left after the
removal of sugar from the mother syrup. This viscous
material is composed of sucrose, glucose and fructose at
total carbohydrate concentration of 45-60% (Mariam et
al., 2009). In Pakistan, sugarcane is grown in the three
zones and is an important and the second largest cash crop
of Pakistan grown over an area of 963,000 hectares with
an average yield of 47 tons/hectare (Ather et al., 2009).
Normally molasses contains about 46% total sugars, 3.0%
crude protein, 0.0% fat with 79.5° brix (Curtin, 1983) and
are used in animal feed as well as for the production of
alcohol through fermentation. As it contains high amount
of sugars (46%), it must not be considered as a waste
material instead researchers should make efforts to
produce value added products from this cheaper source of
carbohydrates/sugars (Rashid & Altaf, 2008).
In this study, attempts were made for lactic acid
production using sugar cane molasses as cheaper
available raw substrate through a fermentation process
with indigenous bacterial
delbrueckii). The fermentation conditions were optimized
considering fermentation time, temperature and substrate
level as main process factors.

Materials and Methods

Strain and growth medium: The indigenous culture of
Lactobacillus delbrueckii, a homofermentative lactic acid
producer, was utilized in the study. The culture was
isolated from yoghurt and was identified by following the
procedure given by Harrigan (1998). The stock cultures
were maintained at 4ºC in 250 mL MRS broth (DeMan’s
Sharpe and Rogosa Broth) with sub-culturing after every
10 days throughout the experiment. The medium for cell
growth contained (g L-1): peptone 10.0, meat extract 10.0,
yeast extract 05.0, d-glucose 20.0, tween-80 01.0,
K2HPO4 02.0, sodium acetate 05.0, tri-ammonium citrate
02.0, MgSO4.7H2O 0.2, MnSO4.4H2O 0.05 in 1000 mL
water.

Inoculum preparation: Lactobacillus delbrueckii cells
from stock cultures were transferred to sterile growth
medium (MRS agar plates) and incubated at 38 ºC for 24
hours. Then the culture was inoculated into MRS broth in
screw capped test tubes, incubated for 24 hours at 38 ºC
and was used for fermentation process.

culture (Lactobacillus

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UMAR FAROOQ ET AL.,

334
Fermentation: The lactic acid fermentation of sugar
molasses was carried out by the Lactobacillus strain at
34ºC, 38ºC and 42ºC with 0, 6, 12, 18 and 24% substrate
levels (Luis et al., 2003). The fermentation media
contained (g 100mL-1); peptone 10.0, meat extract 10.0,
yeast extract 05.0, Tween-80 01.0, K2HPO4 02.0, Sodium
acetate 05.0, tri-ammonium citrate 02.0, MgSO4.7H2O
0.2, MnSO4.4H2O 0.05, with different percent levels of
sugar cane molasses (0, 6, 12, 18, 24) and water 100 mL
(Glucose was replaced with sugar cane molasses).

Analysis: Fermentation medium was filtered through
Whatman filter paper and the filtrate was used for the
estimation of sugars and lactic acid on daily basis up to 8
days of fermentation. Total sugars were estimated using
Lane and Eynon method by titration with Fehling’s
solution (Kirk & Sawyer, 1991). Lactic acid was
estimated by high performance liquid chromatography
(Akalin et al., 2002) as described below:

Sample preparation: Seven mL of fermented media was
added to 40 ml of buffer-acetonitrile mobile phase (0.5 %
(m v-1) (NH4)2HPO4 (0.038 M) - 0.4 % (by volume)
acetonitrile (0.049 M), at pH=2.24 with H3PO4, extracted
for 1 hour in orbital shaker and centrifuged at 6000 x g for
5 min. The supernatant was collected and filtered once
through Whatman #1 filter paper and twice through a 0.45-
µm membrane filter, and then used directly for HPLC
analysis. Triplicate analyses were performed on all samples.

HPLC analysis: Samples for Lactic acid production were
analyzed through HPLC with UV detector (Perkin Elmer-
series 200) at 214 nm using RP-18 column (120 x 4.6 mm).
The operating conditions were: mobile phase, aqueous
0.5% (m v-1) (NH4)2HPO4 (0.038 M) - 0.2% (by volume)
acetonitrile (0.049 M) adjusted to pH=2.24 with H3PO4;
flow rate 0.3 mL min-1; ambient column temperature. The

Table 1. Mean squares for utilization of total sugars and lactic acid production from sugar cane molasses.
mobile phase was prepared by dissolving analytical-grade
(NH4)2HPO4 in distilled deionized water, HPLC-grade
acetonitrile, and H3PO4. HPLC-grade reagents were used as
standards (Sigma Chemical Co., St. Louis, MO). Solvents
were filtered through a 0.45-µm membrane filter and
degassed under vacuum. Quantification was based on
internal standard method.

Statistical analysis: The statistical analyses of the data
were carried by following three factor factorial
experiment and DMR test by the software Minitab (Steel
et al., 1997).

Results

Table 1 indicates the analysis of variance for total
sugar utilization and lactic acid production from sugar
cane molasses. Different levels (0, 6, 12, 18 and 24) of
sugar cane molasses subjected to fermentation at different
temperatures (34, 38 and 42ºC) in a specifically designed
medium revealed that the fermentation time and substrate
levels were highly significant (p<0.05) and effective in
utilizing total sugars present in molasses during the
fermentation process. The effect of temperature was
found to be non-significant. Similarly, the first order
interactions of all the variables were non-significant
except the one between fermentation time and substrate
levels on utilization of total sugars in the media during
fermentation. The results indicated that the lactic acid
production was affected significantly by the selected
fermentation time, temperature and substrate levels.
However, the first order interactions among time x
temperature, temperature x time, and time x temperature x
substrate level were found to be non-significant, except
the interaction of fermentation time and substrate levels,
which was highly significant (p<0.05).
Mean squares
Source of variance df
Utilization of total sugars
356.83**
0.04 NS
541.05**
0.008 NS
23.27**
0.002 NS
0.003 NS
0.055

Lactic acid production
183.34**
0.28**
205.09**
0.03 NS
7.94**
0.05 NS
0.02 NS
0.04

Fermentation time (D)
Temperature (T)
Substrate level (S)
D x T
D x S
T x S
D x T x S
Error
Total
**Highly significant (p<0.01), *Significant (p<0.05), NS Non-significant (p>0.05)

Figure 1 shows the utilization of total sugars with the
passage of fermentation time. Utilization of total sugars
increased as the time of fermentation was increased
decreasing the available sugar content in the media. The
results indicated that 8.16 g/100 ml total sugar contents
were utilized after 8 days of fermentation process with a
gradual increase from the initial point. However, the
utilization of total sugars was lower at the start of
fermentation. This indicates encouraging utility of sugars
during fermentation time period (days).
Figure 2 indicates the proportional increased use of
available sugars by the Lactobacillus delbreukii as the
level of dose was increased from 0.00 to 9.91 g 100mL-1.
8
2
3
16
24
6
48
216
323
The highest utilization (8.04 g/100 ml) of total sugars was
found in 24% substrate level followed by 18% (6.05
g/100 ml) substrate level. However, utilization of total
sugars was not observed in the medium without substrate.
The first order interaction between fermentation time
and substrate levels revealed that the highest utilization of
total sugars occurred after 8 days of fermentation period
whereas, low amount of total sugars was utilized at the
start of experiment regardless of substrates levels.
However, no total sugars utilization was detected in the
media without substrate where no sugar source was added
in the media, throughout the fermentation period. The
utilization of total sugars ranged from 0.00 to 12.95 g/100

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LACTIC ACID PRODUCTION FROM SUGAR CANE MOLASSES
335
ml (Fig. 3, Table 2). The results further showed that per
day utilization of total sugars was the maximum (2.4 g
100mL-1) at 3rd day of fermentation whereas in
subsequent days sugar utilization was continuously
decreased (Fig. 4). It was observed that lactic acid
production persistently increased up to (5.85 g 100mL-1)
with an increase in fermentation time till the completion
of fermentation, although non significant increase was
observed in between 7th and 8th day of fermentation (Fig.

5).The lactic acid production from sugar cane molasses
was significantly higher at 42 than 34 and 38ºC
temperature treatments. The quantity of lactic acid
produced was 3.77 g/100 ml, 3.69 g/100 ml and
3.67g/100 ml at 42, 38 and 34ºC temperature,
respectively. However, the difference in lactic acid
production between 34 and 38ºC temperature was found
statistically non-significant (Fig. 6).

Fig. 1. Effect of fermentation time on utilization of total sugars
during lactic acid production from sugar cane molasses.


Fig. 2. Effect of substrate level on utilization of total sugars
during lactic acid production from sugar cane molasses.

Fig. 3. Effect of fermentation time and substrate level on
utilization of total sugars during lactic acid production from
sugar cane molasses.

Fig. 4. Effect of fermentation time and substrate level on per day
utilization of total sugars during lactic acid production from
sugar cane molasses.


Fig. 5. Effect of fermentation time on lactic acid production
from sugar cane molasses.


Fig. 6. Effect of temperature on lactic acid production
from sugar cane molasses.
Fermentation time (Days)
Fermentation time (Days)
Fermentation time (Days)
Fermentation time (Days)
Substrate level (%)
Temperature (oC)
Utilization for total sugars
(g 100 mL-1)
Utilization for total sugars
(g 100 mL-1)
Utilization for total sugars
(g 100 mL-1)
Utilization for total sugars
(g 100 mL-1)
Lactic acid (g 100 mL-1)
Lactic acid (g 100 mL-1)

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UMAR FAROOQ ET AL.,

336

Table 2. Effect of fermentation time and substrate level on utilization of total sugars (g/100 ml) during lactic
acid production from sugar cane molasses.
Substrate level (%)
Fermentation time
(Days)
6 12
0 0.00 ± 0.00 x 0.00 ± 0.00 x
1 0.38 ± 0.01 w 0.70 ± 0.01 v
2 0.99 ± 0.01 u 2.06 ± 0.02 s
3 1.94 ± 0.01 s 3.80 ± 0.08 p
4 2.55 ± 0.02 r 4.98 ± 0.04 o
5 2.76 ± 0.03 r 5.56 ± 0.09 n
6 3.07 ± 0.02 q 6.12 ± 0.05 m
7 3.30 ± 0.02 q 6.42 ± 0.07 l
8 3.30 ± 0.02 q 6.60 ± 0.05 l
Mean 2.04 ± 0.13 D 4.03 ± 0.26 C
Means carrying same letters are not significantly different
18 24
Mean
0.00 ± 0.00 x
1.03 ± 0.02 u
3.08 ± 0.02 q
5.97 ± 0.04 m
7.49 ± 0.07 k
8.41 ± 0.04 i
9.14 ± 0.07 h
9.53 ± 0.14 g
9.80 ± 0.17 f
6.05 ± 0.40 B
0.00 ± 0.00 x
1.37 ± 0.02 t
4.00 ± 0.01 p
8.01 ± 0.06 j
10.03 ± 0.05 e
11.32 ± 0.08 d
12.20 ± 0.08 c
12.44 ± 0.04 b
12.95 ± 0.23 a
8.04 ± 0.53 A
0.00 ± 0.00 I
0.87 ± 0.07 H
2.53 ± 0.21 G
4.93 ± 0.43 F
6.26 ± 0.53 E
7.01 ± 0.60 D
7.64 ± 0.65 C
7.92 ± 0.66 B
8.16 ± 0.69 A


Table 3 Effect of time and substrate on lactic acid production from sugar cane molasses.
Substrate level (%)
Fermentation time
(Days)
6
0 0.00 ± 0.00 u 0.00 ± 0.00 u
1 0.31 ± 0.01 t 0.57 ± 0.02 s
2 0.81 ± 0.02 r 1.66 ± 0.03 p
3 1.57 ± 0.03 p 3.069 ± 0.07 m
4 2.06 ± 0.05 o 4.04 ± 0.07 k
5 2.19 ± 0.05 o 4.53 ± 0.06 j
6 2.54 ± 0.07 n 4.94 ± 0.10 hi
7 2.65 ± 0.05 n 5.22 ± 0.07 g
8 2.71 ± 0.06 n 5.41 ± 0.09 g
Mean 1.65 ± 0.11 C 3.27 ± 0.22 B
Means carrying same letters are not significantly different
12 18 24
Mean
0.00 ± 0.00 u
0.83 ± 0.02 r
2.58 ± 0.04 n
4.98 ± 0.05 h
6.24 ± 0.11 e
7.10 ± 0.09 c
7.72 ± 0.10 a
7.76 ± 0.08 a
7.73 ± 0.07 a
4.99 ± 0.33 A
0.00 ± 0.00 u
1.14 ± 0.02 q
3.37 ± 0.04 l
4.75 ± 0.13 i
6.01 ± 0.12 f
6.65 ± 0.08 d
7.34 ± 0.09 b
7.59 ± 0.06 a
7.55 ± 0.09 a
4.93 ± 0.30 A
0.00 ± 0.00 H
0.71 ± 0.06 G
2.11 ± 0.18 F
3.59 ± 0.29 E
4.59 ± 0.36 D
5.12 ± 0.40 C
5.63 ± 0.44 B
5.80 ± 0.45 A
5.85 ± 0.44 A


The lactic acid production with 18% substrate levels
was found significantly the highest (4.99 g/100 ml)
although 24% substrate yielded 4.93 g/100 ml, which was
non-significant with the yield obtained from 18% (Fig. 7).
The substrate levels (6 and 12%) proportionally produced
maximum lactic acid. However, no lactic acid was
produced in the medium lacking substrate due to non-
availability of carbon source to the microorganisms.
The interaction of fermentation time and substrate
levels (Fig. 8) showed an increasing trend of lactic acid
production with increase in fermentation time and

substrate levels. Significantly the highest lactic acid
production took place after 7 days of fermentation (7.76 ±
0.08 g/100 ml) in the media containing 18% cane
molasses substrate as carbon source (Table 3). Rapid
increase in lactic acid production was observed at 2nd and
3rd day of fermentation with maximum at 7 days of
fermentation whereas, it persisted uptil 8th with a non
significant difference with the yield of day 7,
corresponding to the utilization of total sugars stated
elsewhere (Fig. 9).

Fig. 7. Effect of substrate level on lactic acid production from
sugar cane molasses.

Fig. 8. Effect of fermentation time and substrate level on lactic
acid production from sugar cane molasses
Lactic acid (g 100 mL-1)
Lactic acid (g 100 mL-1)
Fermentation time (Days)
Substrate level (%)

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LACTIC ACID PRODUCTION FROM SUGAR CANE MOLASSES
337

Fig. 9. Effect of fermentation time and substrate level on per day
lactic acid production from sugar cane molasses.

Discussion

Microorganisms are not only well known due to their
harmful effects such as diseases and food spoilage but
also make key contributions to the welfare of humanity
(Anon., 1990; Tortora et al., 2010). These are widely used
in food industry for production of a large number of
fermented food products and at the same time these are
also very helpful for conversion of food industrial wastes
into value added products (Laufenberg et al., 2003). The
ability of microorganisms to convert large and complex
molecules into the simplest one depends upon the type of
culture and the growth requirements which include both
intrinsic and extrinsic environmental conditions (Lucas et
al., 2007). The selection of a suitable culture to convert
the specific type of substrate into useful products thus
plays a vital role in fermentation technology (Krishna,
2005). For the production of useful products at industrial
level, attempts are being made by the researchers to
identify the cheapest and easily available substrates to
reduce the cost of production (John et al., 2006).
Thus overall 12.95 g/100 ml (97.30%) total sugars
were utilized during fermentation of sugar cane molasses.
The other researcher also found 78 to 89% and 47.2%
total sugars conversion to lactic acid under different
fermentation conditions (Nancib et al., 2001)
working on the effect of supplementation by different
nitrogen sources on the production of lactic acid from
date juice by Lactobacillus casei subsp. Rhamnosus.
Similarly, El-Sherbiny et al., (1986); Mintian et al.,
(2005); Richter & Berthold (1998) also observed 80.7, 71
and 89% total sugar utilization during lactic acid
production from different substrates used during studies
on lactic acid production. In our study, more utilization of
total sugars was observed during fermentation of sugar
cane molasses in comparison to the findings of the
researchers stated therein. The reason might be the
selection of culture and substrate (composition) sucrose to
avail carbon source of energy in sugar cane molasses.
Conversion rate of total sugar to lactic acid basically
depends mainly on substrate for growth of the lactic acid
bacteria/culture (John et al., 2009)
In case of lactic acid production, our results fall within the
limits i.e., 11.27 to 85.03% lactic acid recoveries after 96
while
hours (4 days) of fermentation by Lactobacillus
delbrueckii (Luis et al., 2003). The results are also
supported by the findings of other researcher (Kim et al.,
2003) who, achieved 91% overall yield of lactic acid at
42°C with initial pH=6.0 of the media during lactic acid
production from food wastes through fermentation with
Lactobacillus delbrueckii.
Several other researchers haw observed different
yield production ranging from (51- 97%) the production
of lactic acid using various substrates during optimizing
the fermentation conditions(El-Sherbiny et al., 1986;
Mintian et al., 2005; Richter & Berthold, 1998; Narita et
al., 2004; Aksu & Kutsal, 1986; Garde et al., 2002; Bai
et al., 2003; Lee et al., 2004). The yield of lactic acid
produced in our experiment falls within the range of
previous work done. The differences in lactic acid
recovery during this study with that of some other
researchers might be due to the variation in temperature,
substrate levels, types of substrates and the selection of
suitable culture for fermentation.

Conclusion

The lactic acid production is significantly influenced
by fermentation time, temperature and substrate levels.
The effect of these parameters on lactic acid production is
linear and after 6 days of fermentation stationary phase is
achieved which however depends upon fermentation
conditions. The maximum lactic acid was produced after
7 days of fermentation in media containing 18% substrate
level with a mean value of 7.76 ± 0.08 g/100 ml (77.6 g/l)
at 42ºC. The maximum recovery of lactic acid with
respect to initial total sugar contents of the media (9.91 ±
0.20 g/100 ml) was 78.30%. For continuous production of
lactic acid under these conditions the media should be
reconstituted after 3 days of fermentation as optimum per
day acid production is after 72 hours (3 days)

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of a continuous
(Received for publication 23 April 2010)