Utilization of molasses sugar for lactic acid production by 1

Lactobacillus delbrueckii subspecies delbrueckii mutant Uc-3 in 2

batch fermentation. 3
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Running title: Lactic acid from molasses in batch fermentation 6
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Arti Dumbrepatil, Mukund Adsul, Shivani Chaudhari, Jayant Khire, and 10
Digambar Gokhale* 11
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NCIM Resource Center 14
National Chemical Laboratory 15
Pune 411 008, India 16
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Corresponding Address: Dr. D.V. Gokhale 20
Scientist In-Charge, NCIM 21
National Chemical Laboratory 22
Pune 411 008, maharashtra, India 23
Tel: 91-20-25902670 24
Fax: 91-20-25902671 25
Email: dv.gokhale@ncl.res.in 26
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Copyright 2007, American Society for Microbiology and/or the Listed Authors/Institutions. All Rights Reserved.
Appl. Environ. Microbiol. doi:10.1128/AEM.01595-07
AEM Accepts, published online ahead of print on 2 November 2007

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Abstract 32
Efficient lactic acid production from cane sugar molasses by Lactobacillus 33
delbrueckii mutant Uc-3 in batch fermentation process is demonstrated. Lactic acid 34
fermentation using molasses was not significantly affected by yeast extract 35
concentrations. The final lactic acid increased with the increase of molasses sugar 36
concentrations up to 190 g/liter. The maximum lactic acid concentration of 166 g/liter 37
was obtained at molasses sugar concentration of 190 g/liter with a productivity of 4.15 38
g/liter/h. Such high concentration of lactic acid with high productivity from molasses is 39
not reported so far and hence mutant Uc-3 could be a potential candidate for economical 40
production of lactic acid from molasses at commercial scale. 41
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Utilization of molasses sugar for lactic acid production by Lactobacillus 55
delbrueckii mutant Uc-3 in batch fermentation 56
57
Lactic acid can be used as a preservative, acidulant, and flavor in food, textile and 58
pharmaceutical industries. It could become a commodity chemical for the production of 59
lactate esters, propylene glycol, propylene oxide, acrylic acid, 2,3-pentanedione, 60
propanoic acidacetaldehyde and dilactide (3, 15). It has also been receiving importance as 61
a feedstock for manufacture of polylactic acid (PLA) which could be a good substitute for 62
synthetic plastic derived from petroleum feedstock. Approximately 90% of the total lactic 63
acid produced worldwide is by bacterial fermentation (20). The chemical synthesis of 64
lactic acid always leads to racemic mixture which is major disadvantage. Fermentative 65
production of lactic acid offers great advantage in producing optically pure L- or D-lactic 66
and also DL-lactic acid depending on the strain selected for fermentation. The optical 67
purity of lactic acid is crucial factor to the physical properties of PLA and it is L(+) lactic 68
acid that can be polymerized to a high crystal PLA suited to commercial uses such as 69
fibers and films (14). 70
Most studies within production of lactic acid have focused on the use of pure 71
substrates such as glucose (9, 12) or lactose (5) for the production of lactic acid. The use 72
of natural substrates like starch (4, 10, 11, 18) and cellulose (1, 6, 16) are economically 73
unfavorable because they are much expensive and also require pretreatment in order to 74
release fermentable sugars. The manufacturing cost of lactic acid can be significantly 75
reduced if waste products such as whey or molasses containing fermentable sugars could 76
be used for the production of lactic acid. India is one of the largest countries producing 77
more than 20 million tons of cane sugar from sugar cane. During this process, large 78
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amount of molasses is generated as the byproduct which contains 40-60% sucrose which 79
can be converted to lactic acid by the use of microorganisms. 80
In this paper, we describe the efficient conversion of molasses sugar by a mutant 81
strain, Lactobacillus delbrueckii Uc-3, for lactic acid production. The mutant was isolated 82
by UV mutagenesis followed by selection on the basis of bigger zone of acid formation 83
on sucrose based medium (7). The mutant is reported to utilize glucose preferentially 84
from high concentrations of hydrolyzed cane sugar resulting in co-production of lactic 85
acid and fructose (13). Recently, we have reported the complete utilization of bagasse 86
derived cellulose to lactic acid with an 80% yield using this strain (1) which was 87
attributed to the presence of cellobiose and cellotriose enzymes present in the mutant 88
strain (2). 89
For the evaluation of lactic acid production from cane molasses, experiments were 90
performed in 250-ml, screw-cap flasks at 42
o
C with shaking at 150 rpm. The flask 91
contained 100 ml production medium consisting of hydrolyzed 10 g cane molasses sugar, 92
4.0 g CaCO
3
, and 0.5 g yeast extract. The cane molasses was obtained from Godavari 93
Sugar Mills Limited, Sameerwadi, India. The molasses contained sucrose (31%), glucose 94
(9.5%) and fructose (10%) and nitrogen (0.95%). The cane molasses sugar was 95
hydrolyzed by adding 1 ml of 20% H
2
SO
4
in 100 ml of molasses solution. The acidified 96
molasses solution was heated in boiling water bath for 20 min. The cane molasses 97
contained 46-48% reducing sugar. The pH of the medium was adjusted to 6.5 with 4.0 M 98
KOH prior to sterilization. The flasks were inoculated (5% inoculum) with culture grown 99
in hydrolyzed, sucrose based medium (7). The culture samples harvested after suitable 100
time intervals were centrifuged at 2000 x g for 20 min to separate the cells. The 101
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supernatant was analyzed for sugar and lactic acid and for determining the pH of 102
fermented broth. Lactic acid was analyzed by high pressure liquid chromatography 103
(HPLC) equipped with UV or refractive index detectors using an Aminex HPX-87H 104
column and sugar was analyzed by dinitrosalicylic acid (DNS) method as reported earlier 105
(7). The dry cell weight was determined by a calibration curve related with optical 106
density at 660 nm to dry weight (g/liter). One unit of optical density corresponded to 0.76 107
g/liter of dry cell weight. 108
The parent strain Lactobacillus delbrueckii NCIM 2365 and mutant Uc-3 were 109
evaluated initially for lactic acid production in molasses based fermentation medium 110
using 102 g/liter of cane molasses sugar and varying concentrations of yeast extract . The 111
mutant Uc-3 produced 4.5 times more lactic acid than the parent strain within 24 h using 112
102 g/liter of molasses sugar (Table 1). Lactic acid bacteria are generally fastidious 113
organisms requiring complex nutrients such as amino acids and vitamins for cell growth. 114
Yeast extract is most commonly used nitrogen source which provides vitamin B complex 115
content in addition to organic nitrogen to lactic acid bacteria (19). It was found that 2.5 116
g/liter yeast extract was sufficient to obtain maximum lactic acid with high productivity. 117
The requirement of less amount of yeast extract as nitrogen source could be attributed to 118
the presence of enough nitrogen in molasses. Further experiments were performed using 119
5 g/liter of yeast extract because we used higher molasses sugar concentrations.. In order 120
to investigate the influence of molasses concentration on lactic acid production, 121
Lactobacillus delbrueckii mutant Uc-3 was grown using 110 500 g/liter of molasses 122
(equivalent to 51 240 g/liter of total sugar). As shown in Table 2, the final lactic acid 123
concentration increased with increase in initial molasses sugar concentration up to 190 124
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g/liter. A sharp decrease in lactic acid production was observed at 240 g/liter of molasses 125
sugar concentration. This could be probably due to substrate inhibition, a phenomenon 126
observed in traditional batch fermentation. A maximum lactic acid production (166 127
g/liter) was obtained within 40 h of fermentation with an initial molasses sugar 128
concentration of 190 g/liter. At all molasses sugar concentrations, the lactic acid yields 129
were 0.88 0.96 g/g based on the total sugar consumed and the highest yield (0.96 g/g) 130
and productivity (4.3 g/liter/h) were obtained at 148 g/liter of molasses sugar 131
concentration. 132
The profile of growth (optical density), pH, lactic acid production and molasses 133
sugar utilization is shown in Fig 1. The maximum amount of lactic acid (166g/liter) was 134
produced from 190 g/liter of molasses sugar within 40 h of fermentation, with an increase 135
in optical density from 1.5 to 21.6 and a decrease in pH from 6.0 to 5.3. The decrease in 136
pH of the fermentation is not significant probably due to buffering action of molasses. 137
We observed drastic pH drop from 6.8 to 4.5 of the fermented broth when sucrose based 138
production medium was used for lactic acid production (7). Hence molasses based 139
production medium could be an advantage to maintain the pH of the fermentation 140
medium above 5.3 at which fermentation rates are faster. This could be one of the reasons 141
for high lactic acid productivity observed in molasses based medium. 142
Table 3 summarizes the results obtained from this work and from other literature 143
previously reported on lactic acid concentration, lactic acid yield and productivity in 144
batch fermentation of molasses. Recently, Wee et al (17) reported that maximum lactic 145
acid concentration of 134.9 g/liter was obtained at molasses concentration of 333 g/liter 146
(equivalent to 170 g/liter of total sugar) with productivity of 1.5 g/liter/h using 147
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Enterococcus faecalis. The high productivity of 4.3 was obtained at lower molasses 148
concentrations. However, their requirement of yeast extract is very high (15 g/liter) to 149
obtain highest productivity of lactic acid. Lactobacillus delbrueckii mutant Uc-3 used in 150
this study could produce 166 g/liter of lactic acid from 400 g/liter of molasses (equivalent 151
to 190 g/liter of total sugar). It is noteworthy to mention that we required very small 152
amount of yeast extract (5 g/liter) to obtain high lactic acid productivity even at high 153
molasses concentration. Therefore, the mutant Uc-3 proved to be an efficient strain for 154
production of lactic acid from high concentration of molasses with very high 155
productivity. 156
In conclusion, the Lactobacillus delbrueckii mutant Uc-3 proved to be a 157
promising strain for the production of lactic acid from molasses. The requirement of yeast 158
extract is brought down to minimum because molasses contains enough nitrogen source 159
necessary to grow such fastidious organisms. Also the buffering capacity of the molasses 160
could be an advantage in maintaining the pH of the medium above pH 5.3 during the 161
fermentation. Molasses was also proven to be an economically feasible raw material for 162
industrial production of lactic acid since it is fortified with enough nutrients necessary for 163
growth of lactic acid bacteria. 164
We acknowledge the financial support from NMITLI Division of the Council of 165
Scientific and Industrial Research, New Delhi, India. 166
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REFERENCES 171
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production from cellobiose and cellotriose by Lactobacillus delbrueckii mutant 175
Uc-3. Appl. Environment. Microbiol. 73: 5055 5057. 176
3. Akerberg, C., and G. Zacchi. 2000. An economic evaluation of the fermentative 177
production of lactic acid from wheat floor. Bioresour. Technol. 75: 119-126. 178
4. Altaf, M., B. J. Naveena, M. Venkateshwar, E. V. Kumar, and G. Reddy. 179
2006. Single step fermentation of starch to L(+) lactic acid by Lactobacillus 180
amylophilus GV6 in SSF using inexpensive nitrogen sources to replace peptone 181
and yeast extract optimization by RSM. Proc. Biochem. 41: 465-472. 182
5. Amrane, A., and Y. A. Prigent. 1996. A novel concept of bioreactor: specialized 183
function two-stage continuous reactor, and its application to lactose conversion in 184
to lactic acid. J. Biotechnol. 45: 195-203. 185
6. Chen, R., and Y. Y. Lee. 1997. Membrane-mediated extractive fermentation for 186
lactic acid production from cellulosic biomass. Appl. Biochem. Biotechnol. 63: 187
435-448. 188
7. Kadam, S. R., S. S. Patil, K. B. Bastawde, J. M. Khire, and D. V. Gokhale. 189
2006. Strain improvement of Lactobacillus delbrueckii NCIM 2365 for lactic acid 190
production. Proc. Biochem. 41: 120-126. 191
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8. Kotzamanidis, Ch., T. Roukas, and G. Skaracis. (2002). Optimization of lactic 194
acid production from beet molasses by Lactobacillus delbrueckii NCIMB 8130. 195
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9. Kwon, S., I. K. Yoo, W. G. Lee, H. N. Chang, and Y. K. Chang. 2001. High 197
rate continuous production of lactic acid by Lactobacillus bulgaricus in two-stage 198
membrane cell-cycle bioreactor. Biotechnol. Bioeng. 73: 25-34. 199
10. Linko, Y. Y., and P. Javanainen. 1996. Simultaneous liquefaction, 200
saccharification, and lactic acid fermentation on barley starch. Enzyme Microb. 201
Technol. 19: 118-123. 202
11. Ohkouchi, Y., and Y. Inoue. 2006. Direct production of L(+)-lactic acid from 203
starch and food wastes using Lactobacillus manivotivorans LMG18011. 204
Bioresour. Technol. 97: 1554-1562. 205
12. Olmos-Dichara, A., F. Ampe, J. L. Uribelarrea, A. Pareileux, and G. Goma. 206
1997. Growth and lactic acid production by Lactobacillus casei spp. Rhamnosus 207
in batch and membrane bioreactor: influence of yeast extract and tryptone 208
enrichment. Biotechnol. Lett. 19: 709-714. 209
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Gokhale. 2006. Production of lactic acid and fructose from media with cane sugar 211
using mutant of L. delbrueckii NCIM 2365. Lett. Appl. Microbiol. 43: 53-57. 212
14. Sodegard, A., and M. Stolt. 2002. Properties of lactic acid based polymers and 213
their correlation with composition. Prog. Polym. Sci. 27: 1123-1163. 214
15. Vardarajan, S., and D.J. Miller. 1999. Catalytic upgrading of fermentation 215
derived organic acids. Biotechnol. Prog. 15: 845-854. 216
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16. Venkatesh, K. V. 1997. Simultaneous saccharification and fermentation of 217
cellulose to lactic acid. Bioresour. Technol. 62: 91-98. 218
17. Wee, Y. J., J. N. Kim, J. S. Yun, and H. W. Ryu. 2004. Utilization of sugar 219
molasses for economical L(+)-lactic acid production by batch fermentation of 220
Enterococcus faecalis. Enzyme Microb. Technol. 35: 568-573. 221
18. Xiodong, W., G. Xuan, and S. K. Rakshit. 1997. Direct fermentative production 222
of lactic acid on cassava and other starch substrates. Biotechnol. Lett. 19: 841- 223
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19. Yoo, I. K., N. N. Cheng, E. G. Lee, Y. K. Cheng, and S. H. Moon. 1997. Effect 225
of B vitamin supplementation on lactic acid production by Lactobacillus casei. J. 226
Ferment. Bioeng. 84: 172-175. 227
20. Zhou, S., K. T. Shanmugam, L. P. Yomano, T. B. Grabar, and L. O. Ingram. 228
2006. Fermentation of 12% (w/v) glucose to 1.2 M lactate by Escherichia coli 229
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Table 1. Lactic acid production by parent and mutant strain in cane molasses sugar 240
241
Parent strain

Mutant Uc-3 Molasses
sugar
(g/L)
Yeast
extract
(g/L) Dry cell
weight
(g/liter)
Lactic
acid
(g/liter)
Productivity
(g/liter/h)
Dry cell
weight
(g/liter)
Lactic
acid
(g/liter)
Productivity
(g/liter/h)
0 4.50.3 8.00.7 0.330.05 5.80.6 43.63.5 1.800.1
2.5 5.00.4 14.01.2 0.580.06 9.20.8 74.05.0 3.080.2
5 5.70.3 16.41.1 0.680.04 8.70.6 73.03.7 3.040.1


102
10 5.80.3 16.81.1 0.700.04 9.80.7 73.24.0 3.050.2
242
The cultures were grown anaerobically at 42
o
C with shaking (150 rpm). The samples 243
were removed after 24 h of fermentation and analyzed for growth and lactic acid. The 244
values are average of three independent experiments. 245
246
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Table 2. Effect of initial hydrolyzed molasses sugar concentration on fermentation time 248
required, lactic acid production, dry cell weight and lactic acid productivity of mutant 249
Uc-3 250
251
Molasses sugar
(g/L)
Lactic acid
(g/liter)
Dry cell weight
(g/liter)
Lactic acid
yield (g/g)
Productivity
(g/liter/h)
51 40.02.4 (16) 7.420.4 0.88 2.500.2
102 84.63.8 (30) 8.750.3 0.94 2.820.1
123 105.06.5 (30) 14.00.9 0.95 3.500.2
148 129.07.0 (30) 14.00.8 0.96 4.300.2
190 166.07.5 (40) 15.10.8 0.95 4.150.2
240 88.25.0 (48) 2.700.2 0.94 1.830.1
252
The values are average of three independent experiments. 253
The values in parenthesis indicate time in hours when the samples were analyzed. 254
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Table 3: Comparison of different lactic acid overproducing strains grown on molasses in 255
batch fermentation. 256
257
Strains used Lactic
acid
(g/liter)
Molasses
sugar
(g/liter)
Lactic
acid
yield
(g/g)
Fermentation
time
(h)
Productivity
(g/liter/h)
References
Lactobacillus
delbrueckii
NCIMB 8130*
90.0 100 0.98 24 3.8 8
Enterococcus faecalis
RKY1**
95.7 102 0.95 24 4.0 17
Enterococcus faecalis
RKY1**
127.0 136 0.95 60 2.1 17
Enterococcus faecalis
RKY1**
134.9 170 0.93 90 1.5 17
Lactobacillus
delbrueckii
Uc-3***
129.0 148 0.96 30 4.3 This work
Lactobacillus
delbrueckii
Uc-3***
166.0 190 0.95 40 4.15 This work
258
Yeast extract concentration used with the various strains was as follows. 259
260
* 50 g/liter ** 15 g/liter *** 5g/liter 261
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0
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200
0 10 20 30 40
0
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25
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H

279
280
281
FIG. 1. Profile of lactic acid production, growth, pH, and sugar utilization during 282
fermentation by Lactobacillus delbrueckii mutant Uc-3 using molasses sugar 283
concentration of 190 g/liter. 284
Molasses sugar (), Lactic acid (), Growth (), pH () 285
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