Journal of Microbiology and Biotechnology Research
Scholars Research Library
J. Microbiol. Biotech. Res., 2012, 2 (1): 90-93
(http://scholarsresearchlibrary.com/archive.html)
ISSN : 2231 –3168
CODEN (USA) : JMBRB4
Microbial Production of Lactic Acid by Using Crude Glycerol from Biodiesel
Bhushan Choubisa, Hiren Patel, Mayank Patel, Bharatkumar Dholakiya*
Department of Applied Chemistry, Sardar Vallabhbhai National Institute of Technology,
(SVNIT), Surat, Gujarat, India
______________________________________________________________________________
ABSTRACT
Biodiesel derived raw glycerol represent a copious and inexpensive source which can be used as
raw material for lactic acid conversion. Lactobacillus delbrueckii strain when grown with 20 g
raw glycerol l-1, produced 4.37 g lactic acid l-1 after 2 days and Lactococcus lactis subsp.la
strain with pure glycerol produced 2.26 g lactic acid l-1
Keywords Biodiesel, crude glycerol, lactic acid.
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INTRODUCTION
Biodiesel which is derived from the transesterification reaction of a triglyceride (fat/oil) with an
alcohol to form ester and glycerol [1, 2]. Synthesis of biodiesel generates glycerol as a byproduct is yielded at about 10% of biodiesel production. [3]. As per the reported study, the crude
glycerol represents a copious and inexpensive source which can be converted to value added
products such as 1, 3-propanediol, polyhydroxyalkanoate, hydrogen, epichlorohydrin and also
lactic acid. [4, 5]. The higher revenue from the byproduct, which benefits itself from the
economies of scale available in a large biofuels plant, would improve the economics of biofuel
production.
Lactic acid (2-hydroxyproponoic acid) has wide range of application in food, leather, cosmetic,
& pharmaceutical industries [6, 7]. Recently it emerged as an important feedstock for production
of biopolymers & chemicals such as polylacticde (PLA), acetaldehyde, acrylic acid, 2,3pentadione [8]. Lactic acid can be produced by chemical synthesis or microbial fermentation [9,
10]. The microbial process is better because it lead to the production of optically pure lactic acid
while chemical route produce racemic mixture with the requirement of high temperature &
pressure [11]. Pure glycerol conversion to lactic acid using Escherichia coli AC-521 have been
practiced earlier [12]. Lactic acid production from various substrates like corn [13], sugarcane
[14], cassava [15], beets [16], wheat [17], and paper sludge [18] has been studied in past decades
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Bharatkumar Dholakiya et al
J. Microbiol. Biotech. Res., 2012, 2 (1):90-93
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to meet the increasing demands for lactic acid. In the current study we have evaluated the growth
of all the six strains on the two carbon substrates: pure glycerol and crude glycerol from
biodiesel production and also perform batch fermentation for lactic acid production by using a
lower- cost carbon substrate, namely crude glycerol.
MATERIALS AND METHODS
Materials
Crude glycerol was obtained from biodiesel plant of Central Salt & Marine Chemical Research
Institute Bhavnagar, Gujarat India. All other chemicals were obtained commercially and of
analytical grade.
Microorganism and media
The following strains were used in this study : four were Lactobacillus casei 2125, Lactobacillus
delbrueckii 2025, Lactobacillus pentosus 2912, Bacillus laevolacticus2464, NCIM Pune and two
strain were Paenibacillus pabuli 2912, Lactococcus lactis subsp.la 3041, MTCC Chandigarh.
Screening & Enrichment of the strains
All the strains were first screen for the efficient utilization of raw glycerol & production of lactic
acid. For screening, prepare a suspension of all the strain. Take 1 ml of 106-fold diluted culture
broth and spread the suspension on agar plates containing yeast extract 2 g L−1, beef extract 5 g
L−1, peptone 3 g L-1, glycerol 20 g L−1, NaCl 3 g L−1, K2HPO4 2 g L−1, Agar 20 g L-1, CaCO3 10
g L-1 at pH-7. Glycerol in the medium was used as substrate to favor the growth of glycerolutilizing microorganisms. Incubated the plate at 37 ◦C for 12 h. When acid was produced during
growth of the strain, it could react with CaCO3 Clear zone was observed around the colony.
Selected strains were used for lactic acid fermentation. The composition of preculture medium
was K2HPO4 3g L−1, (NH4)2SO4 5g L-1, yeast extract 4 g L−1, peptone 5 g L-1, glycerol 20 g L−1.
The colonies were transferred into a 250 ml flask with 50 ml preculture medium and incubated in
a rotary shaker at 37 ◦C and 150 rpm for 12 h.
Batch flask fermentation
Screened strains were first cultured in preculture medium to identify if the lactic acid was a
major metabolic product of the strains. Then took a 10 ml of preculture medium and inoculate in
to the batch flask fermentation medium. The medium used for batch flask fermentation contained
the following components: yeast extract 4 g L−1, peptone 7 g L−1, glycerol 20 g L−1, K2HPO4 10
g L−1, (NH4)2SO4 10g L-1 and then incubated in a rotary shaker at 42 ◦C and 200 rpm with
uncontrolled pH for 2 days. Then pH was maintained by adding NaOH at regular intervals. The
data are the averages of triplicate experiments
Analytical methods
Lactic acid production was confirmed by TLC method. Further qualitative and quantitative
analysis was carried out by HPLC using Supelcogel 610H column. Glycerol was analyzed
system with a waters 2414 Refractive index (RI) detector and lactic acid was analyzed by a 2690
UV detector at 210 nm.
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Bharatkumar Dholakiya et al
J. Microbiol. Biotech. Res., 2012, 2 (1):90-93
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RESULTS AND DISCUSSION
In this experiment, both pure glycerol and raw glycerol were used as sole carbon source in
fermentation medium for all the six strain. From table 1 it could revels that except strain
Lactobacillus casei for crude glycerol all other are subjected to lactic acid conversion. In both
medium pure glycerol and crude glycerol lactic acid acts as major byproduct. The initial glycerol
concentration was 20 g L-1 maintained throughout the experiment. But trace amount of ethanol
also found in raw glycerol carbon source. In the fermentation medium containing pure glycerol,
maximum production of lactic acid was found with strain Bacillus laevolacticus 2.35g L-1,
byproduct acetic acid 0.74 35g L-1 and glycerol 9.09 g L-1. Same for the fermentation medium
containing raw glycerol, maximum production of lactic acid was obtained with strain
Lactobacillus delbrueckii 4.37 g L-1 byproduct acetic acid 0.68 g L-1, and glycerol 9.63 g L-1.
Previously from the reported study Escherichia coli AC-521 suitable for producing lactic acid
from pure glycerol with high concentration and productivity [12].The HPLC chromatogram of
crude glycerol showed some peals of impurities. Since this glycerol was derived from biodiesel,
these peaks may be of diglyceride, monoglyceride and some free fatty acid. In comparison with
the crude glycerol to pure glycerol the productivity was observed far more in crude glycerol
rather than in pure glycerol. The fundamental reason behind it was the presence of impurities like
diglyceride, monoglyceride and some free fatty acid acted as a nutrient for microorgamism
growth.
Table I: Comparative results of various products produced in medium containing Pure Glycerol and Raw glycerol by
various Bacterial strains. Initial glycerol concentration 20 g L−1, fermentation time 48 h, uncontrolled pH.
Strain
Lactic acid Acetic acid
g L-1
g L-1
1-a
Lactobacillus casei
(PG)
0.99
0.66
Lactobacillus delbrueckii (PG)2-a
1.68.
0.53
1.12
0.38
Lactobacillus pentosus
(PG)3-a
Bacillus laevolacticus
(PG)4-a
2.35
0.74
1.70
2.82
Paenibacillus pabuli
(PG)5-a
Lactococcus lactis subsp.la (PG)6-a
2.26
2.33
Lactobacillus casei
(CG)1-b
Lactobacillus delbrueckii (CG)2-b
4.37
0.68
Lactobacillus pentosus
(CG)3-b
1.43
0.67
Bacillus laevolacticus
(CG)4-b
0.75
0.70
Paenibacillus pabuli
(CG)5-b
0.66
0.42
Lactococcus lactis subsp.la (CG)6-b
1.40
2.13
a
PG = Pure Glycerol, b CG = Raw Glycerol
Ethanol
g L-1
0.067
0.056
0.124
Glycerol
g L-1
9.226
9.25
9.00
9.09
8.83
8.97
3.35
9.63
8.71
8.3
8.64
7.55
CONCLUSION
Present work revels that microbial conversion of raw glycerol to lactic acid is possible in
presence of suitable microorganism. From the HPLC chromatogram it showed that Lactobacillus
delbrueckii (CG) organism have maximum yield of lactic acid from raw glycerol among all six
organisms. However the overall lactic acid productivity is not the highest. Therefore the level of
productivity of strain Lactobacillus delbrueckii can be further improved by optimizing the
production conditions as well as mutation of strain. A crude glycerol to lactic acid fermentation
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Bharatkumar Dholakiya et al
J. Microbiol. Biotech. Res., 2012, 2 (1):90-93
______________________________________________________________________________
model potentially provides an additional feedstock for production of biodiesel while offering a
lower-cost carbon substrate and eliminating crude glycerol disposal. These criteria are important
for the bioconversion of industrial byproducts into valuable products.
Acknowledgment
We wish thank to the Department of Applied Chemistry, SVNIT, Surat for providing laboratory
facilities and Biotechnology Department of South Gujarat University Surat , Bioscience.
Analytical facilities providing by the Central Salt & Marine Chemicals Research Institute,
Bhavnagar, Gujarat, India are gratefully acknowledged.
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