International Journal of Science and Research (IJSR)

ISSN (Online): 2319-7064

Index Copernicus Value (2015): 78.96 | Impact Factor (2015): 6.391

Isolation and Identification of Lipase-producing

Bacteria from Oil-Contaminated Soils
Khaing Phyo Wai1, Weine Nway Nway Oo2
1
Department of Biotechnology, Mandalay Technological University, Mandalay Region, Republic of the Union of Myanmar
2
Department of Biotechnology, Technological University (Kyaukse), Kyaukse, Republic of the Union of Myanmar

Abstract: In the present study, the indigenous microbes from oil-contaminated soil samples of oil processing mills were isolated based
on their lipolytic activities. The isolated bacteria were screened on lipid-enriched media with different substrates and were selected the
strains showing lipolytic activity. To be confirmed the lipase activity, the strains were further examined on olive oil and rhodamine B
media. And then, the best lipase producer strains were further identified by 16S rDNA sequencing method. Based on the 16S rDNA
sequencing results, the strains were close to Acinetobacter species and Stenotrophomonas species.

Keywords: oil-contaminated soil, lipase, olive oil, 16S rDNA sequencing method

1. Introduction 2. Materials and Methods

The large part of the earth’s biomass is represented by lipids. A. Chemicals
Lipids are essential to all living systems. They are the most Tributyrin and p-nitrophenyl palmitate (pNPP) were
important source of energy, play structural roles in purchased from sigma (USA). The other chemicals were also
membranes and are involved in signaling events. To be able of analytical grade. Olive oil used for lipase production was
to carry out these functions, lipids require lipolytic enzymes commercially available in local market.
during their metabolism. Lipolytic enzymes catalyze the
turnover of these water-insoluble compounds [1]. They also B. Methods
breakdown lipids and make them mobile within the cells of
individual organisms [2]. Lipolytic enzymes are grouped into 1) Collection of sample
3 main categories, which are esterases, phospholipases and Oil-contaminated soil samples from the local oil mills in
lipases [3]. Myanmar were collected and stored in sterile plastic bags.
The samples were transferred to the laboratory under sterile
Lipases are defined basically as fat-splitting enzymes that conditions and stored at 4ºC until examination.
catalyze the hydrolysis of long-chain triacylglycerols to from
glycerols and fatty acids in the presence of excess water. 2) Isolation of lipolytic bacteria
Also, they can catalyze the reverse reaction, synthesis of The isolation was primely processed by serial dilutions of
triacylglycerols, under non-aqueous conditions [4]. Lipases samples and spread plate method on tributyrin agar medium.
occur widely in nature, but commercially useful lipases are The composition of tributyrin agar medium is (per liter) 5 g
usually obtained from microorganisms that produce a wide of peptone, 3 g of yeast extract, 10 ml of tributyrin and 15 g
variety of extracellular lipases. Microbial lipases are more of agar. The pH of the medium was adjusted to 7 with 0.1M
widely applied in industries due to their shorter generation NaOH. The culture plates were incubated at 35ºC for 48 hrs.
time; ease of bulk production which is further enhanced with This medium was chosen for selectively isolating colonies
advancement in fermentation technologies; and ease of capable of growing on lipid enriched medium and showing
manipulation, either genetically or environmentally. Lipase- clear zones around them. Colonies showing clear zone
producing microorganisms have been found in diverse diameters were picked up as lipolytic positive strains and
habitats such as industrial wastes, vegetable oil processing streaked onto the nutrient media as pure cultures. Then, these
factories, diaries, soil contaminated with oil, etc [5]. Many strains were also examined on different enriched media with
microorganisms such as bacteria, yeast and fungi are known two different substrates such as egg- yolk and tween 80. On
to secrete lipases. Of all these, bacterial lipases are more these media, 14 out of 28 strains showed lipolytic activities.
economical and stable [6]. Bacterial lipases are used These potential lipolytic strains were observed under the
extensively in food and dairy industry for the hydrolysis of microscope and then used for further processes.
milk fat, cheese ripening, flavor enhancement and lipolysis of
butter fat. The industrial demands for new sources of lipases 3) Screening of lipase producing bacteria
with different enzymatic characterstics that could create In addition to lipases, esterases are also grouped into
novel applications stimulate the isolation and selection of hydrolases. Esterases break ester bonds of short-chain fatty
new strains of lipolytic microorganisms. We were interested acids whereas lipases catalyze the hydrolysis of long-chain
in this topic because isolation of new lipase-secreting fatty acids that are insoluble or poor soluble. Therefore, the
bacteria and study of their enzyme production, purification lipase must be capable of identifying an insoluble or
and characterization could provide new lipase with better aggregated substrate. Esterase activity is found to be highest
quality and wider range of applications. towards more water soluble substrate [7].Olive oil has the
Volume 6 Issue 7, July 2017
www.ijsr.net
Licensed Under Creative Commons Attribution CC BY
Paper ID: ART20175449 DOI: 10.21275/ART20175449 1171
 International Journal of Science and Research (IJSR)
ISSN (Online): 2319-7064
Index Copernicus Value (2015): 78.96 | Impact Factor (2015): 6.391
advantage of including high concentration of oleic acid and Totally, 14 strains showed lipolytic activity on these
being more economical. The lipolytic positive strains were screening media and their microscopic morphology was
further screened for lipase activity by using the modified shown in (Table 1).
rhodamine-B agar media consisting of olive oil as substrate
at 35ºC. The lipase activity was detected by the presence of (a)
pink or orange colored colonies and by fluorescence under
UV light.

4) Characetrization and molecular identification of

bacteria
The cultures were then characterized morphologically based
on Bergey’s manual of systemic bacteriology. The
identifications were confirmed by using nucleotide sequence
analysis of 16S rDNA genes. The genomic DNA was isolated
from the bacteria by the DNA extraction method described (b) (c)
by Hosek et al. [8]. 16S rRNA gene of isolated strain was
amplified from its genomic DNA using a pair of universal
bacterial primer, 10F (5’- AGTTTGATCCTGGCTC - 3’) as
forward primer and 800R (5’- CTACCAGGGTATCTAAT -
3’) as reverse primer. The highly purified DNA was then
amplified in a thermocycler at conditions: 35 cycles of 95ºC
for 30 seconds, 56ºC for 30 seconds and 72ºC for 1 min [9].
PCR products were visualized by agarose gel electrophoresis.
DNA sequencing was performed in a highly automated gene
sequencer. These sequences had been submitted to the Figure 1: Screening of Lipolytic activity on (a) tributyrin
GenBank database (BLASTN) and compared with the other agar medium (b) egg-yolk medium (c) tween 80 medium
sequences to analyze the bacterial classes and their
phylogenies. Table 1: Cell morphology and physiology of the isolates
Isolate Shape Gram’s stain
L1 Rod Positive
3. Results and Discussion L3 Diplococcoid rod Negative
L5 Rod Positive
3.1 Isolation of lipolytic strains L8 Cocci Positive
L10 Cocci Positive
Soil samples taken from oil mills in Myanmar were examined L11 Rod Positive
for the presence of lipolytic strains. Oil-contaminated soils L12 Rod Positive
were chosen as sample sources because they could be good L13 Rod Positive
environment for the habitats of lipid degrading bacteria and L14 Diplococcoid rod Negative
are rich of lipid. A total of 28 morphologically distinct strains L16 Rod Negative
(L1- L28) which showed clear zones around the colonies L17 Rod Negative
were isolated from oil-contaminated soils by using tributyrin L19 Rod Negative
agar media (Fig.1 (a)). L20 Diplococcoid rod Negative
L25 Rod Negative
The egg-yolk suspension allows for the detection of
lecithinase and lipase activity. Lipase destroys the fats within 3.2 Screening of lipase positive bacteria
the egg yolk, which results in greenish blue colour of the
colony surface when flooded with copper II sulphate solution Lipase producer strains were identified by the formation of
(Fig 1 (b)). orange fluorescent halos around the colonies when olive oil
rhodamine B spread plates incubated at 35ºC were exposed
Tweens (fatty acid esters of polyoxyethylene sorbitan) have to UV light at 350nm. Olive oil is used as lipase substrate
been the most widely used substrates for the detection of and rhodamine B is the indicator of lipase activitiy. This
lipase/esterase producing microorganisms in agar media. method is not sensitive to pH changes and does not inhibit
Screening using tween agar plates shows precipitation around the growth of bacteria. Seven out of 14 strains were shown
the lipase/esterase producing micro-organisms. The method the best lipase activity (Fig.2). These rhodamine B-olive oil
is based on the precipitation as the calcium salt of the fatty media can identify lipase-producing bacteria from esterase-
acids released by hydrolysis of tweens. Liberated fatty acids producing bacteria. They were selected as the best lipase
bind with the calcium incorporated into the medium. The producer strains and used for further identification. Some
calcium complex is visible as insoluble crystals around the biochemical properties of the selected bacteria were also
inoculation site. On Tween 80 agar plates, formation of shown in (Table 2).
precipitation by the isolates also confirmed that this bacterial
strains possessed lipolytic activity (Fig 1 (c)).

Volume 6 Issue 7, July 2017

www.ijsr.net
Licensed Under Creative Commons Attribution CC BY
Paper ID: ART20175449 DOI: 10.21275/ART20175449 1172
 International Journal of Science and Research (IJSR)
ISSN (Online): 2319-7064
Index Copernicus Value (2015): 78.96 | Impact Factor (2015): 6.391
L16 10F Stenotrophomonas 99%
A 800R maltophilia K279a complete 99%
genome, strain K279a
NCBI Reference Sequence:
NC_010943.1
L17 10F Stenotrophomonas 99%
800R maltophilia K279a complete 99%
genome, strain K279a
NCBI Reference Sequence:
B NC_010943.1
L19 10F Stenotrophomonas 99%
Figure 2: Screening of lipase positive strains under the UV 800R maltophilia K279a complete 99%
light (A) lipase positive control (B) lipase negative control genome, strain K279a
NCBI Reference Sequence:
Table 2: Some biochemical properties of the isolates NC_010943.1
Strain Catalase Oxidase Lecithinase L20 10F Acinetobacter baumannii 96%
L3 + - + 800R strain AB030, complete 97%
L14 + - + genome
NCBI Reference Sequence:
L16 + - +
NZ_CP009257.1
L17 + - +
L25 10F Stenotrophomonas 99%
L19 + - +
800R maltophilia K279a complete 99%
L20 + - + genome, strain K279a
L25 + - + NCBI Reference Sequence:
NC_010943.1
3.3 Characterization of lipase positive strains
4. Conclusion
Upon the amplification of 16S rDNA sequence using the
primers, the amplified products of 800 bp were obtained Nowadays, the use of lipases in industries is becoming
(Fig.3) which were then sequenced and compared with the increasingly important. Extensive and persistent screening for
Genbank databases using BLASTN program. It was found to new indigenous microorganisms with improved lipase
have 95-98% identity with L3 and L14 strains to abilities will lead to faster ways to upgrade the process. With
Acinetobacter pitti, L16, L17, L19 and L25 strains to the findings, we obtained three strains of best lipase
Stenotrophomonas maltophilia, L20 strain to Acinetobacter producers from oil-contaminated soils in oil mills. To obtain
baumannii in (Table 3). maximum production of extracellular lipases, we need to
control the best physico-chemical environment that greatly
influences the enzyme production. So, the present study
requires the optimization of the lipase-production conditions
for further industrial applications.

5. Acknowledgements
Special thanks to the Department of Biotechnology,
Mandalay Technologial University and Biotechnology
Research Department, Kyaukse for giving the opportunity to
perform this study and for providing the laboratory facilities.

Figure 3: Colony PCR result. Line1: 1kb DNA ladder. References

Line2: ~800bp PCR products
[1] Gilham, D., and R. Lehner. 2005. Techniques to measure
Table 3: Sequencing result lipase and esterase activity in vitro. Methods 36: 139-
Sample Primer Sequence accession Sequence 147.
code (10-800bp) description identity [2] Beisson, F., V. Arondel, and R. Verger. 2000. Assaying
L3 10F Acinetobacter pittii PHEA-2 97% Arabidopsis lipase activity. Biochemical Society
800R chromosome, complete 98% Transactions 28 (6): 773-775.
genome [3] Arpigny, J. L., and K.E. Jaeger. 1999. Bacterial lipolytic
NCBI Reference Sequence: enzymes: classification and properties. Biochem. J. 343:
NC_016603.1 177-183.
L14 10F Acinetobacter pittii PHEA-2 97% [4] Jaeger, K.E, S. Ransac, B. W. Dijkstra, C. Colson,M.
800R chromosome, complete 98%
van Heuvel, and O. Misset. 1994. Bacterial Lipases.
genome
NCBI Reference Sequence:
FEMS Microbiology Reviews 15: 29-63.
NC_016603.1 [5] K.V.V.S.N. Bapiraju, P. Sujatha, P. Ellaiah, and T.
Ramana, Afr. J. of Biot., 2004, 3 , 618-621.

Volume 6 Issue 7, July 2017

www.ijsr.net
Licensed Under Creative Commons Attribution CC BY
Paper ID: ART20175449 DOI: 10.21275/ART20175449 1173
 International Journal of Science and Research (IJSR)
ISSN (Online): 2319-7064
Index Copernicus Value (2015): 78.96 | Impact Factor (2015): 6.391
[6] T. Achamman, M.K. Monoj, A. Valsa, S. Mohan, and R.
Manjula, Ind. J. Microbiol., 2003, 43, 67 – 69.
[7] Fojan, P., P.H. Jonson, M.T.N. Petersen, and S.B.
Petersen. 2000. What distinguishes an esterase from a
lipase: A novel structural approach. Biochimie 82: 1033-
1041.
[8] J. Hosek, P. Svastova, M. Moravkova, I. Pavlik and M.
Bartos, Veterinarni Medicina, 2006, 51, 180–192.
[9] Pissamai Powedchagun, Hideyuki Suzuki and Sirirat
Rengpipat, J. Sci. Technol, 2011, 33, 1-8.
[10] E. Mobarak-Qamsari, R. Kasra-Kermanshahi, Z.
Moosavi-nejad, Iran. J. Microbiol., 2011, 3, 92-98.
[11] K.E. Jaeger, T .Eggert, Curr. Opinion Biotechnol.,1998,
13, 390-397.

Author Profile
Ms. Khaing Phyo Wai is a PhD student at the
department of Biotechnology, Mandalay Technological
University. She was born in Myittha, Kyaukse
Township, Mandalay Region, Republic of the Union of
Myanmar. Her date of birth is September 23, 1985 and
she is now 32 years old. She received the Degree of Bachelor of
Science (Biotechnology) from Yangon Technological University,
Myanmar in 2006. She also held Master Degree from Mandalay
Technological University, Myanmar in 2010. Currently, she is
doing her PhD research at the department of Biotechnology,
Mandalay Technlogical University. She also has work experiences
in the fields of “Microbial Enzyme Activity of Trichoderma sp.”
and “Antimicrobial Activity of Alcaligenes sp.”.

Volume 6 Issue 7, July 2017

www.ijsr.net
Licensed Under Creative Commons Attribution CC BY
Paper ID: ART20175449 DOI: 10.21275/ART20175449 1174