ISOLATION OF LIPASE PRODUCING BACTERIA FROM OIL AND

MILK CONTAMINATED SOIL

A
RESEARCH PROJECT
PRESENTED
TO
THE FACULTY OF LIFE SCIENCES
IN PARTIAL FULFILLMENT OF THE REQUIRMENT
FOR THE DEGREE OF
BACHELOR OF SCIENCES
IN
BIOTECHNOLOGY (Hons.)
(2017-2020)
UNDER THE SUPERVISION OF
Dr. MONIKA
SUBMITTED BY
RAMNEET KAUR
Roll no - 3350

DEPARTMENT OF BIOTECHNOLOGY
MATA GUJRI COLLEGE, FATEHGARH SAHIB -140406 (PUNJAB)
1. INTRODUCTION

Industrial biotechnology is a rapidly growing field which involves the use of enzymes and
microorganisms. Enzymes have been used by men since biblical times either as vegetables rich
in enzymes or as microorganisms and their products (in brewing processes, baking and
production of alcohol). Beer and yoghurt also owe their flavour and texture to a range of
enzyme- producing organisms that were domesticated many years ago. Enzymes are natural
catalysts which play a diversified role in many aspects of everyday life.

1.1 Hydrolases

The hydrolase family includes a group of enzymes that catalyse bond cleavage by reacting with
water. Amongst others, these consist of lipases, proteases, amidases, epoxide hydrolases and
glycosidases. Hydrolases are placed in Class 3 according to the IUB classification of enzymes
by the Enzyme Commission, and these are further classified by the type of bond hydrolysed;
for instance, lipases are classified as EC 3.1.1.3 as they hydrolyse the carboxyl ester bonds of
triacylglycerols. Currently, the biotechnological applications of hydrolases are of special
interest as they have some advantageous characteristics which make them ideally suited for
industrial use. Strikingly, most of the enzymes used in industry are microbial enzymes,
originating either from bacteria, fungi and yeasts.

1.2 Lipase

Lipase (triacylglycerol acyl hydrolase, EC 3.1.1.3) catalyses the hydrolysis of the carboxyl
ester bonds in triacylglycerols to produce diacylglycerols, monoacylglycerols, fatty acids and
glycerol. In addition, lipases catalyse the hydrolysis and transesterification of other esters as
well as the synthesis of esters. Many lipases exhibit enantioselective properties.

A small amount of lipase, called gastric lipase, is made by cells in stomach. The main source
of lipase in digestive tract is our pancreas, the pancreas is an organ located just below to
stomach toward the back. Its role is to break down specific components of dietary fats.
Lipase is secreted from the pancreas via a duct that empties into the gastrointestinal tract at
the duodenum. It thus acts on food that has already been partially digested in the stoma ch.
First, bile made in liver and released into intestine converts dietary fat into small fatty globules.

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Pancreatic lipase, acts on these fat globules, converting them into fatty acids and glycerol,
which are small, energy-dense molecules used by all our cells. Fatty acids and glycerol travel
in blood and lymph vessels to reach all parts of our body.

Triglycerides cannot across the intestinal wall directly. These consist of a glycerol
"backbone" with three fatty acids attached, one to each of glycerol's carbon atoms. Lipase
specifically converts triglycerides to two free fatty acids and a monoglyceride .

Lipoprotein lipase (LPL) is a member of the lipase gene family, which includes pancreatic
lipase, hepatic lipase and endothelial lipase. LPL is water soluble enzyme that hydrolyzes
triglycerides in lipoproteins, which is found in chylomicrons and very low-density lipoproteins.
Lipoprotein lipase which is a hydrolytic enzyme produced by many tissues and is rate-limiting
for the removal of lipoprotein triglycerides from the circulation. It also has other important
roles in many normal tissues, as well as in certain metabolic diseases including obesity.

In additions they also serve as biocatalyst for acidolysis, esterification and amionlysis. The
common mode of lipases as a biocatalyst is shown below in fig 1.1.

Fats or oils +Water →fatty acid + Glycerol

Fig 1.1 Hydrolysis or synthesis (acylglycerols/esters) reactions catalyzed by lipases.

Lipases of microbial origin have greater industrial attraction because they are available
in large quantities and can be produced with high yields. Numerous lipases have been
characterized and efforts have been made to improve their stability in organic solvents for
varied applications. Cost effectiveness, the main concern of industries is also found suitable in
lipase catalyzed process as compared to traditional downstream processing where major
bottleneck are energy consumption and toxic byproducts (Parihar et al., 2012).
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Bacterial lipases are used extensively in food and dairy industry for the hydrolysis of milk fat,
cheese ripening, flavor enhancement and lipolysis of butter fat. The industrial demands for new
sources of lipases with different enzymatic characteristics that could create novel applications
stimulate the isolation and selection of new strains of lipolytic microorganisms. In wake of
recent advancements in microbiology and biotechnology, lipases have emerged as key enzymes
owing to their multifaceted properties which find use in a wide array of industrial applications.
and pharmaceutical industries and industrial wastes management Lipase (Triacylglycerol
lipases, EC 3.1.1.3) is an enzyme used in numerous biotechnological processes including food,
leather, cosmetic, detergents.

1.3 Production of Lipase

Lipases are industrially important enzymes that catalyze the hydrolysis of triglycerides to free
fatty acids and glycerol at lipid-water interfaces, involving interfacial adsorption and
subsequent catalysis. These unique enzymes are widely found in plants, animals, and
microbials, being used increasingly in food industry, detergents, cosmetics, and
pharmaceuticals. Lipase is produced by many microorganisms and eukaryotes, but the most
commercially useful are the ones, created by microorganisms. In the industry, due to exclusive
properties, special attention has been paid to microbial lipase. Microbial enzymes are also more
stable than the 120 enzymes in plants and animals with easier and safer production (Griebeler
et. al., 2011).

Lipases are produced by microorganisms (bacteria and fungi), plants and animals, However,
microbial lipase especially from bacteria are more useful than their plant and animal origin.
since they have great variety of catalytic activities and microorganisms are easy to manipulate
genetically and capable of rapid growth on inexpensive media. the microbial lipases are
commercially most important mainly are secreted into the culture medium by many of
microbial species are belong to bacteria, fungi, yeasts and actinomyces. Lipases play a vital
role in the manufacturing and services sectors for the mankind . Microbial lipases have gained
special industrial attention due to their selectivity, stability and broad substrate specificity.
Microbial enzymes are also more stable than their corresponding animal and plant enzymes
and their production is more convenient and safer. Generally, bacterial lipases are
glycoproteins but some extracellular bacterial lipases are lipoproteins. The production of
extracellular lipases from bacteria is greatly influenced by medium composition besides
physicochemical factors such as temperature, pH and dissolved oxygen . the major factor for

3
the expression of lipase activity has always been reported as the carbon source, since lipase are
inducible enzymes. These enzymes are generally produced in the presence of a lipid such as
oil or any other inducer, such as triacylglycerols, fatty acids, hydrolysable esters, Tweens,
glycerol and bile salts. However, nitrogen sources and essential micronutrients should also be
carefully considered for growth and production optimization. Many microorganisms, such as
bacteria, yeasts, and fungi have been known as producers of extracellular lipases, which are
produced by many different species of bacteria, like Bacillus and Pseudomonas (Kiran et al.,
2008; Wang et al., 2009). Over 160 strains of microorganisms were screened for exogenous
lipolytic activity. The highest lipase activity was found in Streptomyces sp., Bacillus sp., and
Pseudomonas sp. Many industrial microbial lipases are derived from fungi and bacteria. Since
the fungi can secrete enzymes into their environments, purification of this enzyme is easier. In
modern processes, fungal genes are expressed in bacterial systems. Lipase-producing
microorganisms can be found in various habitats such as industrial waste, oil plants, petroleum
contaminated soil, oil seeds, and rotted food. Medium with soybean meal-0.77% (w/v);
(NH4)2SO4-0.1 M; KH2PO4-0.05 M; rice bran oil-2% (v/v); CaCl2-0.05 M; PEG 6000-0.05%
(w/v); NaCl-1% (w/v); inoculum-1% (v/v); pH 3.0; incubation temperature 35 °C and
incubation period of five days have been identified as optimal conditions for maximal lipase
production. Enzyme production is strongly influenced by the age of the culture. Tributyrin and
olive oil have proven to be the best sources of carbon for lipase-producing strains (mayam et
al.,(2012)

1.4 Uses of lipase

The increasing global demand for oils raises waste production, which in turns renders
environmental pollution with waste oils one of the major problems in today's industrial world
which will lead to environmental destruction. As a result, easy and affordable methods should
be employed to minimize the amount of waste. There have been numerous studies on the use
of lipase for disposal of oily waste water, though these enzymes are commonly unstable in
environmental conditions. Hence, the use of bacteria, compatible with soil and wastewater and
capable of producing enzymes in the soil and oils waste, is a good choice to treat this type of
wastewater.

Lipases have been used for the degradation of wastewater contaminants such as olive oil from
oil mills. The treatment process involved the cultivation of lipase-producing microbial strains
in the effluents. The microbial treatment of waste from fast-food restaurants for the removal of

4
fats, oils and greases. They cultivated pure and mixed microbial flora known to produce lipases
and other enzymes. Acinetobacter sp. was the most effective of the pure cultures, typically
degrading 60 65% of the fatty material.

Lipases affect a variety of substrates, including natural oils, fatty acids esters, and synthetic
triglycerides. Many agro-industrial residues can be used as potential substrates to produce
enzymes. Resistant to solvents, they can be used in a wide range of biotechnological processes,
among which the new examples, such as biopolymer synthesis, production of biodiesel, and
treatment of wastewater containing oils, has been successfully completed.

The high potential of lipases in food technology applications and related issues reflect the need
to develop new affordable technologies to increase production, while enhancing the scale and
purity of these enzymes. The use of lipases has been expanded greatly with new programs
being studied in the food industry, continuously. There are various ongoing scientific
investigations in the field of developing enzymatic hydrolysis processes to precede traditional
biological treatment. Lipases properties have improved by both protein and genetic
engineering. Innovations in enzyme stabilization plays an important role in using this enzyme
as a biocatalyst in effective food processing technology.

Apart from food industry, lipases are being employed in various industries such as production
of biodiesel, bio-polymer, detergent, pulp and paper, health products, and pesticides. Properties
and application of lipase as a catalyst for reactions with commercial potential may greatly
expand industrial biotechnology. Lipases are the most important group of biocatalysts for
biotechnological applications. In order to optimize these enzymes for utilization in the industry.

The alkaline thermophilic lipases find application in detergent industry many fatty food stains
and human serum contain triglycerides which are hydrolyzed by lipases to produce fatty acids,
monoglycerides and diglycerides, which are easier to remove than unhydrolyzed triglycerides
(Ram Reddy and Pallavi, 2012).

Lipases were generally added to the detergents primarily in combination with proteases and
cellulases. However, other enzymes such as amylases, peroxidases and oxidases are also
reported to be added in detergent preparations. Removal of oil/fatty deposits by lipase is
attractive owing to its suitability under milder washing conditions. To be a suitable additive in
detergents, lipases should be both thermostable as well as alkalophilic and capable of
functioning in the presence of the various components of washing powder formulations (Ram
Reddy and Pallavi, 2012).

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Microbial lipases are industrially important enzymes. A number of commercial products have
been launched successfully worldwide. In 1994, Novo Nordisk introduced the first commercial
recombinant lipase ‘Lipolase’ from the fungus Thermomyces lanuginosus and was expressed
in Aspergillus oryzae. In 1995, two bacterial lipases were introduced – ‘Lumafast’ from
Pseudomonas mendocina and ‘Lipomax’ from Pseudomonas alcaligenes by Genencor
International.

1.5 Aims and objectives of project work

The current work aimed to use microbes as bioreactors to produce the required enzyme lipase
in laboratory conditions. The goals of this project were isolation lipase producing bacteria from
Oil and milk contaminated soil. To achieve aims, the work was divided into following sections:

➢ Isolation of bacterial colonies from milk and oil contaminated soil

➢ Screening of lipase producing bacteria
➢ Production of lipase producing bacteria
➢ Enzyme isolation and purification with ammonium salt
➢ Qualitative estimation of lipolytic activity

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2. REVIEW OF LITRATURE

The literature pertaining to lipases has been reviewed by several investigators (Gupta et al.,
2004; Hasan et al., 2006; Hasan et al., 2009; Jaeger and Eggert, 2004; Kapoor et al., 2012;
Salihua et al., 2012). First lipase was discovered in pancreatic juice in the year 1856 by Claude
Bernard. Animal pancreatic extracts were traditionally used as the source of lipase for
commercial applications. However, microbial sources of lipase were explored when industrial
potential of lipases enhanced and their demand could not be met by the supply from animal
sources. The number of available lipases has increased mainly as a result of achievements made
in the cloning and expression of enzymes from microorganisms, as well as of an increasing
demand for these biocatalysts with novel and specific properties such as specificity, stability,
pH, and temperature (Bornscheuer et al., 2002; Menoncin et al., 2009).

Lipases are widely distributed in animal, plants and microorganisms (Bornscheuer, 2002),
however, microbial lipases are commercially most important mainly because of the ease of
their cultivation and genetic manipulation to obtain higher yield. Commercially important
microbial lipases are produced from bacteria, fungi and yeast (Abada, 2008; Babu and Rap,
2007).

Lipases have emerged as one of the leading biocatalysts with proven potential for contributing
to the multibillion dollar underexploited lipid technology bio-industry and have been used in
in situ lipid metabolism and ex situ multifaceted industrial applications. The number of
available lipases has increased since the 1980s. This is mainly a result of the huge achievements
made in the cloning and expression of enzymes from microorganisms, as well as of an
increasing demand for these biocatalysts with novel and specific properties such as specificity,
stability, pH, and temperature. Lipases are produced by animals, plants, and microorganisms.
Microbial lipases have gained special industrial attention due to their stability, selectivity, and
broad substrate specificity. Many microorganisms are known as potential producers of
extracellular lipases, including bacteria, yeast, and fungi. Fungal species are preferably
cultivated in solid state fermentation (SSF), while bacteria and yeast are cultivated in
submerged fermentation. The importance of lipases can be observed by the great number of
published articles recently. In fact, over the last few years, there has been a progressive increase
in the number of publications related to industrial applications of lipase-catalyzed reactions,
performed in common organic solvents, ionic liquids, or even in non-conventional solvents.

7
The present review is focused on lipase production discussing the main microorganisms,
substrates, and process operations used in this specific field.

2.1 Microbial Sources of Lipases

Lipases are ubiquitous in nature and are produced by several plants, animals, and
microorganisms. Lipases of microbial origin represent the most widely used class of enzymes
in biotechnological applications and organic chemistry. A review of the most recent (from 2004
to the present) potential microorganisms for lipase production in both submerged and solid-
state fermentations are reported in Table 2.1 and Table 2.2

2.1.1 Bacteria

Among bacterial lipases being exploited, those from Bacillus exhibit interesting properties that
make them potential Candidates for biotechnological applications. Bacillus subtilis, Bacillus
pumilus, Bacillus licheniformis , Bacillis coagulans, Bacillus stearothermophilus, and Bacillus
alcalophilus are the most common bacterial lipases. In addition, Pseudomonas sp.,
Pseudomonas aeruginosa, Burkholderia multivorans, Burkholderia cepacia, are also reported
as bacterial lipase producers.

Ertugrul et al.,(2007) isolated 17 bacterial strains that could grow on media based on OMW
and selected the most promising strain for lipase production. After screening in tributyrin agar
medium, a strain of Bacillus sp. was identified as the best lipase producer. After the medium
optimization, the intracellular activity found was 168 U mL−1.

Kiran et al.,(2008) isolated 57 heterotrophic bacteria from the marine sponge Dendrodoris
nigra, of which 37% produced a clear halo around the colonies on tributyrin agar plates for
lipase production. Particularly, the strain Pseudomonas MSI057 exhibited large clean zones
around the colonies. Then, this strain was selected for further studies, and after optimization, a
maximum lipase activity was found as 750 U mL−1.

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Table 2.1 List of some lipase producing bacteria

Table 2.2 List of some lipase producing fungal and yeast

Sources: Gupta et al., 2004; Treihel et al., 2010

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Carvalho et al., (2008) isolated a bacterium strain from petroleum-contaminated soil and
codified as Biopetro-4. After investigation of several inducers on lipase activity, the maximum
value obtained was 1,675 U mL−1 after 120 h of fermentation.

Abada (2008) produced lipase from a strain of B. stearothermophilus AB-1 isolated from air
and obtained a maximum lipase activity of 1,585 U mL−1 in 48 h of fermentation.

Takaç and Marul(2008) isolated microbial cultures from soil enriched by periodic sub-
culturing of samples in nutrient broth containing 1% (v/v) tributyrin. The isolation process was
performed by serial dilution samples on tributyrin agar (TBA) plates. Bacillus sp. was selected
towards producing the largest opaque halo. Active colonies were re-streaked on TBA agar for
purification.

Shariff et al.,(2007) isolated a thermophilic bacterium, Bacillus sp. strain L2 from a hot spring
in Perak, Malaysia. An extracellular thermostable lipase activity was detected through plate
and broth assays at 70 °C after 28 h of fermentation. In most cases, bacteria are preferably
cultivated in SmF, due to the high water activity required by the microorganisms (higher than
0.9). There are few exceptions for bacteria grown in SSF. However, when the bacteria are well
adapted to this solid medium, the production is normally high.

Mahanta et al., (2008) obtained a maximum lipase activity of 1,084 U gds−1 using a solvent
tolerant P. aeruginosa PseA strain.

Alkan et al., (2007) produced an extracellular lipase by B. coagulans and obtained a maximum
lipase activity of 149 U gds−1 after 24 h of fermentation.

Fernandes et al.,(2007) (obtained a maximum lipase activity of 108 U gds−1 after 72 h of

fermentation by B. cepacia.

2.1.2 Fungi

Most commercially important lipase-producing fungi are recognized as belonging to the genera
Rhizopus sp., Aspergillus sp., Penicillium sp.,

Geotrichum sp., Mucor sp., and Rhizomucor sp.Lipase production by fungi varies according to
the strain, the composition of the growth medium, cultivation conditions, pH, temperature, and
the kind of carbon and nitrogen sources Fernandes et al., (2004) . The industrial demand for new
sources of lipases with different catalytic characteristics stimulates the isolation and selection
of new strains. Lipase producing microorganisms have been found in different habitats such as

10
industrial wastes, vegetable oil processing factories, dairy plants, and soil contaminated with
oil and oilseeds among others B. Vishnupriya et al.,(2010).

Vishnupriya et al., (2006) studied the lipase production by Sterptomyces grisesus and obtained
maximum enzyme activity of 51.9U/ml.

Colen et al.,(2010) isolated 59 lipase-producing fungal strains from Brazilian savanna soil
using enrichment culture techniques. An agar plate medium containing bile salts and olive oil
emulsion was employed for isolating and growing fungi in primary screening assay. Twenty
one strains were selected by the ratio of the lipolytic halo radius and the colony radius. Eleven
strains were considered and, among them, the strain identified as Colletotrichum
gloesporioides was the most productive.

In another work, Cihangir and Sarikaya ,(2004) isolated a strain of Aspergillus sp. from soil
samples from the different regions of Turkey and obtained an expressive activity of 17 U
mL−1. In SmF, Teng and Xu (2008) investigated the lipase production by Rhizopus chinensis
and obtained, at the optimized experimental conditions, a maximum lipase activity of 14 U
mL−1.

Bapiraju et al.,(2005) optimized the lipase production by the mutant strain of Rhizopus sp. and
the optimum activity was 29 U mL−1.

Kaushik et al.,(2006) studied the production of an extracellular lipase from Aspergillus

carneus and obtained a maximum activity of 13 U mL−1. In SSF, Kempka et al., (2008)
investigated the lipase production by Penicillium verrucosum and the optimum activity was
about 40 U gram of dry substrate−1 (gds).

Vargas et al., (2008) studied the lipase production by Penicillium simplicissimum and obtained
an activity of 30 U gds−1. Both P. verrucosum and P. simplicissimum were isolated from the
babassu oil industry.

A quantitative comparison between SmF and SSF is difficult due to the difference in the
methods used for determining the lipase activity. However, some qualitative information
presented in the literature can be of interest. For example, extracellular lipases were obtained
using Rhizopus homothallicus with lipase activities of 1,500 U gds−1 and 50 U mL−1, by SSF
and SmF, respectively .

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Azeredo et al.,(2000) obtained lipase activities of 17 U gds−1 and 12 U mL−1 for SSF and
SmF, respectively, by cultivation of Penicillium restrictum. Recently, some works reporting
the use of immobilized whole biomass of filamentous fungi have also been published. The
immobilization is advantageous since it can avoid biomass washout at high dilution rates. Also,
high cell concentration in the reactor could be achieved and the separation of biomass from the
medium is favored .

Wolski et al., (2005) reported the use of response surface methodology to optimize the lipase
production by submerged fermentation using immobilized biomass of a newly isolated
Penicillium sp. At the optimized experimental conditions, the authors reached a lipase activity
around 21 U mL−1, higher than the activity obtained by the same microorganism before
immobilization.

Yang et al., (2004) studied the repeated-batch lipase production by immobilized mycelium of
Rhizopus arrhizus in submerged fermentation. The lipase productivity increased from 3 to 18
U mL−1 h−1, changing the process from batch to repeated-batch mode.

Ellaiah et al., (2006) used the whole immobilized biomass of Aspergillus niger to produce
lipase and obtained similar activities for both free and immobilized biomass cultivations (4 U
mL−1).

2.1.3 Yeast

According to Vakhlu and Kour.,(2007) the main terrestrial species of yeasts that were found
to produce lipases are: Candida rugosa, Candida tropicalis, Candida antarctica, Candida
cylindracea, Candida parapsilopsis, Candida deformans, Candida curvata, Candida valida,
Yarrowia lipolytica, Rhodotorula glutinis, Rhodotorula pilimornae, Pichia bispora, Pichia
mexicana, Pichia sivicola, Pichia xylosa, Pichia burtonii, Saccharomycopsis crataegenesis,
Torulaspora globosa, and Trichosporon asteroids.

The genes that encode lipase in Candida sp., Geotrichum sp., Trichosporon sp., and Y.
lipolytica have been cloned and over-expressed . Although lipases from C. rugosa and C.
antarctica have been extensively used in different fields.

Potumarthi et al., (2008) collected marine soil samples near an oil extraction platform in the
Arabian Sea. After the isolation of the colonies, they were transferred to plates containing 2%

12
tributyrin and incubated at 35 °C for 3–4 days. The colonies that showed the largest hydrolysis
halos zone were selected. The most effective strain for lipase production was identified as
Rhodotorula mucilaginosa (MTCC 8737) by its phenotypic characteristics.

Kumar and Gupta., (2008) isolated 15 yeasts from petroleum and oil sludge areas in Delphi
(India). The isolates were purified and checked for their lipolytic potential. Among these yeast
strains, one strain was selected for further studies, based on the largest halo of lipolysis. On the
basis of sequence homology, this strain was found to belong to Rhodotorula mucilaginosa
genus and share 99% homology with the already existing database.

Ciafardini et al., (2006) have discovered that freshly produced olive oil is contaminated by a
rich microflora, capable of conditioning the physicochemical and organoleptic characteristics
of the oil, through the production of enzymes. Among the microorganisms that were isolated
from this oil, several strains of yeasts were identified as Saccharomyces cerevisiae, Candida
wickerhamii, Williopsis californica, and Candida boidinii, of which S. cerevisiae and W.
californica showed good potential to produce lipase. The lipase activity in S. cerevisiae was
noted to be intracellular, and extracellular in W. californica. The three-phase olive oil
extraction process generates a dark-colored effluent, usually termed olive oil mill wastewater
(OMW).

D’Annibale et al., (2006) investigated the valorization of oil mill waste (OMW) by its use as
a possible growth medium for the microbial production of extracellular lipase.

Among the 12 strains tested, the most promising strain was C. cylindracea. Candida sp. is the
most potential lipase producer from yeasts reported in the literature. He and Tan (2006) used
the response surface methodology to optimize culture medium for lipase production by the
strain Candida sp. 99-125. After optimization, the authors reported the optimum lipase activity
as 6,230 and 9,600 U mL−1 in shaken flasks and in a 5-L bioreactor, respectively. In a 30-L
bioreactor, Tan et al.,(2003) reached a maximum lipase activity of 8,300 U mL−1, thus
showing that lipase activity values are highly influenced by the microorganism, substrates, and
the operational conditions. In contrast to the high activities reached in the above-mentioned
works, Rajendran et al., (2008) reported the optimum lipase activity of 3.8 U mL−1 by C.
rugosa.

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2.2 Screening of lipase- producing microorganisms

Lipolysis could be detected directly by changes in the appearance of the substrate such as
tributyrin and triolein, which were emulsified mechanically in various growth media and
poured into Petri dishes. Lipase production was indicated by the formation of clear halos
around the colonies grown on tributyrin containing agar plates. Bacillus strains were screened
for lipolytic activity on agar plates containing tributyrin or Tween 20 or Tween 80 (1%, w/v)
and 2% agar-agar. Lipolytic Bacillus sp. LBN 4 was isolated on tributyrate agar medium using
glycerol tributyrate as substrate. Lipolytic activity on solid media could be visualized by using
dyes such as Victoria blue B, Spirit blue, Nile blue sulfate and Night blue. The drop in pH due
to the fatty acids released as a result of hydrolysis was observed by change in the colour of
indicators used. There was a linear relationship between the diameter of the fatty acid diffusion
spot and the logarithm of the enzyme concentration. This technique is very convenient for rapid
screening of lipolytic microorganisms but acidification of the medium due to the generation of
acidic metabolism other than free fatty acids, which are released by microbial lipases, can give
false results. The fluorescent dye Rhodamine B could also be used in plate assay containing
emulsified olive oil to detect lipolytic organisms where substrate hydrolysis caused the
formation of orange fluorescent halos around bacterial colonies visible upon UV irradiation
and the lipase activities ranged from 1 to 30 nkat (Maryam et al.,2012)

2.3 Lipase production and parametric optimization

Enzymes can be produced by submerged fermentation (SmF) or by a solid state

fermentation (SSF). SmF, which involves the growth of the microorganism as a suspension in
nutrient enriched liquid medium, is the preferred method for production of commercial
enzymes, principally because sterilization and process control are easier to engineer in these
systems. On the other hand, SSF is the growth of microorganisms on most substrates in the
absence of free-flowing water. The advantages of SSF processes over liquid batch fermentation
include smaller volumes of liquid required for product recovery, cheap substrate, low
cultivation cost for fermentation, and lower risk of contamination. Fungal species are
preferably cultivated in SSF, while bacteria and yeast are cultivated in SmF (Dutra et al., 2008).
Lipase production in SmF has been reported using batch, repeated batch, fed-batch and
continuous fermentation.

Many studies have been undertaken to define the optimal culture and nutritional requirements
for lipase production by submerged culture. Microbial lipases production is greatly influenced

14
by medium composition besides physicochemical factors such as inoculum size and age,
temperature, pH, dissolved oxygen concentration etc.. The type and concentration of carbon
and nitrogen sources influence lipase production. The major factor for lipase activity has
always been reported as the carbon source, since lipases are inducible enzymes. Lipidic carbon
sources seem to be essential for obtaining a high lipase yield; however, a few authors have
produced good yields in the absence of fats and oils. These enzymes are generally produced in
the presence of a lipid such as oil or any other inducer, such as diacylglycerols, fatty acids,
hydrolysable esters, tweens, bile salts, and glycerol.

Nitrogen sources and essential micronutrients should also be carefully considered for growth
and production optimization. These nutritional requirements for microbial growth are fulfilled
by several alternative media as those based on defined compounds like sugars, oils, and
complex components such as peptone, yeast extract, malt extract media, and also agro-
industrial residues containing all the components necessary for microorganism development.
Generally, high productivity has been achieved by culture medium optimization. An improper
optimization of these factors leads to a lower production of the enzyme. Fermentation
conditions for enzyme production can be optimized either by considering one variable at a time
approach or by statistical approach (Maryam et al.,2012).

2.3.1 Effect of inoculum size and age

The preliminary requirement for mass production of microbial cells as well as enzymes
involves determination of suitable inoculum size and age. Initially, microbial cell growth as
well as enzyme activity increase with increase in inoculum size, but declines after a certain
limit. The effect of inoculum size on lipase activity can be correlated with total dissolved
oxygen in the medium. Any variation in inoculum size from the optimum concentration results
in reduced enzyme yield. Generally, 1-10 % (v/v) inoculum of bacterial culture is sufficient for
SmF reactions. Lee et al. (1999) reported that 1% inoculum size was optimum for lipase
production by Bacillus thermoleovorans ID-1 while Hasan et al. (2006) reported 5 % in case
of Bacillus sp. FH5. The maximum lipase yield reached up to 251.78 U/ml by G.
stearothermophilus B-78 at an inoculum size of 2.5 ml per 20 ml (Maryam et al.,2012).

A survey of the literature revealed that 12-24 h old inoculum would be best suited for
fermentation reactions of majority of microbial strains. A higher inoculum age is not preferred
at the industrial level. An inoculum of 18 h was found to be optimum for lipase production by

15
Thermoactinomyces vulgaris whereas 6 h and 24 h old culture led to the maximum lipase
production for Bacillus sp.

2.3.2 Effects of incubation period, temperature and pH

Incubation period is the time taken by the inoculated culture for synthesis of the desired product
(enzyme) utilizing the medium nutrients. However, the duration of incubation depends up on
the type of fermentation. Generally, the incubation period required for production under SSF
is higher as compared to SmF. Further, duration of incubation also varies according to the type
of microorganism used. The optimum period for bacterial lipase production in SmF was
reported to vary from 18 to 48 h (Ebrahimpour et al., 2008; El-Shafei and Rezkallah, 1997;
Handelsman and Shoham, 1994; Hasan et al., 2006). Fungal strains and yeast strain require
more incubation time as compared to bacterial strain. Candida rugosa (Song et al., 2001)
produced maximal titre of lipase after 60 h of incubation whereas Rhizopus oryzae (Hiol et al.,
2000) and Penicillium wortmanii (Costa and Peralta., 1999) required 4 and 5 days of incubation
respectively.

Temperature is an important physical parameter affecting the production of enzyme from a

given microbial culture. Different types of microorganisms require different optimum
fermentation temperature for maximum enzyme production. Some microorganisms such as
Bacillus cereus (Dutta and Ray, 2009), Acinetobacter radioresistens (Chen et al., 1998),
Enterobacter agglomerans (Zhen-qian and Chun-yun, 2009) and Pseudomonas sp. 7323
(Zhang and Zeng, 2008) showed 30 °C as the optimum temperature for growth and lipase
production. Hasan et al. (2006) reported the optimum temperature of 37 °C for lipase
production by Bacillus sp. FH5.

The pH of the growth medium is very important factor for microbial growth and enzyme
production. Depending upon the pH requirement, a microorganism may be acidophilic,
alkalophilic or both. Small variation from the optimum pH may lead to significant drop in
growth and enzyme production. Largely, bacteria prefer pH around 7.0 for best growth and
lipase production, such as in case of Bacillus sp. (Sugihara et al., 1991), Acinetobacter sp.
(Barbaro et al., 2001) and Burkholderia sp. (Rathi et al., 2001). On the other hand, bacterial
lipases were reported to prefer alkaline pH (>7.0) for growth and enzyme production (Dong et
al., 1999; Sharma et al., 2002 ; Wang et al., 1995).

16
2.3.3 Effects of carbon and nitrogen sources on lipase production

The major factor for the expression of lipase activity has always been carbon source. Sugihara
et al. (1991) reported lipase production from Bacillus sp. in the presence of 1 % olive oil in the
culture medium whereas modest enzyme activity was observed in the absence of olive oil even
after prolonged cultivation. Production of lipase can be significantly influenced by other carbon
sources such as sugars, sugar alcohol, polysaccharides, whey, casamino acids and other
complex sources.

However, lipases from Pseudomonas aeruginosa EF2 (Gilbert et al., 1991a) and Acinetobacter
calcoaceticus (Mahler et al., 2000) were repressed in the presence of long chain fatty acids,
such as oleic acid. Kanwar et al. (2002) reported the production of a Pseudomonas sp. G6
lipase in the presence of n-alkane substrates, with a maximum production of about 25 U/ml
when n-hexadecane was the sole carbon source. Olive oil and n-hexadecane were employed as
the carbon source for producing an alkaline lipase from Acinetobacter radioresistens (Liu and
Tsai, 2003). A thermophilic Bacillus strain A30-1 (ATCC 53841) produced maximal levels of
thermostable alkaline lipase when corn oil and olive oil (1 %) were used as carbon source
(Wang et al., 1995).

Lin et al. (1996) produced an alkaline lipase from Pseudomonas pseudoalcaligenes F111 in a
medium that contained both olive oil (0.4%) and Triton X-100 (0.2%). The addition of Triton
X-100 enhanced the alkaline lipase production by 50-fold compared to using olive oil alone.
Kim et al. (1998) reported production of a highly alkaline thermostable lipase by Bacillus
stearothermophilus L1 in a medium that contained beef tallow and palm oil. Salihu et al. (2011)
reported the use of palm oil mill effluent for lipase production by Candida cylindracea with an
activity of 20.26 U/ml under the optimized conditions. Besides carbon sources, nitrogen
sources have significant effect on lipase production as it is directly related to the cell growth
and division of microbial strains.

17
Table 2.3 The incubation period, temperature and pH for some of the lipolytic
microorganisms:

Sources: Hasan et al., 2004; Treichel et al., 2010

18
Table 2.4 Carbon and nitrogen sources used for lipase production from different
microorganisms

Sources: Gupta et al., 2004; Hasan et al., 2004; Treichel et al.,2010.

Nitrogen source can be provided in either inorganic (ammonium, sodium salts etc.) or organic
form (proteins, amino acids and urea etc.). Generally, organic nitrogen is preferred, Microbial
strain Incubation time.

such as peptone and yeast extract, which have been used as nitrogen source for lipase
production by various Bacillus sp. such as Bacillus strain A30-1, Bacillus alcalophilus and
Bacillus licheniformis strain H1. Inorganic nitrogen sources such as ammonium chloride and
diammonium hydrogen phosphate have also been reported to be effective in some microbes
(Bradoo et al., 1999; Dong et al., 1999; Gilbert et al., 1991a, 1991b; Rathi et al., 2001).
Kempka et al. (2008) reported corn steep liquor, yeast extract and peptone as best nitrogen

19
sources for lipase production from Penicillium verrucosumin while Mahanta et al. (2008)
reported peptone, NH4Cl and NaNO3 in case of Pseudomonas aeruginosa PseA.

2.4 Biotechnological Applications of Lipases

The industrial applications of lipases have been reviewed by many researchers .Lipases can be
used as biocatalyst in a variety of applications such as resolution of drugs,
esterification/transesterification reactions etc. The biosynthesis of esters is currently of much
commercial interest because of the increasing popularity and demand for natural products
amongst consumer. Biotransformations and enzymatic methods of ester synthesis are more
effective when performed in non-aqueous media.

2.4.1 Food industry

Lipases are employed in situ, and sometimes together with other enzymes, during the
elaboration of bread, cheese, and other foods to improve their shelf-life and their rheological
properties, or to produce aromas. Moreover, they are used ex situ to produce flavours, and to
modify the structure or composition of acylglycerols by inter- or transesterification, in order to
obtain acylglycerols with an increased nutritional value, or suitable for parenteral feeding.

2.4.2 Organic chemistry

Organic chemistry is the most important application of lipases after the food industry. They are
used to produce specific products that cannot be produced chemically, or whose elaboration by
classical chemical means is difficult or expensive. For example, they are used in
pharmaceutical and agrochemical industries for the modification or synthesis of antibiotics,
anti-inflammatory compounds, pesticides, etc., and for the production of enantiopure
compounds or the resolution of racemic mixtures.

2.4.3 Chiral resolution

Chirality is a geometrical attribute. An object that is not superimposable on its mirror image is
said to be chiral. The most common type of chiral organic molecule contains a tetrahedral
carbon atom attached to four different groups. Such a carbon is said to be a stereogenic center
and such a molecule exists in two stereoisomeric forms. Chirality is not a prerequisite for
bioactivity but in bioactive molecules where a stereogenic center is present, great differences
are observed in the activity of the enantiomers. This is general phenomenon and applies to all
bioactive substances, such as drugs, insecticides, herbicides, flavors, fragrances and food

20
additives. Conventional chemical synthesis of drugs containing a chiral centre generally yields
equal mixtures of enantiomers. During the past decade, many studies have shown that racemic
drugs usually have the desired therapeutic activity residing mainly in one of the enantiomers
and the other enantiomers might interact with different receptor sites, which can cause
unwanted side effects. The lipases have been reported to accept a wide range of substrates for
the production of compounds in high enantiomeric excess which could be used as chiral
building blocks for the synthesis of compounds of pharmaceutical interest.

2.4.4 Lipase as biosensor

The quantitative determination of triacylglycerol is of great importance in clinical diagnosis

and in food industry. The lipid sensing device as a biosensor is rather cheaper and less time
consuming as compared to the chemical methods for the determination of triacylglycerols. An
analytical biosensor was developed for the determination of lipids for the clinical diagnosis
(Masahiko et al., 1995). C. rugusa lipase biosensor from Candida rugosa has been developed
as a DNA probe.

2.4.5 Lipases in bioremediation

Oil spills in refinery, shore sand and processing factories could be handled by the use of lipases
from different origins (Demarche et al., 2011). It has been also used for the degradation of
wastewater contaminants such as olive oil from oil mills. Another important application has
been reported for the degradation of polyester waste, removal of biofilm deposits from cooling
water systems and also to purify the waste gases from factories .

2.4.6 Detergency and cleaning

An important application of lipases resistant to high temperatures, proteolysis, and denaturation

by surfactants, is their use in laundry detergents along with proteases to improve the removal
of lipid stains. They are also used in the synthesis of surfactants for soaps, shampoos and dairy
products.

2.4.7 Paper industry

Lipolytic enzymes are used to remove pitch, the lipid fraction of wood that interferes with the
elaboration of paper pulp. They also help in the removal of lipid stains during paper recycling
and to avoid the formation of sticky materials.

21
3. MATERIALS AND METHOD

To isolate the lipase producing bacteria from oil and milk contaminated soils, the following
materials and methods are used :
3.1 Samples collection

Isolation of lipolytic bacteria was made from three types of soil samples: 1. garage soil , 2.
Milk contaminated soils and 3. oil contaminated soil. For the first type of soil, samples from a
depth of 2cm was collected from garage where the soil was contaminated with lubricants and
second type of soil was collected from shiv temple (Fatehgarh Sahib, Punjab) contaminated
with milk .The third type, soil samples were collected from oil mill (kiratpur Sahib, Punjab ).

3.2 Isolation

Three samples of different soils considered in study. Samples of soils were diluted serially
from 10-1 up to 10-16 in sterile distilled water, each dilution was cultured on nutrient agar
plates by Spread plate method to obtain isolated colonies after 24 hours of incubation (Patel et
al., 2018).

3.3 Screening of the Isolates for Lipase Activity

Bacterial isolates were screened for lipase production were streaked on Tween 80 agar Plate
and phenol red media incubated at 37˚C for 48 hours.(Kumar et al.,2012 and Lee et al.,2015)

3.4 Lipase Production

The production of lipase enzyme is done by growing the isolate in inoculation and production
media .

3.4.1 Inoculation media

The isolate with higher lipase activity was inoculated in the 15 ml of inoculum media (20 gm
glucose, 10 gm yeast extract, 10 gm peptone, 10 gm CH3COONa.3H2O, 0.09 gm MgSO4,
0.03 gm MnSo4, 1.5 mg CuSO4.5H2O, 0.5 gm KCL, 5 ml Olive oil, 1000 ml Distilled water
and pH 10.8). The inoculum flasks were then incubated at 37°C temperature for overnight on
rotary shaker at 120 rpm (Patel et al.,2016)

3.4.2Production media

The composition of production medium used in this study was Peptone 0.2gm, NH4H2PO4

22
0.1gm, NaCl 0.25gm, MgSO4.7H2O 0.04gm, CaCl2.2H2O 0.04gm, Olive oil 2ml, Tween 20
2-3 drops. (Gaherwal et al.,2015). Submerged microbial cultures were incubated in two 250
ml Erlenmeyer flasks containing 50 ml of liquid medium each on a rotary shaker (120 rpm)
and incubated at 37°C for 48 and 92 hours respectively (Ullah et al.,2015) and pH 5,6,7 and 8
respectively.

3.5 Enzyme Isolation and purification by Ammonium salt precipitation

After incubation, the culture was centrifuged at 6000rpm for 10 minutes to obtain clarified
supernatant. The cell free culture supernatant fluid was used as the sources of extracellular
enzyme. Further purification was performed using Ammonium sulphate salt precipitation.
According to this method, ammonium sulphate salt was slowly added to crude enzyme solution
under continuous stirring at 0°C until the required saturation percentage was reached (50-
100%). The solution was then incubated at 40˚C overnight. Then, the solution was centrifuged
6000rpm for 10 minutes and the precipitates obtained were re-dissolved in phosphate buffer
saline pH7.5 (Poddor and Singha.,2015).

3.6 Qualitative determination of lipolytic Activity

Lipase activity was measured by titrimetric method using olive oil as a substrate.10 ml Olive
oil and 2ml of Cacl2 (0.6%) is dissolved in 5ml potassium phosphate buffer (0.2M) pH 7.2 .
1ml of enzyme was added to the solution and incubated at 125 rpm for 15, 20, 30 minutes at
35°C. The reaction was stopped by addition of 20ml of acetone: ethanol solution (1:1) ( Kumar
et al., 2012).

3.6.1 Effect of pH on lipase activity

The optimum pH for lipase production was selected by varying the pH of the production medium as 5,
6,7 and 8 respectively. The amount of fatty acids liberated were estimated by titrating with 0.1M
NaOH until pH 10.2 using a phenolphthalein indicator (Kumar et al.,2012)

𝑣𝑜𝑙𝑢𝑚𝑒 𝑜𝑓 𝑎𝑙𝑘𝑎𝑙𝑖 𝑐𝑜𝑛𝑠𝑢𝑚𝑒𝑑 × 𝑛𝑜𝑟𝑚𝑎𝑙𝑖𝑡𝑦 𝑜𝑓 𝑁𝑎𝑂𝐻

𝑙𝑖𝑝𝑎𝑠𝑒 𝑎𝑐𝑡𝑖𝑣𝑖𝑡𝑦 =
𝑡𝑖𝑚𝑒 𝑜𝑓 𝑖𝑛𝑐𝑢𝑏𝑎𝑡𝑖𝑜𝑛 × 𝑣𝑜𝑙𝑢𝑚𝑒 𝑜𝑓 𝑒𝑛𝑧𝑦𝑚𝑒 𝑠𝑜𝑙𝑢𝑡𝑖𝑜𝑛

23
4. RESULTS AND DISCUSSION

4.1 Isolation and screening

Three different soil samples were taken in the study. Samples of soils were diluted serially
from 10-1 up to 10-16 in sterile distilled water, each dilution was cultured on nutrient agar
plates by Spread plate method to obtain isolated colonies after 24 hours of incubation.Colonies
obtained were picked from nutrient agar plate termed as sample no 1-20.To test isolates as a
lipase producer, streaked on tween 80 agar plate (fig 4.1) and phenol red media containing
olive oil (fig 4.3) .

Tween have been the most widely used substrates for the detection of lipase producing
microorganisms in agar media. Screening using tween agar plates shows precipitation around
the lipase producing micro-organisms. The method is based on the precipitation as the calcium
salt of the fatty acids released by hydrolysis of tweens. Liberated fatty acids bind with the
calcium salt incorporated into the medium.

Fig 4.1 Tween 80 agar plates showing precipitation of calcium salt .

24
The isolates capable of lipase production were further screened to isolate the best possible
lipase producing bacteria based on color change in Phenol Red media with olive oil as a
substrate(fig 4.2). production of yellow color indicates lipase producer in Phenol red media
due to change in pH of the medium as the liberation of fatty acid( fig 4.4), the data was used
for further production of enzyme.

Fig 4.2 Phenol red media cultured with different isolates . showing different color due to lipase
activity

25
Fig 4.3 Phenol red culture media of sample 1 Fig 4.4 Phenol red culture media of sample
Showing no lipase activity . 12 showing most lipase activity .

Fig 4.5 Phenol red culture media of sample 8 showing less Lipase activity.

26
4.2 Production
Out of 20 bacterial isolates only 9 isolates were lipase producer. In which sample 12 show the
most color change that means most enzyme activity then other samples. so, sample 12 was used
for further production of enzyme by production media (3.4.2) and divided into two parts, one
was incubated for 2 days labeled as 12a and another for 4 days labeled as 12b.

4.3 Qualitative estimation of lipase enzyme by lipase assay titrimetric method

Titrimetric method was used for check quality and efficiency. The amount of fatty acids
liberated were estimated by titrating with 0.1M NaOH until pH 10.2 using a phenolphthalein
indicator.

𝑣𝑜𝑙𝑢𝑚𝑒 𝑜𝑓 𝑎𝑙𝑘𝑎𝑙𝑖 𝑐𝑜𝑛𝑠𝑢𝑚𝑒𝑑 × 𝑛𝑜𝑟𝑚𝑎𝑙𝑖𝑡𝑦 𝑜𝑓 𝑁𝑎𝑂𝐻

𝑙𝑖𝑝𝑎𝑠𝑒 𝑎𝑐𝑡𝑖𝑣𝑖𝑡𝑦 =
𝑡𝑖𝑚𝑒 𝑜𝑓 𝑖𝑛𝑐𝑢𝑏𝑎𝑡𝑖𝑜𝑛 × 𝑣𝑜𝑙𝑢𝑚𝑒 𝑜𝑓 𝑒𝑛𝑧𝑦𝑚𝑒 𝑠𝑜𝑙𝑢𝑡𝑖𝑜𝑛

4.3.1 Effect of time on enzyme quality

The optimum time period for lipase activity was selected by varying the time of incubation.
The substrate olive oil broth media was added to 1 ml of sample 12a and 12b and incubated for
15, 20, 30 minutes at 35°C. The amount of fatty acids liberated were estimated by titrating with
0.1M NaOH until pH 10.2 using a phenolphthalein indicator.

Table 4.1 Effect of time on enzyme activity.

s.no sample 12a sample 12b blank incubation time(in

minutes)
volume of alkali consumed (ml)
1.) 5.4 50.2 4 15
2.) 5.3 53.1 4.2 20
3.) 5 63.2 4 30

Sample 12a didn’t show enzyme activity as same as blank. sample 12b shows the different
enzyme activity according to time change as shown in following table 4.2

Table 4.2 Results of enzyme activity at different incubation time.

Time (in min.) Enzyme activity (IU/ml)

15 0.1666

20 0.1327
30 0.1053

27
 70

60
alkali consumed (in ml)

50

40

30

20

10

0
15 mins. 20 mins. 30 mins.
time (mins.)
12a 12b blank

Fig 4.1 Graphical representation of given values in table 4.1

4.3.2 Effect of pH on Enzyme quality

The optimum pH of both samples was selected by varying the pH of the olive oil broth medium
as 5, 6 ,7 and 8 respectively, incubated for 15 minutes at 35˚C . The amount of fatty acids
liberated were estimated by titrating with 0.1M NaOH until pH 10.2 using a phenolphthalein
indicator.
Table 4.3 Effect of pH on enzyme activity .

s.no sample 12a sample 12b blank pH

volume of alkali consumed (ml)
1.) 4.1 40.2 4.3 8
2.) 5 49.6 4 7
3.) 5.3 39 5.5 6
4.) 5.1 31 5 5

Sample 12a didn’t show enzyme activity as same as blank .sample 12b shows the different
enzyme activity according to pH change as shown in following table 4.4

28
 60

50

40
Alkali consumed

30 12a
12b
20 blank

10

0
8 7 6 5
pH of olive oil broth

Fig 4.2 Graphical representation of given values in table 4.3

Table 4.4 Result of lipase activity at different pH.

pH of olive oil broth Lipase activity (IU/ml)

8 0.134
7 0.163
6 0.13
5 0.103

4.5 DISCUSSION

In present study, Oil and milk contaminated soil used for isolation of lipase producing bacteria
Sagar et al.,( 2013) from area of Fatehgarh Sahib and Kiratpur Sahib, Punjab, India. lipolytic
organisms were isolated and identified using preliminary and confirmatory tests. Total 20
isolates obtained by primrary screening on Nutrient agar plate grown in the selective medium
like Phenol red broth media and Tween-80 agar plate similar to Kumar et al.(2012) and Lee et
al.(2015) were found to produce lipase. Their growth showed that they can use olive oil and
Tween 80 as carbon source and showed the lipase producing feasibility. Isolate no 12 obtained
from oil mill site soil sample of Kiratpur sahib area of Kiratpur sahib, Punjab, India showing
maximum lipolytic activity which were further used for production. In recent study, Inoculum
media used similar to Patel et al. (2016) which was used for inoculation of Production media

29
which was similar to Gaherwal S. et al.(2015).The maximum production of enzyme obtained
after 96hr of incubation at 37°C and pH 7.0 in Rotary shaker at 120 rpm which is similar to
Nadeem Ullah et al.(2015) except of production time of 48hr and 92hr . The culture was
centrifuged at 6000rpm for 10 minutes to obtain clarified supernatant. Further purification was
performed using Ammonium sulphate salt precipitation similar to Poddor and Singha., (2015).
Lipase activity was measured by titrimetric method using olive oil as a substrate as similar to
Kumar et al. (2012)

30
5. CONCLUSION

The current work was aimed to isolate lipase producing soil bacteria isolated from oil and milk
contaminated soil. The bacteria that was positive towards lipase production was prepared as
pure culture and used for further analysis. Production media was prepared by incorporating
tween 80 and olive oil in the basic media. The production media was incubated for 2 and 4
days and the enzyme thus produced was extracted and purified. The pure enzyme was thus
assayed qualitatively and the efficiency was found to be 0.166 IU/ml. The study was further
focused to study the optimal conditions for the maximum enzyme production. The optimum
time for production, incubation time for olive oil broth and pH was calculated to be 4 day (92
hours), 15 minutes and 7 respectively. The enzyme thus produced was maximum under these
conditions. This bacteria can be used for further molecular and biochemical characterization.

31
6. SUMMARY

Lipase catalyses the hydrolysis of the carboxyl ester bonds in triacylglycerols to produce
diacylglycerols, monoacylglycerols, fatty acids and glycerol. In addition, lipases catalyse the
hydrolysis and transesterification of other esters as well as the synthesis of esters. Lipase is
secreted from the pancreas via a duct that empties into the gastrointestinal tract at the
duodenum. It thus acts on food that has already been partially digested in the stomach. First,
bile made in liver and released into your intestine converts dietary fat into small fatty globules.
Pancreatic lipase, acts on these fat globules, converting them into fatty acids and glycerol,
which are small, energy-dense molecules used by all our cells. Fatty acids and glycerol travel
in blood and lymph vessels to reach all parts of our body.

The project entitled “Isolation of lipase producing bacteria from oil and milk contaminated
soil”.

• It deals with the introduction highlighting the objectives of the research work
undertaken, aim of the study and method of isolation, screening and production of lipase
producing bacteria.
• The review of literature summarizes the literature highlighting the production,
identification and characterization of various lipase producing microbes and there
parametric optimization.
• Material and methods employed for isolation and screening of lipase producing
microbes, preparation of inoculation and production media, extraction and purification
of lipase enzyme and qualitative determination of lipolytic activity.
• Result and discussion compiled the experimental investigations and data were
represented in the form of figures and tables. Results showed that isolate 12 showed
most lipase activity after the incubation of 92 hours at 37°C and pH 7, the lipase
efficiency was found to be 0.166 IU/ml.

32
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