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Effect of Carbon and Nitrogen Sources on Lipase
Production by Isolated Lipase-Producing Soil
Yeast
Thanagrit Boonchaidung and Thidarat Papone Graduate School, Khon Kaen University, Khon Kaen 40002, Thailand
Department of Microbiology, Faculty of Science, Khon Kaen University, Khon Kaen 40002, Thailand
Email: [email protected]; [email protected]
Ratanaporn Leesing Department of Microbiology, Faculty of Science, Khon Kaen University, Khon Kaen 40002, Thailand
Research Group for Development of Microbial Hydrogen Production Process from Biomass, Khon Kaen University,
Khon Kaen 40002 Thailand
Email: [email protected]
Abstract—Microbial lipase can be applied in many
industrial, such as detergent formulation, fat and oil
degradation, pharmaceutical synthesis, cosmetics
production and biodiesel preparation. Increasing in demand
for lipase, increasing attention has been focused to how to
produce it efficiently and economically. In this work, the
effect of carbon and nitrogen sources and pH on lipase
production by isolated soil yeast Candida sp. KKU-PH2-15
was investigated by shaking flask batch culture. The
maximum biomass of 18.77g/L was obtained using glucose
and palm oil as carbon source and inducer, yeast extract as
nitrogen source at pH 6.0, 120 rpm, and 30◦C for 60h of
cultivation time. The highest extracellular lipase activity of
lipase-producing yeast isolate was achieved at 1.2 U/mL
with specific activity of 0.65U/mg.
Index Terms—Lipase-producing yeaset, lipase, microbial
lipase
I. INTRODUCTION
Lipase, (EC 3.1.1.3; triacylglycerol acylhydrolases) are
enzymes that catalyze hydrolysis and synthesis of esters,
and transesterification reactions. Lipases catalyze a wide
range of commercial and industrial processes, such as the
synthesis of chemicals, the preparation of specialty esters
for the food and cosmetic industries, treatment of fatty
effluents, pharmaceuticals and leather industry and
lipase-catalyzed transesterification for biodiesel fuel
production [1]-[3].
Microbial lipases have a great potential for commercial
applications due to their selectivity and broad substrate
specificity. Nowadays, most lipases produced
commercially are currently obtained from fungi and
yeasts. Microbial lipases are produced by Candida
antarctica, Pseudomonas cepacia, Thermomyces
lanuginosus have been employed as biocatalysts in
biodiesel synthesis [4], [5]. However, the locally lipase-
producing yeast Candida sp. KKU-PH2-15 has proved to
produce extracellular lipase efficiently that stabilizing in
organic solvent up to 300 µL of ethanol. This is a great
feature on its use as a catalyst for biodiesel production via
transesterification processes [6]. Lipase production is
influenced by the type and concentration of carbon and
nitrogen sources, the culture pH medium, the growth
temperature, and the dissolved oxygen concentration [7].
Lipidic carbon sources seem to be generally essential for
obtaining a high lipase yield; however, a few authors
have produced good yields in the absence of fats and oils.
In this paper, the effect of carbon and nitrogen sources on
lipase production by isolated lipase-producing yeast
isolate KKU-PH2-15 is described.
II. MATERIALS AND METHODS
A. Microalgae Strains and Culture Condition
The lipase-producing yeast isolate KKU-PH2-15
isolated from soil samples collected in the area of Roi-Et
province, north-eastern Thailand, was used for lipase
production. The seed culture was pre-cultivated in basal
medium supplemented with 20 g/L glucose at 30°C in an
incubator shaker at a shaking speed of 120 rpm for 24h.
Basal medium was consisted of (g/L): glucose 3.0, yeast
extract 2.0, MgSO4.7H2O 0.75, K2HPO4 1.40, palm oil
12.5 % (V/V). Batch cultures were investigated to
determine the effect of carbon and nitrogen sources on
lipase production, lipase-producing yeast isolated was
grown in 250-mL Erlenmeyer flask containing 100mL of
basal medium and incubated at 30C in an incubator
shaker at a shaking speed of 120 rpm for 72h. Samples
were taken every 12h for determination of growth, lipase
activity and protein concentration.
B. Effect of Different Carbon and Nitrogen Sources on
Lipase Production
Glucose, fructose, sucrose, xylose, lactose, galactose
and starch, were added to the basal medium
2013 Engineering and Technology Publishing
Journal of Life Sciences and Technologies Vol. 1, No. 3, September 2013
176doi: 10.12720/jolst.1.3.176-179
Manuscript received June 10, 2013; revised August 30, 2013.
supplemented with palm oil in order to study the effect of
carbohydrate carbon sources on lipase production. Palm
oil, soybean oil, corn oil, sunflower oil and rice bran oil
were added to the basal medium supplemented with
glucose in order to study the effect of lipidic carbon
sources on lipase production. Yeast extract, peptone,
tryptone, casein acid and ammonium nitrate, were used to
study the effect of nitrogen source on lipase production.
C. Analytical Methods
Triplicate samples were analyzed for cell dry weight,
lipase activity and protein concentration. The culture
broth was centrifuged at 8,000 rpm at 4C for 20 min.
The obtained supernatant was analyzed for lipase activity
and protein concentration. Harvested biomass was
washed twice with distilled water and then dried at 90°C
to constant weight. The biomass was determined
gravimetrically. Protein determination was performed
according to Lowry (1951) using bovine serum albumin
as standard.
Lipase activity was determined by measuring the
increase in the absorbance at 405 nm in a visible
spectrophotometer caused by the release of p-nitrophenol
(pNP) after hydrolysis of p-nitrophenyl palmitate (pNPP)
as substrate [8]. The substrate mixture (0.5 mM) was
prepared by dissolving p-NPP in ethanol. The reaction
mixture was conducted by addition of 0.5mL of culture
supernatant in 1.0mL of substrate mixture, and then
incubated at 35◦C, 15 min. The reaction was stopped by
adding 2mL of 0.25 m Na2CO3 solution and measured the
absorbance at 405 nm. One unit of lipase activity was
defined as the amount of enzyme that liberated 1 µmol of
pNP from pNNP per milliliter per minute under standard
assay condition. The calibration curve was prepared using
pNP as standard.
III. RESULTS AND DISCUSSION
A. Effect of Different Carbon Sources
Figure 1. Effect of carbohydrate carbon sources on growth and lipase
production by Candida sp. KKU-PH2-15 at 30C, 120 rpm for 60h.
The effect of carbohydrate and lipidic carbon sources
on lipase production of Candida sp. KKU-PH2-15 was
investigated at 30◦C and 120 rpm throughout 72h of
cultivations. It is apparent that all of carbon sources were
used mainly for cell growth at the beginning of
cultivation. As shown in Fig. 1, it was concluded that
good growth with 17.58 and 17.50 g/L of biomass using
glucose and lactose as carbohydrate carbon source,
respectively. High lipase activity of 1.045 and 0.984
U/mL was obtained on media supplemented with glucose
and sucrose, respectively, while the activity was low in
basal medium supplemented with xylose (0.654 U/mL)
and galactose (0.521 U/mL). The production medium
without carbohydrate carbon sources (control) led to low
growth and lipase activity.
Figure 2. Effect of lipidic carbon sources on growth and lipase
production by Candida sp. KKU-PH2-15 at 30C, 120 rpm for 60h
Lipidic carbon sources or inducers such as fats and
vegetable oils seem to be generally essential for obtaining
a high lipase yield, vegetable oils was used for inducers
on lipase production of lipase-producing microorganism.
The effect of lipidic carbon sources on lipase production
was investigated at 30◦C and 120 rpm for 72h. As
indicated in Fig. 2, palm oil and sunflower oil were the
effective lipidic carbon sources for cell growth and lipase
production. A biomass of 14.5 and 16.9g/L were obtained
on basal medium supplemented with palm oil and
sunflower oil, respectively. A biomass of 11.4, 7.2, 6.0
and 7.12g/L were obtained using soy bean oil, corn oil,
rice bran oil and without vegetable oil as lipidic carbon
sources, respectively. Lipase activities were 0.876 and
1.069 U/mL higher with palm oil and sunflower oil,
respectively at 60h of cultivation time when compared
with soy bean oil, corn oil and rice bran oil. Hence,
sunflower oil and palm oil could be effectively used for
the production of lipase from yeast isolate Candida sp.
KKU-PH2-15. The production medium without lipidic
carbon sources (control) led to low growth and low lipase
activity.
B. Effect of Different Nitrogen Sources
The effect of nitrogen sources on growth and lipase
production was investigated at 30◦C and 120 rpm using a
mixture of glucose and palm oil as carbon source.
Reports in the literature show that the lipase production
could be improved by inorganic nitrogen sources while
the cell growth was influenced by organic ones [9]. In
this work, yeast extract, peptone, casein acid and tryptone
were used as the organic nitrogen source and ammonium
nitrate was used as inorganic source. The obtained result
indicated that yeast extract was effective nitrogen source
for both of growth and lipase production by Candida sp.
KKU-PH2-15. Maximum lipase activity of 1.134 U/mL
was significantly observed by yeast extract when added
2013 Engineering and Technology Publishing
Journal of Life Sciences and Technologies Vol. 1, No. 3, September 2013
177
as a nitrogen source, while lipase activities of 0.782,
0.656, 0.463 and 0.464 were obtained when peptone,
casein, tryptone and ammonium nitrate were used,
respectively, (Fig. 3).
Figure 3. Lipase activity (A), protein concentration (B), specific activity
(C) and biomass (D) of Candida sp. KKU-PH 2-15 cultivated in basal medium supplemented with different source of nitrogen using glucose
as carbon source at 30C, 120 rpm for 60h.
Among the nitrogen sources tested, yeast extract and
peptone supported the maximum biomass of 16.4 and
12.2g/L, respectively, while a biomass of 7.29, 6.32, and
6.3g/L was obtained using casein, tryptone and
ammonium nitrate, respectively. In this study, inorganic
nitrogen source was not suitable for cell growth and
lipase production. High yields of the enzymatic activity
were obtained when the culture medium was added with
nitrogen sources, while low titer of lipase activity was
observed without the addition of nitrogen sources
(control).
It is seen that lipase production occurred
simultaneously to the cell growth and continued after the
stationary phase was reached. However, it is seen that the
lipase produced by selected yeast isolate Candida sp.
KKU-PH2-15 is not very high titer compared with the
other source therefore increasing of lipase activity should
be investigated such as mutation of yeast by mutagen,
optimizing the culture condition via statistical method via
respond surface methodology (RSM). Identification of
selected yeast Candida sp. KKU-PH2-15 will be
performed by sequence analysis of the variable D1/D2
domain of the large subunit (26S) ribosomal DNA.
In order to reduce the enzyme production costs, some
strategies can be applied like the use of agro-industrial
residues i.e. sugarcane molasses as carbon or inducer
sources for lipase production to evaluate the feasibility of
lipase application in biodiesel production. Lipase-
catalyzed transesterification for biodiesel production
should be investigated for further study.
ACKNOWLEDGMENT
This work was financially supported by The Center for
Alternative Energy Research and Development (AERD)
and Incubator Researcher’s Project, Khon Kaen
University, Khon Kaen, Thailand and Human Resource
Development in Science Project, Office of the Higher
Education Commission through Science Achievement
Scholarship of Thailand (SAST).
REFERENCES
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[6] T. Boonchaidung and R. Leesing, “Lipase production by isolated
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[7] M. Elibol and D. Ozer, “Influence of oxygen transfer on lipase
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Thanagrit Boonchaidung graduated the Bachelor Degree of Science
(B.Sc.) in Microbiology from Naresuan University, Thailand, obtained his Master degree of Science (M.Sc.) in Microbiology from Khon Kaen
2013 Engineering and Technology Publishing
Journal of Life Sciences and Technologies Vol. 1, No. 3, September 2013
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University, Thailand in 2012. Since 2013, he is a technical lecturer at
the Office of General Education, Khon Kaen University, Thailand.
Thidarat Papone got Bachelor of Science (B.Sc.) in Biology from Mahasarakam University, Thailand. Currently she is a PhD student at
the Department of Microbiology, Faculty of Science, Khon Kaen
Univesity Thailand.
Ratanaporn Leesing graduated PhD from the University of
Montpellier 2, France in 2006. She is assistant professor at the
Department of Microbiology, Faculty of Science, Khon Kaen University,
Khon Kaen, Thailand and researcher at Research Group for Development of Microbial Hydrogen Production Process from Biomass,
Khon Kaen University. The research area is biofuel production from
microalgae and oleaginous yeast.
2013 Engineering and Technology Publishing
Journal of Life Sciences and Technologies Vol. 1, No. 3, September 2013
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