Effect of Carbon and Nitrogen Sources on Lipase Production ...

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



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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

[1] R. Sharma, Y. Chisti, and U. C. Banerjee, “Production, purification, characterization, and applications of lipases,”

Biotechnol Adv, vol. 19, pp. 627–62, 2001. [2] K. E. Jaegerm and T. Eggert, “Lipases for biotechnology,”

Current Opinion in Biotechnology, vol. 13, pp. 390–397, 2002.

[3] S. Ertugrul, G. Donmez, and S. Takac, “Isolation of lipase producing bacillus sp. from olive mill wastewater and improving

its enzyme activity,” Journal of Hazardous Materials, vol. 149, pp.

720-724, 2007. [4] A. Robles-Medina, P. A. Gonzalez-Morino, L. Esteban-Cerdan,

and E. Molina-Grima, “Biocatalysis: Towards ever greener

biodiesel production,” Biotechnology Advanced, vol. 27, pp. 398–408, 2009.

[5] S. Shah, S. Sharma, and M. N. Gupta, “Biodiesel preparation by

lipase catalysed transesterification of jatropha oil,” Energy Fuels, vol. 18, pp. 154, 2004.

[6] T. Boonchaidung and R. Leesing, “Lipase production by isolated

lipase-producing yeast,” in Proc. International Conference on Microbial Taxonomy, Basic and Applied Microbiology, Khon

Kaen, Thailand, 2012, pp. 371-379.

[7] M. Elibol and D. Ozer, “Influence of oxygen transfer on lipase

production by rhizopus arrhizus,” Process Biochem, vol. 36, pp.

325-9, 2000.

[8] N. R. Sonare and V. K. Rathod, “Transesterification of used sunflower oil using immobilized enzyme,” Journal of Molecular

Catalysis B: Enzymatic, vol. 66, pp. 142–7, 2010.

[9] D. Papaparaskevas, P. Christakopoulos, D. Kekos, and B. J. Macris, “Optimizing production of extracellular lipase from

rhodotorula glutinis,” Biotechnol Lett, vol. 14, pp. 397-402, 1992.

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



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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.



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