GHTMDD - 373

Received: July 3, 2000Accepted: April 2, 2001

Bulletin of the Chemists and Technologists of Macedonia, Vo!. 20, No. 1, pp. 39-43 (2001)ISSN 0350 - 0136

DDC: 663.152.311 : 582.28

Original scientific paper

LIPASE PRODUCTION BY GEOTRICHUM CANDIDUM-M2

Irina Mladenoska*, Aco Dimitrovski

Faculty of Technology and Metallurgy, The "Sv. Kiril & Metodij" University,Rudjer Bos'kovic16, MK-lOOO Skopje, Republic of Macedonia

*E-mail: irinaetf@mt.net.mk

Investigation on the effects of fermentation conditions and culture medium composition on lipase productionby the yeast-like fungus Geotrichum candidum-M2 has been performed. The sunt10wer oil sediment was tested ascarbon source and compared to several other oils and carbohydrates. It was proven as the best carbon source, whereasthe yeast extract was the best nitrogen source. The culture medium selected as optimal one contained each of them inconcentration of 1 %. Maximum lipolytic activity of 0.45 D/ml was achieved under the following conditions: dura-tion of the process 48 h, inoculum concentration 5 % (v/v), inoculum age 48 h, initial pH 9.0 and agitation rate 100rpm. G. candidum lipase showed good termostability at temperatures about 50 0 C and pH stability at alcaline pH 9.

Key words: Geotrichum candidum; lipase production; submerged fermentation; sunt10weroil sediment;environmental parameters

INTRODUCTION

Upases (EC 3.1.1.3) or triacyl glycerol hy-drolases are widely distributed in nature. Lipasebiosynthesis occurs in animals, plants and micro-organisms. Microbiallipases are receiving particu-lar attention because of their actual and potentialindustrial application. They are widely used infood and pharmaceutical industry as well as in oiland fat industry. Cheese ripening, preparation ofcocoa butter substitutes and flavor production aresome of their applications in food industry [1].They can hydrolise oils and fats even at very lowtemperatures, which makes them particularly suit-able for application in washing detergent [2]. Li-pase ability of catalyzing reactions other than hy-drolysis, such as esterification and interesterifica-tion, can be exploited in the production of some

specific compounds, such as tailor made lipids andbioemulsifiers [3,4].

Moulds are the most exploited among the mi-croorganisms as lipase producers. Aspergillus ni-ger and Rhisopus arrhisus are producers of someof the best sold commercial lipase preparations [5].Maybe the most specific one is the fungus Geo-trichum candidum, which reacts only with fattyacids with a eis-double bond in the 9-position [6].

In this paper, the optimal composition ofgrowth medium for lipase production by locallyisolated strain of Geotrichum candidum was de-

termined. The effect of different environmental pa-rameters on lipase synthesis was also investigated.

EXPERIMENTAL

Microorganism

The microorganism was isolated in our labo-ratory from spoiled milk. It was identified as Geo-trichum candidum penicillatum. The identificationwas performed on miniAPI apparatus. Strips for

the biochemical tests were supplied from Biome-rieux - France. The microorganism was depositedat a Faculty culture collection under the name Geo-trichum candidum-M2. The culture was maintainedat 4 °C on malt extract agar slants.

40 I. Mladenoska, A. Di11Iitrovski

Culture medium

The mineral medium had the following compo-sition (g/l): KH2P04 2, MgS04'7H2O 1, CaCh'H2O 1and NaCI 1. It was supplemented with carbon andnitrogen sources. Altering the type of carbon andnitrogen source one at a time, a selection of thebest medium for lipase production was carried out.Several carbohydrates and lipids were examined ascarbon sources. One of the lipids was the sun-flower oil sediment, a complex substrat with thefollowing composition (%): oil 63, water 3.9, ni-trogen 2.59 and suspended solids, 30.5. The me-dium was sterilized at 121 QCfor 35 minutes.

Growth conditions

A lO-ml portion of seed culture (3.5 x 106spores/m!) was inoculated into 500-ml Erlenmeyerflasks containing 100 ml of the culture medium.When medium composition was investigated, cul-tivation lasted 72 hours at 30 QC,and was carriedout on a rotary shaker at 170 rpm. When duration ofthe process was an experimental variable, period ofincubation was prolonged to 96 h. Selected processdurationof 48 h was used in subsequentexperiments.

When the effects of the concentration and in-oculum age on lipase and biomass production wereinvestigated, inoculum inconcentration 5, 10 and15 % v/v of the culture old 48, 72 and 96 h, wasused. In this experiment pH of the medium was

unadjusted. When the effect of initial pH was in-vestigated, inoculum in concentration of 10 % v/vwas used, obtained from the culture old 72 h. Ini-tial pH was adjusted with HCI and NaOH solu-tions. In this experiment pH interval between 4 and10 was used.

In the experiment for selection of the optimalagitation rate, agitation of 100, 150 and 200 rpmwas used. All other parameters were the optimalones selected from the previous experiments.

Separation of lipase

At the end of the cultivation, the culture brothwas filtered and centrifuged at 4000 rpm for 50minutes. The supernatant was used as the crudeenzyme for the estimation of lipase activity.

Analytical methods

Lipase activity was measured by the methodof Kwon and Rhee [7]. One unit (1U) of lipolyticactivity is the amount of enzyme, which liberates1 /lmol free fatty acids per minute under the assayconditions. Oil content in the sunflower oil sedi-

ment was determined gravimetrically after three-fold extraction by petrolether. Nitrogen content inthe sediment was determined by the Kjeldahlmethod. Water content was determined gravimetri-cally by drying at 105 QC.

Effect of carbon source

RESULTS AND DISCUSSION

Several carbohydrates and lipids were testedfor their effect on lipase production by Geotrichumcandidum-M2. Yeast extract of 1 % (w/v) wasused as a nitrogen source. The highest lipolyticactivity of 0.28 U/ml was obtained by the utiliza-tion of sunflower oil sediment (Table 1). Biomassproduction in this case was 9.67 g/!. Maximumgrowth of 11.70 g/l was achieved in the mediumwith olive oil as a sole carbon source. This lipidsubstrate was well used for growth but much lessfor the lipase production, the activity of which was0.02 Ulm!. Regarding the carbohydrates, they didnot stimulate the biosynthetic activity of the fun-gus. The conclusion that lipids were better carbonsources for lipase production than carbohydrates,confirmed the results reported by many authors [8,9].

Table 1

Effect of carbon and nitrogen source on growth and

lipase production by Geotrichum candidum-M2

Source Biomas (g/l) Lipase activity (U/ml)CarbonCarbohydrates (1 %w/v)

GlucoseLactoseFructoseGalactoseSoluble starch

LipidsSunflower oilOlive oilSunflower oil sediment

Nitrogen (I % w/v)UreaYeast extractSoybean flourNH4N03(NH4)2HP04(NH4)2S04

Bull. Chem. Technol. Macedonia, 20, 1,39-43 (2001)

7.57 0.042.73 0.026.57 0.015.76 0.001.92 0.01

2.93 0.0111.7 0.029.67 0.28

3.91 0.016.51 0.288.96 0.042.43 0.045.36 0.077.40 0.02

Lipase production by Geotrichum candidum-M2 41

Effect of nitrogen source

A number of organic and inorganic nitrogensources were tested in their effect on lipase produc-tion. In this experiment sunflower oil sediment wasused as a sole carbon source, in concentration of

1% (v/v). The highest lipase activity of 0.28 V/mlwas obtained in a medium with yeast extract (Ta-ble 1). Its intrinsic effect on growth and biosyn-thetic activity of the fungus can be result of thedifferent amino acids and growth factors present inlarge quantity in the yeast extract [l0]. The bestgrowth of the fungus was obtained with the soy-bean flower as a nitrogen source, but the lipaseproduction was only 0.03 U/mI. The utilization of

inorganic nitrogen sources did not stimulate lipaseproduction by G. candidum-M2. Medium with

sunflower oil sediment as carbon source and yeastextract as nitrogen source was used in all subse-quent experiments.

Selection of the process duration

In order to determine the effect of dynamicsof the lipase production, the fungus was cultivated96 hours, and samples were taken every 24 hours.In Figure 1, the lipolytic activity and the biomassconcentration versus incubation period are plotted.One can see that the enzyme production was notfollowed by the growth. The maximum growth of12.30 g/l was obtained after 48 hours of fermenta-tion, when the curve of lipase productionpassesthrough minimum. Although the highest lipase ac-tivity of 0.43 V/ml was obtained after 24 h of fer-mentation, the 48-hour process was chosen as theoptimal one because of easier downstream process-ing. Namely, by th<).ttime most of the lipid materi-als disturbing the isolation of the enzyme, werespent. Lipase activity achieved for this period was0.25 U/mI.The slight rise of lipolytic activity ap-

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peared after 48th hour. This may be due to a re-lease of the membrane-bounded lipase as a resultof cell autolysis. Process duration of 48 h was usedin subsequent experiments.

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Fig. 1. Time course of lipase (0) and biomass ( . )production on selected culture medium containing sunflower

oil sediment (1 %v/v) and yeast extract (1 %w/v)

Effect of inoculum age and its concentration

In order to see the effect of thesetwo parame-ters on growth and lipolytic activity of the fungus,inoculum obtained from culture old 48, 72 and 96hours, was used in concentration of 5, 10 and 15 %v/v.

The highest biomass concentration of 13.20g/l was achieved with the lowest concentration of

inoculum of 5% (v/v) and the youngest culture 48hours old (Fig. 2). At this concentration of inocu-lum, the biomass production decreased with in-creasing age of the culture. The effect was the

same for the lipase production. The highest lipo-lytic activity of 0.36 V/ml was achieved when theyoungest culture was used. It seems that the bio-

synthetic activity declines as the age of the cultureincreases. Increase of inoculum concentration

showed similar effect as increase of the age. Boththe growth and the biomass production decreasedwith increasing inoculum concentration.

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Fig. 2. Effect of concentration and age of inoculum on lipase (0) and biomass (.) production.The inoculum was added in concentrations (ml/100 ml medium): 5, 10 and 15, with age of: A) 48 h , B) 72 hand C) 96 h

fJIac. xeM. TeXHOJI. MaKelloHllja, 20, 1,39-43 (2001)

A B C

42 I. Mladenoska. A. Dimitrovski

Effect of initial pH

To observe the effect of initial pH on the li-pase production by G. candidum-M2, pH of themedium was varied from 4 to 11. Other parameterswere unaltered.

The highest lipolytic activity and growth ofthe culture were obtained at the pH values out ofthe neutral area. The maximum lipolytic activity of0.40 Ulml was achieved at the initial pH value of9.0 (Fig. 3). The next highest value, 0.36 V/ml wasobtained at pH 4.0. As it can be seen from Fig. 3,the media with initial pH 7.0 and 8.0 are inconven-ient both for growth and lipase production. Similarresults were reported for solid state fermentation ofthis strain on rice bran [11].

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Another interesting feature of G. candidum-M2 is that the physiological and biosyntheticchanges, influenced by pH, had the reflection onthe culture's morphology. Namely, in the fermen-tation broth of G. candidum-M2 cultivated at pH7.0 and 8.0, spherical formations with diameter of3 mm were found. In the microscopic photographyof the fungus cultivated at pH 8.0 appearance ofchlamidospores, the form of defending spores, canbe seen. On the other hand, the fungus cultivated atpH 9.0 distinguishes itself with budding blasto-spores. It is obvious that, under influence of pH,two different forms of the fungus appeared. Theeffect of environmental conditions on the mor-phology and physiology is also confirmed in otheryeast-like fungi [12, 13].

Some preliminary experiments that we haveperformed, showed that the lipase produced byGeotrichum candidum-M2 posses two very impor-tant predispositions for detergent application, suchas stability at high temperatures about 50° C and atalkaline pH range about 9. Since these experimentsare still not completed, we suggest that much fu-ture work can be done in that direction.

Effect of agitation rate

In this experiment mixing rates of 100, 150and 200 rpm were used. The initial pH of mediumwas adjusted to 9.0. The highest lipolytic activityof 0.45 Ulml was obtained at the lowest mixingrate of 100 rpm (Fig. 4). The best result for growthof the fungus of 13 g/l was achieved for the lowestagitation rate.

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Fig. 4. Effect of the agitation rate on growth (.)and lipase (D) production

This is not the first report which gave evi-dence of the negative effect of the higher mixingrates on the lipase production by Geotrichum can-didum. Alford and Smith [14] reported that the li-pase yields reduced for 60 % as a result of the mix-ing at low rates and even more at the higher ones.Wouters [15], similarly to the previous case, re-pOtted that the growth and the lipase production byGeotrichum candidum decreased as the aeration oragitation rate of the culture medium increased. Ar-ends et al. [16] reported the mechanical disruptionof the micel1ium as a reason for the decreasedli-

pase production by Aspergillus awamory, when itwas cultivated at increased agitation rates.

CONCLUSIONS

Selection of medium composition and deter-mination of some important environmental para-meters on lipase production by Geotrichum can-

didum-M2, were performed. This locally isolatedstrain utilizes sunflower oil sediment as a sole car-bon source and as an inducer for lipase production

Bull. Chem. Technol. Macedonia. 20, 1,39-43 (2001)

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Lipase production by Geotric/zul1l candidul1l-M2 43

at the same time. The yeast extract has the intrinsiceffect on the biosynthetic activity of this fungus.

The lowest concentration of 5 % v/v inocu-lum from the culture with inoculum age of 48 hshould be used for lipase production by the fungus.

This microorganism showed the highest lipase ac-tivity at initial pH 9.0 and under the lowest agita-tion rate of 100 rpm.

Aknowledgement:We aknowledgethe financialsupport of the Macedonian Ministry of Science.

REFERENCES

[1]A. M. McKay. Left. Appl. Microbiol., 16, 1 (1993).

[2] R. T. Nielsen, Austral Patent 29764/84.

[3] J. A. Arcos, M. Bernabe and C. Otero, Biotechnol. Bio-eng., 57, 505 (1998).

[4] A. Ducret, A. Giroux, M. Trani and R. Lortie. Biotechnol.Bioeng., 48,214 (1995).

[5] G. M. Frost & D. A. Moss. In Biotechnology, ed. H. J.Rehm & G. Reed. VCH Publisher, Weinheim, Germany,1987, pp. 113-12l.

[6] A. R. Macrae and R. C. Hammond. Biotechnol. Gen.Engin. Rev., 3,193 (1985).

[7]D. Y. Kwon and J. S. Rhee. JADCS, 63 (1), 89 (1986).

[8] M. W. Baillargeon, R. G. Bistline and P. E. Sonnet. Appl.Microbiol. Biotechnol., 30, 92 (1989).

[9] P. Christacopoulos, C. Tzia, D. Kekos and B. J. Macris.,Appl. Microbiol. Biotechnol., 38, 194 (1992).

[10] S. S. Shchelokova, M. J. Tabak and M. Z. Zakirov, Prik-lad. Biochem. Microbiol., 14 (4), 494 (1976).

[11] M. Dimitrovska and A. Dimitrovski. Poster presented atthe 8thEuropean Congress on Biotechnology, Budapest,Hungary, 17-21 August 1997.

[12] 1. PIa, C. Gil, F. Monteoliva, F. Navarro-Gracia, M.Sances and C. Nombela. Yeast, 12, 1677 (1996).

[13] 1. S. Zvjaginceva and E. L. Ruban. Microbiol., 42 (4),743 (1978).

[14] J. A. Alfrod, J. L. Smith. JADCS, 42, 1038 (1965).

[15] J. T. M. Wouters. In: Biotechnology, ed. H. J. Rehm andG. Reed, VCH Publisher, Weinheim, Germany, 1987, pp.113-12l.

[16] 1. M. Arends, V. V. Dorokhov, T. M. Turochkina and T.G. Borisova. Priklad. Biochem. Microbiol., 16 (5), 691(1975).

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