Bulletin of the Chemists and Technologists of Macedonia, Vo!. 20, No. 1, pp.

39-43 (2001)
GHTMDD - 373 ISSN 0350 - 0136
Received: July 3, 2000 DDC: 663.152.311 : 582.28
Accepted: April 2, 2001
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 production
by the yeast-like fungus Geotrichum candidum-M2 has been performed. The sunt10wer oil sediment was tested as
carbon source and compared to several other oils and carbohydrates. It was proven as the best carbon source, whereas
the yeast extract was the best nitrogen source. The culture medium selected as optimal one contained each of them in
concentration 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 100
rpm. 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; sunt10wer oil sediment;
environmental parameters

INTRODUCTION

Upases (EC 3.1.1.3) or triacyl glycerol hy- specific compounds, such as tailor made lipids and
drolases are widely distributed in nature. Lipase bioemulsifiers [3,4].
biosynthesis occurs in animals, plants and micro- Moulds are the most exploited among the mi-
organisms. Microbiallipases are receiving particu- croorganisms as lipase producers. Aspergillus ni-
lar attention because of their actual and potential ger and Rhisopus arrhisus are producers of some
industrial application. They are widely used in
of the best sold commercial lipase preparations [5].
food and pharmaceutical industry as well as in oil Maybe the most specific one is the fungus Geo-
and fat industry. Cheese ripening, preparation of
trichum candidum, which reacts only with fatty
cocoa butter substitutes and flavor production are acids with a eis-double bond in the 9-position [6].
some of their applications in food industry [1].
They can hydrolise oils and fats even at very low In this paper, the optimal composition of
temperatures, which makes them particularly suit- growth medium for lipase production by locally
able for application in washing detergent [2]. Li- isolated strain of Geotrichum candidum was de-
pase ability of catalyzing reactions other than hy- termined. The effect of different environmental pa-
drolysis, such as esterification and interesterifica- rameters on lipase synthesis was also investigated.
tion, can be exploited in the production of some

EXPERIMENTAL

Microorganism the biochemical tests were supplied from Biome-

rieux - France. The microorganism was deposited
The microorganism was isolated in our labo- at a Faculty culture collection under the name Geo-
ratory from spoiled milk. It was identified as Geo- trichum candidum-M2. The culture was maintained
trichum candidum penicillatum. The identification at 4 °C on malt extract agar slants.
was performed on miniAPI apparatus. Strips for
40 I. Mladenoska, A. Di11Iitrovski

Culture medium unadjusted. When the effect of initial pH was in-

vestigated, inoculum in concentration of 10 % v/v
The mineral medium had the following compo- was used, obtained from the culture old 72 h. Ini-
sition (g/l): KH2P04 2, MgS04'7H2O 1, CaCh'H2O 1 tial pH was adjusted with HCI and NaOH solu-
and NaCI 1. It was supplemented with carbon and tions. In this experiment pH interval between 4 and
nitrogen sources. Altering the type of carbon and 10 was used.
nitrogen source one at a time, a selection of the
In the experiment for selection of the optimal
best medium for lipase production was carried out.
agitation rate, agitation of 100, 150 and 200 rpm
Several carbohydrates and lipids were examined as
was used. All other parameters were the optimal
carbon sources. One of the lipids was the sun-
ones selected from the previous experiments.
flower oil sediment, a complex substrat with the
following composition (%): oil 63, water 3.9, ni-
trogen 2.59 and suspended solids, 30.5. The me- Separation of lipase
dium was sterilized at 121 QCfor 35 minutes.
At the end of the cultivation, the culture broth
was filtered and centrifuged at 4000 rpm for 50
Growth conditions
minutes. The supernatant was used as the crude
enzyme for the estimation of lipase activity.
A lO-ml portion of seed culture (3.5 x 106
spores/m!) was inoculated into 500-ml Erlenmeyer
flasks containing 100 ml of the culture medium. Analytical methods
When medium composition was investigated, cul- Lipase activity was measured by the method
tivation lasted 72 hours at 30 QC,and was carried
of Kwon and Rhee [7]. One unit (1U) of lipolytic
out on a rotary shaker at 170 rpm. When duration of activity is the amount of enzyme, which liberates
the process was an experimental variable, period of
1 /lmol free fatty acids per minute under the assay
incubation was prolonged to 96 h. Selected process conditions. Oil content in the sunflower oil sedi-
durationof 48 h was used in subsequentexperiments.
ment was determined gravimetrically after three-
When the effects of the concentration and in- fold extraction by petrolether. Nitrogen content in
oculum age on lipase and biomass production were the sediment was determined by the Kjeldahl
investigated, inoculum inconcentration 5, 10 and method. Water content was determined gravimetri-
15 % v/v of the culture old 48, 72 and 96 h, was cally by drying at 105 QC.
used. In this experiment pH of the medium was

RESULTS AND DISCUSSION

Effect of carbon source Table 1

Effect of carbon and nitrogen source on growth and
Several carbohydrates and lipids were tested lipase production by Geotrichum candidum-M2
for their effect on lipase production by Geotrichum
Source Biomas (g/l) Lipase activity (U/ml)
candidum-M2. Yeast extract of 1 % (w/v) was Carbon
used as a nitrogen source. The highest lipolytic Carbohydrates (1 %w/v)
activity of 0.28 U/ml was obtained by the utiliza- Glucose 7.57 0.04
tion of sunflower oil sediment (Table 1). Biomass Lactose 2.73 0.02
Fructose 6.57 0.01
production in this case was 9.67 g/!. Maximum Galactose 5.76 0.00
growth of 11.70 g/l was achieved in the medium Soluble starch 1.92 0.01
with olive oil as a sole carbon source. This lipid Lipids
substrate was well used for growth but much less Sunflower oil 2.93 0.01
for the lipase production, the activity of which was Olive oil 11.7 0.02
Sunflower oil sediment 9.67 0.28
0.02 Ulm!. Regarding the carbohydrates, they did
Nitrogen (I % w/v)
not stimulate the biosynthetic activity of the fun- Urea 3.91 0.01
gus. The conclusion that lipids were better carbon Yeast extract 6.51 0.28
sources for lipase production than carbohydrates, Soybean flour 8.96 0.04
confirmed the results reported by many authors [8, NH4N03 2.43 0.04
9]. (NH4)2HP04 5.36 0.07
(NH4)2S04 7.40 0.02

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

 Lipase production by Geotrichum candidum-M2 41

Effect of nitrogen source peared after 48th hour. This may be due to a re-
lease of the membrane-bounded lipase as a result
A number of organic and inorganic nitrogen of cell autolysis. Process duration of 48 h was used
sources were tested in their effect on lipase produc- in subsequent experiments.
tion. In this experiment sunflower oil sediment was
used as a sole carbon source, in concentration of 0.6
25 :::::J
1% (v/v). The highest lipase activity of 0.28 V/ml 0.5 E
was obtained in a medium with yeast extract (Ta- ~ 20 :3
-9 0.4 ~

~
ble 1). Its intrinsic effect on growth and biosyn- <J)
<J)
15 0.3~
ro ()

thetic activity of the fungus can be result of the E 10 -" 0.2 ~

0
different amino acids and growth factors present in cc 5"-
~
0.18-
large quantity in the yeast extract [l0]. The best
0 0 ::;
growth of the fungus was obtained with the soy- 0 12 24 48 96
bean flower as a nitrogen source, but the lipase Time (h)
production was only 0.03 U/mI. The utilization of
Fig. 1. Time course of lipase (0) and biomass ( . )
inorganic nitrogen sources did not stimulate lipase production on selected culture medium containing sunflower
production by G. candidum-M2. Medium with oil sediment (1 %v/v) and yeast extract (1 %w/v)
sunflower oil sediment as carbon source and yeast
extract as nitrogen source was used in all subse-
Effect of inoculum age and its concentration
quent experiments.

In order to see the effect of thesetwo parame-

Selection of the process duration ters on growth and lipolytic activity of the fungus,
inoculum obtained from culture old 48, 72 and 96
In order to determine the effect of dynamics hours, was used in concentration of 5, 10 and 15 %
of the lipase production, the fungus was cultivated v/v.
96 hours, and samples were taken every 24 hours. The highest biomass concentration of 13.20
In Figure 1, the lipolytic activity and the biomass g/l was achieved with the lowest concentration of
concentration versus incubation period are plotted. inoculum of 5% (v/v) and the youngest culture 48
One can see that the enzyme production was not hours old (Fig. 2). At this concentration of inocu-
followed by the growth. The maximum growth of lum, the biomass production decreased with in-
12.30 g/l was obtained after 48 hours of fermenta- creasing age of the culture. The effect was the
tion, when the curve of lipase productionpasses same for the lipase production. The highest lipo-
through minimum. Although the highest lipase ac- lytic activity of 0.36 V/ml was achieved when the
tivity of 0.43 V/ml was obtained after 24 h of fer- youngest culture was used. It seems that the bio-
mentation, the 48-hour process was chosen as the synthetic activity declines as the age of the culture
optimal one because of easier downstream process- increases. Increase of inoculum concentration
ing. Namely, by th<).ttime most of the lipid materi- showed similar effect as increase of the age. Both
als disturbing the isolation of the enzyme, were the growth and the biomass production decreased
spent. Lipase activity achieved for this period was with increasing inoculum concentration.
0.25 U/mI.The slight rise of lipolytic activity ap-
14 0.4
A B C
12 0.35

~
10 0.3 ?
::;
~8
'"
0.25 z:
'S;
'"
E
0 6
0.2 g
0
iD 0.15~
4 g,
0.1 :::;
2 0.05

0 0
5 10 15 5 10 15 5 10 15
Concentration of inocuium (%v/v)

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)

42 I. Mladenoska. A. Dimitrovski

Effect of initial pH Some preliminary experiments that we have

performed, showed that the lipase produced by
To observe the effect of initial pH on the li- Geotrichum candidum-M2 posses two very impor-
pase production by G. candidum-M2, pH of the tant predispositions for detergent application, such
medium was varied from 4 to 11. Other parameters as stability at high temperatures about 50° C and at
were unaltered. alkaline pH range about 9. Since these experiments
The highest lipolytic activity and growth of are still not completed, we suggest that much fu-
the culture were obtained at the pH values out of ture work can be done in that direction.
the neutral area. The maximum lipolytic activity of
0.40 Ulml was achieved at the initial pH value of Effect of agitation rate
9.0 (Fig. 3). The next highest value, 0.36 V/ml was
obtained at pH 4.0. As it can be seen from Fig. 3, In this experiment mixing rates of 100, 150
the media with initial pH 7.0 and 8.0 are inconven- and 200 rpm were used. The initial pH of medium
ient both for growth and lipase production. Similar was adjusted to 9.0. The highest lipolytic activity
results were reported for solid state fermentation of of 0.45 Ulml was obtained at the lowest mixing
this strain on rice bran [11]. rate of 100 rpm (Fig. 4). The best result for growth
of the fungus of 13 g/l was achieved for the lowest
12 -. 0.4 agitation rate.
10 0.35 :g
~

...J 0.3 :3 14. 0.6

:§ 8~- - 0.25 :;; 12 0.5 ::J
<J)

~ 6 -.- -~ 0.2 ~
(j 10.-
E
E 0.15 ro ::J 0.4 2-
.Q 4 (j
:§ 8.- ~
ca 0.1 ~
2
<J)
Cl)
0.3 :~
t)
- 0.05 8. ro 6 ro
E 0.2 g>.
0 0 :.:::i 0
jjj 4
4 5 6 7 8 9 10
2 .~ 0.1 ~
...J
Initial pH
0 0
Fig. 3. Lipase (D) and biomass (.) production under 100 150 250
influence of initial pH
Agitationrate (rpm)
Fig. 4. Effect of the agitation rate on growth (.)
Another interesting feature of G. candidum- and lipase (D) production
M2 is that the physiological and biosynthetic
changes, influenced by pH, had the reflection on
This is not the first report which gave evi-
the culture's morphology. Namely, in the fermen-
dence of the negative effect of the higher mixing
tation broth of G. candidum-M2 cultivated at pH
rates on the lipase production by Geotrichum can-
7.0 and 8.0, spherical formations with diameter of
didum. Alford and Smith [14] reported that the li-
3 mm were found. In the microscopic photography
pase yields reduced for 60 % as a result of the mix-
of the fungus cultivated at pH 8.0 appearance of
ing at low rates and even more at the higher ones.
chlamidospores, the form of defending spores, can
Wouters [15], similarly to the previous case, re-
be seen. On the other hand, the fungus cultivated at
pOtted that the growth and the lipase production by
pH 9.0 distinguishes itself with budding blasto- Geotrichum candidum decreased as the aeration or
spores. It is obvious that, under influence of pH,
agitation rate of the culture medium increased. Ar-
two different forms of the fungus appeared. The
effect of environmental conditions on the mor- ends et al. [16] reported the mechanical disruption
of the micel1ium as a reason for the decreasedli-
phology and physiology is also confirmed in other
pase production by Aspergillus awamory, when it
yeast-like fungi [12, 13].
was cultivated at increased agitation rates.

CONCLUSIONS

Selection of medium composition and deter- didum-M2, were performed. This locally isolated
mination of some important environmental para- strain utilizes sunflower oil sediment as a sole car-
meters on lipase production by Geotrichum can- bon source and as an inducer for lipase production

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

 Lipase production by Geotric/zul1l candidul1l-M2 43

at the same time. The yeast extract has the intrinsic This microorganism showed the highest lipase ac-
effect on the biosynthetic activity of this fungus. tivity at initial pH 9.0 and under the lowest agita-
The lowest concentration of 5 % v/v inocu- tion rate of 100 rpm.
lum from the culture with inoculum age of 48 h Aknowledgement: We aknowledgethe financial
should be used for lipase production by the fungus. support of the Macedonian Ministry of Science.

REFERENCES

[1] A. M. McKay. Left. Appl. Microbiol., 16, 1 (1993). [10] S. S. Shchelokova, M. J. Tabak and M. Z. Zakirov, Prik-
[2] R. T. Nielsen, Austral Patent 29764/84. lad. Biochem. Microbiol., 14 (4), 494 (1976).
[3] J. A. Arcos, M. Bernabe and C. Otero, Biotechnol. Bio- [11] M. Dimitrovska and A. Dimitrovski. Poster presented at
eng., 57, 505 (1998). the 8th European Congress on Biotechnology, Budapest,
Hungary, 17-21 August 1997.
[4] A. Ducret, A. Giroux, M. Trani and R. Lortie. Biotechnol.
Bioeng., 48,214 (1995). [12] 1. PIa, C. Gil, F. Monteoliva, F. Navarro-Gracia, M.
Sances and C. Nombela. Yeast, 12, 1677 (1996).
[5] G. M. Frost & D. A. Moss. In Biotechnology, ed. H. J.
Rehm & G. Reed. VCH Publisher, Weinheim, Germany, [13] 1. S. Zvjaginceva and E. L. Ruban. Microbiol., 42 (4),
1987, pp. 113-12l. 743 (1978).
[6] A. R. Macrae and R. C. Hammond. Biotechnol. Gen. [14] J. A. Alfrod, J. L. Smith. JADCS, 42, 1038 (1965).
Engin. Rev., 3,193 (1985). [15] J. T. M. Wouters. In: Biotechnology, ed. H. J. Rehm and
[7] D. Y. Kwon and J. S. Rhee. JADCS, 63 (1), 89 (1986). G. Reed, VCH Publisher, Weinheim, Germany, 1987, pp.
113-12l.
[8] M. W. Baillargeon, R. G. Bistline and P. E. Sonnet. Appl.
Microbiol. Biotechnol., 30, 92 (1989). [16] 1. M. Arends, V. V. Dorokhov, T. M. Turochkina and T.
G. Borisova. Priklad. Biochem. Microbiol., 16 (5), 691
[9] P. Christacopoulos, C. Tzia, D. Kekos and B. J. Macris., (1975).
Appl. Microbiol. Biotechnol., 38, 194 (1992).

Pe3IIMe

npOII3BOLJ;CTBO HA JIIInA31I CO GEOTRICHUM CANDIDUM-M2

IIpHlla MJIa/l,eHOCKa*, Al\o )J;HMHTpOBCKH

TeXHO/lOUlKO-Mez7la/lYPUlKU cpaKY/lLTieiu, YHu6ep3uz7lez7l "C6.KupWl U Mez7loouj",

Pyiep EOUlK06UK16, MK-1 000 CKoiije, Peuy6/lUKa MaKeOOftuja
*E-mail: irinaetf@mt.net.mk

KJIY'IUH 36opOBH: Geotrichum candidum; npO/l,YKlJ,Hja Ha mllIa3H; cy6Mep3Ha <pcpMCHTaIJ,uja;

TaJIOr 0/1, cOH'WrJIC/I,oBo MaCJIO; Ha/l,BOpCrnHU napaMCTpu Ha KYJITUBUpau,c

I1CITUTYBaHU cc BJIujaHHjaTa Ha cocTaBOT Ha xpaH- /l,O/l,CKa KBaCO'IHIIOT cKcTpaKT 6cmc Haj/l,06ap U3BOp lIa
JIUBaTa nO/l,JIora U YCJIOBUTC Ha KYJITUBHpau,c BP3 npo- a30T. OnTHMu3HpaHuoT MC/I,HYM ru CO/l,p)j(u TaJIOrOT 0/1,
/l,YKlJ,ujaTa Ha JIUna3U Kaj KBaCHaTa ra6a Geotrichum can- COlI'IOrJIc/I,oBo MaCJIO U KBaCO'lHHOT cKcTpaKT BO KOHlJ,CH-
didum-M2. TaJIOrOT 0/1, COWIOrJIC/I,OBO MaCJIO 6crnc ucnu- TpalJ,HU 0/1, 1 %. MaKcHMaJIHa JIHnOJIHTll'IKa aKTHBHOCT 0/1,
TYBall BO O/l,HOC Ha HcrOBaTa norO/l,HOCT /l,a CJIY)j(U KaKO 0.45 V/ml C /l,06UCHa npu CJIe/l,HHBC YCJIOBH: BpCMCTpaCu,c
II3BOp Ha jarJICpo/l" a HanpaBCHa e U HcrOBa cnopc/I,6a co Ha npOlJ,CCOT 48 h, KOHI~CHTpaI~uja Ha UHOKYJIYMOT 5%
HeKOJIKY jarJICXII/I,paTHU U JIUnU/I,HU 1I3BOpU Ha jarJICpo/l,. v/v, CTapoCT Ha KYJITypaTa 48 h, nOT.JCTHOpH 9.0 H 6p3HHa
TaJIoroT cc nOKa)j(a KaKO Haj/l,06ap U3BOp Ha jarJICpo/l" lIa MCrnau,c 100 vrt/min.

fJIac. XCM.TCXHOJI.MaKc).\oHIIja, 20,1,39-43 (2001)