Vol.3, No.6, 466-470 (2011) Natural Science
http://dx.doi.org/10.4236/ns.2011.36064
Influence of detergent on metabolic activity of fungi
Aspergillus niger
Jelica Stojanović1, Violeta Jakovljević1, Ivana Matović1, Olgica Gajović2, Zoran Mijušković3,
Tomislav Nedeljković2
1Faculty of Science, University of Kragujevac, Kragujevac, Serbia; *corresponding author:
2Medical Faculty, University of Kragujevac, Kragujevac, Serbia
3Military Medical Academy, Belgrade, Serbia
Received 1 April 2011; revised 28 April 2011; accepted 10 May 2011.
ABSTRACT
The aim of this study was to find out, among
grate variety of fungi species from wastewater
these which are resistant to effects of detergent
and its component, ethoxyl-oleyl-cetyl alcohol
and sodium tripolyphosphate. On inoculated
fungi specie grown in vitro condition, in the
presence of mentioned pollutant, the metabolic
changes of bioproduction of different organic
compounds, in various aging step of fungi, have
been investigated. The results indicated sig-
nificant changes in bioproduction of amino ac-
ids and proteins of Aspergillus niger cultivated
in the presence of detergent and its component,
compared with control experiment. The results
suggest that bioremediation by Aspergillus ni-
ger are promising for biodegradation of deter-
gents in aquatic systems.
Keywords: Aspergillus Niger; Detergent;
Bioproduction; Wastewater; Biodegradation
1. INTRODUCTION
Detergents are formulations designed to have clean-
ing/solubilisation properties. These formulations consist
of surface-active agents (surfactants) together with sub-
sidiary components including builders (e.g. tripolyphos-
phate), boosters, filters and auxiliary compounds. Sur-
factants are a group of organic compounds achieved by
chemical synthesis and characterized for specific behav-
ior in solution that makes them especially suitable for
many human activities. Surfactant is an abbreviation for
a surface-active agent that refers to its ability to reduce
the interfacial tension between two phases. This behav-
ior is caused by the molecular composition in the sur-
factant, which has a hydrophobic part, composed of al-
kyl chains, and another part that is an anionic or hydro-
philic group [1].
A massive stream of surfactants is directed to the aq-
uatic environment. Surfactants are probably the largest
supplier of artificial organic carbon to the aquatic envi-
ronment [2]. Even though surfactants are essentially
non-toxic to man at the concentrations likely to be met in
wastewaters, there is wide agreement that their presence
both in natural freshwater sources and in other ecosys-
tems is undesirable. The principal criterion for the eco-
logical behavior of surfactant is their biodegradability
[3].
Many researchers fortified that microorganisms, par-
ticularly some kind of fungi, can act as potential degrad-
ers of detergents [4,5]. Among the fungi which have
such ability, filamentous fungi (Deuteromicotina) are
especially distinguished due to their physiological and
biochemical characteristics [6]. Specificity in apical
growth of these fungi enables penetration in solid sub-
strates and excretion of extracellular enzymes from vesi-
cles on the top of hyphae to environment. Under the in-
fluence of these enzymes complex organic compounds
are decomposed to simpler which fungi can use for
growth and development of mycelia and biomass accu-
mulation [7-9].
The Aspergillus fungus was first recognized as an or-
ganism in 1729 by Micheli [10]. The genus Aspergillus
is found worldwide and consists of more than 180 offi-
cially recognized species, and comprises a particularly
important group of filamentous ascomycete species [11].
Although it includes the major filamentous fungal pa-
thogen of humans, Aspergillus fumigatus [12], most of
the members are useful microorganisms in nature for
degradation of plant polysaccharides [13], and they are
important industrial microorganisms for the large-scale
production of both homologous and heterologous en-
zymes [14-18]. Among them, Aspergillus oryzae and
Aspergillus niger are on the Generally Recognized as
Safe list of Food and Drug Administration in the United

J. Stojanović et al. / Natural Science 3 (2011) 466-470 467
States [19]. Aspergillus niger is one of the most impor-
tant microorganisms used in biotechnology [20,21]
which produces many extracellular enzymes.
Detergents have severe effects on wildlife and human
health due to their toxicological properties [22]. Linear
alkylbenzene sulphonate (LAS) is anionic surfactant
most widely used as a major ingredient in domestic and
industrial detergents. It can easily be degraded by mi-
croorganisms in wastewater treatment plants using aero-
bic processes [23,24], however the intermediates are less
biodegradable [25]. Such metabolites may be toxic to
higher biotrophic members of the ecosystem, but they
also offer a potential carbon and energy source for mi-
croorganisms capable of breaking them down further.
LAS degradation may be performed by specific micro-
organisms that use it as a sole carbon source [26] or as a
co-metabolic transformation [27]. Balson and Felix [28]
described biodegradation as the destruction of a chemi-
cal by the metabolic activity of microorganisms. When
reviewing the literature concerning the degradation of
surfactants it is apparent that studies quote figures for
primary and/or ultimate biodegradation [29]. Primary
degradation can be defined as to have occurred when the
structure has change sufficiently for a molecule to lose
its surfactant properties. Ultimate degradation is said to
have occurred when a surfactant molecule has been ren-
dered to CO2, CH4, water, mineral salts and biomass.
Primary degradation of LAS on activated sludge is grater
then 99% [30-32]. Traces of LAS in natural waters and
soil continuo rapid degradation (half-live of LAS is
about 0.15-0.5 days), but total biodegradation still re-
quires several days [33-37].
Surfactants and water miscible organic solvents are
frequently used to increase the bioavailability of envi-
ronmental contaminants for degradation [38]. LAS is
used as a mediator in polyaromatic hydrocarbons degra-
dation catalyzed by the extracellular enzyme system of
some fungi [39]. Also, it was found that addition of de-
tergents have enhancing effects on extracellular produc-
tion of some metabolites with microorganisms [40] and
may be useful method for over-production of hydropho-
bic compounds by means of biological process.
Herein we describe our studies on influence of deter-
gent on metabolic activity of fungi Aspergillus niger.
2. MATERIAL AND METHODES
The experiments were performed using monosporial
culture of the fungi Aspergillus niger van Tiegheme iso-
lated from the river Lepenica (Serbia) on wastewater
outpouring site. Identification of the culture was done on
Faculty of biology, Belgrade, in Laboratory for algae,
fungi and lichens. Monosporial culture of the fungi was
obtained by the method of exhaustion on a poor po-
tato-dextrose agar [41].
The detergent powder used was packaged household
synthetic detergent of domestic Merix brand (Merima,
Kruševac). Ethoxyl-oleyl-cetyl alcohol and sodium tri-
polyphosphate are also supplied by Merima.
The method can be summarized as follows: fungi were
inoculated into a flask that contained a chemically-de-
fined microbial growth medium and the surfactant to be
tested. The fungi were grown in the sterile liquid nutrient
medium according to Czapek consisted of: 3 g NaNO3, 1
g K2HPO4, 1 g MgSO4, 0.25 g MgSO4x7H2O, 0.01 g
FeSO4x7H2O and 30 g saccharose, dissolved in 1000 ml
of distilled water. Detergent designated D, ethoxyl-oleyl-
cetyl alcohol (AOC) and sodium tripolyphosphate (TPP)
was added (1%) and the flasks incubated for 4-8 days.
The flasks containing 200 cm3 of medium were uni-
formly and constantly shaken on Kinetor shaker at room
temperature in condition of alternate light-dark cycles
[42]. The sterility of the nutrient medium was tested us-
ing mesopeptone agar.
For the determination of free organic acids 10 ml of
medium was taken and mixed with 50 ml of ethanol.
After incubation at 70˚C in a water bath for 1-1.5 hours,
the mixture was filtered through a special filter. The fil-
trate then was concentrate at 50˚C - 60˚C and reduced
pressure to the volume of 40 ml, transferred to a volu-
metric flask and made up to 100 ml after addition of a
teaspoon of the active charcoal. After standing in a water
bath for 30-45 min at 70˚C, 10 ml aliquots of filtrate
were taken for the determination of the free organic ac-
ids by titration with 0.1 M NaOH in the presence of
phenolphthalein as indicator [43-45].
The monosaccharides, glucose and fructose, were de-
termined after cation-exchange chromatography on a
column of AmberliteIR-120 followed by descending
chromatography on Whatman No 1 paper. After reaction
with suitable reagent to produce a green-blue complex,
the amount of glucose and fructose were determined
spectrofotometrically using a red filter in comparison
with the appropriate standard curves [43].
The qualitative and quantitative content of amino
acids was determined by standard method described by
Sparkman [46] using aminoanalyzer BECKMAN mod-
el 120 C. Measuring conditions: stationary phase—
LiChroCART 250-4, mobile phase—0.10 M acetic
buffer (pH 4.4) and acetonitrile in 70:30 ratio, mobile
phase rate 1.0 ml/min, fluorescent detector (Ex 263 nm,
Em 313 nm).
The amount of proteins was determined by Kjeldahl
method on the basis of amount of nitrogen present in
fungi [47] using equation:
Amount of proteins = 6.25 x amount of nitrogen (mg)

J. Stojanović et al. / Natural Science 3 (2011) 466-470 468
3. RESULTS AND DISCUSION
This paper conducted a research on influence of de-
tergent Merix (Merima, Kruševac, Serbia) and its com-
ponents (ethoxyl-oleyl-cetyl alcohol and sodium tri-
polyphosphate) on metabolic activity of fungi Asp ergil-
lus niger. The metabolic changes of bioproduction of
different organic compounds in various aging step of
fungi in the presence of pollutant has been investigated.
This fungus was isolated from the river Lepenica (Serbia)
on wastewater outpouring site and was chosen because it
was the most abundant there. The fungi was then grown
in the liquid nutrient medium according to Czapek,
where 1% of detergent or its component was added,
during the incubation period of 4-8 days after inocula-
tion. Metabolic activity of Aspergillus niger grown in
such liquid nutrient medium and control medium is mo-
nitored over following biochemical parameters: amounts
of free organic acids and monosaccharides on 4th and
8th day of incubation period, amount of proteins on each
day of incubation period and qualitative and quantitative
content of amino acids on the last day of incubation pe-
riod.
Aspergillus niger did not produce carbohydrates on
the 4th day of incubation period. At the end of examina-
tion period, fungi produced different amount of carbo-
hydrates (glucose and fructose) depending on the com-
position of nutrient medium. Compared with the control
medium, production of fructose was increased in all ver-
sion of nutrient medium, except in the case when 1% of
detergent was added (Figure 1).
Production of free organic acids was increased in all
cases at the end of experimental period, but it was dra-
matically increased in nutrient medium with sodium
tripolyphosphate added (Figure 2).
On the last day of experimental trial, in the control
nutrient medium Aspergillus niger produced 15 essential
amino acids while in the nutrient medium with 1% of
detergent added 14 essential amino acid were produced.
The detergent manifested extremely stimulating effect
on bioproduction of lysine, arginine, aspartic and glu-
Figure 1. Amount of carbohydrates (%) produced by A.
niger on 8th day in different liquid nutrient mediums.
Figure 2. Amount of free organic acids (%) produced by
A. niger on the 4th and 8th day in different liquid nutrient
mediums.
tamic acid, valine, leucine, isoleucine and tyrosine. Bio-
production of other amino acids was less intensity, but
still increased in comparison with those produced in
control medium. Only alanine was not produced in the
presence of detergent (Figure 3).
Amount of proteins produced by Aspergillus niger in
different liquid nutrient mediums was monitored on each
day of 4-8 days incubating period and had differed de-
pending on the liquid nutrient medium used and the ag-
ing step of fungi (Figure 4).
With the aging of fungal culture production of pro-
Figure 3. Qualitative and quantitative content of amino
acids produced by A. niger on the 8th day in liquid nu-
trient medium with 1% detergent added.
Figure 4. Amount of proteins (g) produced by A. niger during
4-8. day of incubating period in different liquid nutrient me-
diums.

J. Stojanović et al. / Natural Science 3 (2011) 466-470 469
teins decreased in all cases, except in the medium with 1%
of detergent added in which production was a little bit
higher compared with control experiment. The highest
production of proteins was in the medium with 1% eth-
oxyl-oleyl-cetyl alcohol added on 7th day, while the low-
est was in the medium with 1% sodium tripoly- phosphate
added on 4th day of incubation. Sodium tripolyphosphate
exhibited the highest inhibitory effect on bioproduction of
proteins during the entire incubation period.
4. CONCLUSIONS
On the basis of the results obtained it can be con-
cluded that detergent and its components added to liquid
nutrient medium according to Czapek changed metabolic
activity of fungi Aspergillus niger on a different ways,
what reflected on growth and development of fungi.
The results of amino acid analyses indicated that As-
pergillus niger is resistant to effect of detergent in con-
centration applied. It produced a variety of amino acids,
14 overall, and all of them were 2-10 times more abun-
dant then in control experiment.
Bioproduction of proteins, at the end of examination
period, was stimulated a little by detergent and ex-
tremely inhibited by sodium tripolyphosphate and par-
tially by ethoxyl-oleyl-cetyl alcohol.
Detergent also exhibited high inhibitory effect on bio-
production of free organic acids while sodium tripoly-
phosphate and ethoxyl-oleyl-cetyl alcohol exhibited
considerably stimulating effect.
Detergent powder and its components highly influ-
ence metabolic activity of Aspergillus niger. Thus this
fungus, which is most abundant on wastewater outpour-
ing site of river Lepenica, can metabolize detergent
components for growth and biomass accumulation and
play an important role in purification of river waters.
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