Influence of detergent on metabolic activity of fungi Aspergillus niger

doi:10.4236/ns.2011.36064

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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: [email protected]

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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