Environ Sci Pollut Res (2013) 20:29122923

DOI 10.1007/s11356-012-1193-5

RESEARCH ARTICLE

Optimization of decolorization of palm oil mill effluent

(POME) by growing cultures of Aspergillus fumigatus
using response surface methodology
Chin Hong Neoh & Adibah Yahya & Robiah Adnan &
Zaiton Abdul Majid & Zaharah Ibrahim
Received: 18 July 2012 / Accepted: 11 September 2012 / Published online: 29 September 2012
# Springer-Verlag Berlin Heidelberg 2012

Abstract The conventional treatment process of palm oil

mill effluent (POME) produces a highly colored effluent.
Colored compounds in POME cause reduction in photosynthetic activities, produce carcinogenic by-products in drinking water, chelate with metal ions, and are toxic to aquatic
biota. Thus, failure of conventional treatment methods to
decolorize POME has become an important problem to be
addressed as color has emerged as a critical water quality
parameter for many countries such as Malaysia. Aspergillus
fumigatus isolated from POME sludge was successfully
grown in POME supplemented with glucose. Statistical
optimization studies were conducted to evaluate the effects
of the types and concentrations of carbon and nitrogen
sources, pH, temperature, and size of the inoculum. Characterization of the fungus was performed using scanning
electron microscopy, Fourier transform infrared (FTIR)
spectroscopy, and Brunauer, Emmet, and Teller surface area
Responsible editor: Vinod Kumar Gupta
C. H. Neoh : Z. Ibrahim (*)
Department of Biological Sciences, Faculty of Biosciences
and Bioengineering, Universiti Teknologi Malaysia,
81310 Skudai, Johor, Malaysia
e-mail: zaharah@fbb.utm.my
A. Yahya
Department of Industrial Biotechnology, Faculty of Biosciences
and Bioengineering, Universiti Teknologi Malaysia,
81310 Skudai, Johor, Malaysia
R. Adnan
Department of Mathematics, Faculty of Sciences,
Universiti Teknologi Malaysia,
81310 Skudai, Johor, Malaysia
Z. Abdul Majid
Department of Chemistry, Faculty of Sciences,
Universiti Teknologi Malaysia,
81310 Skudai, Johor, Malaysia

analysis. Optimum conditions using response surface methods at pH 5.7, 35 C, and 0.57 % w/v glucose with 2.5 % v/v
inoculum size resulted in a successful removal of 71 % of
the color (initial ADMI of 3,260); chemical oxygen demand,
71 %; ammoniacal nitrogen, 35 %; total polyphenolic compounds, 50 %; and lignin, 54 % after 5 days of treatment.
The decolorization process was contributed mainly by biosorption involving pseudo-first-order kinetics. FTIR analysis revealed that the presence of hydroxyl, CH alkane,
amide carbonyl, nitro, and amine groups could combine
intensively with the colored compounds in POME. This is
the first reported work on the application of A. fumigatus for
the decolorization of POME. The present investigation suggested that growing cultures of A. fumigatus has potential
applications for the decolorization of POME through the
biosorption and biodegradation processes.
Keywords Color removal . Palm oil mill effluent .
Polyphenolic compounds . Biosorption . Lignin .
Optimization . Pseudo-first-order kinetics

Introduction
The palm oil industry is the largest agro-based industry in
Malaysia. As the second world largest palm oil producer,
Malaysia produced 10.6 million tonnes of palm oil in 1999
and increased to 17.7 million tonnes of palm oil in 2008
(Kushairi and Parveez 2009). This figure is expected to rise
as the demand for palm oil increases since it is one of the
most important vegetable oils in the worlds oil and fats
market. Consequently, palm oil mills will also generate a
huge amount of highly polluting and colored effluent, at
about 2.5 tonnes of raw palm oil mill effluent (POME)
generated for every tonne of crude palm oil produced
(DOE 1999). With this large volume of POME, the palm

Environ Sci Pollut Res (2013) 20:29122923

oil mill industry in Malaysia has been identified as the

largest contributor to the pollution load in rivers throughout
the country. The effluent is colored due to the presence of
lignin and its degraded products, tannin, and humic acids
from crushed palm nut and also lipids and fatty acids released during steam extraction process (Oswal et al. 2002).
Discharge of colored effluents imparts color to receiving
waters and thus inhibits the growth of marine organisms
by reducing the penetration of sunlight, with a consequent
reduction in photosynthetic activity. The colored compounds may chelate with metal ions and thus become directly toxic to aquatic biota (Mohan and Karthikeyan 1997).
Besides, the humic substances will react with chlorine in
drinking water treatment and produces carcinogenic byproducts such as trihalomethanes (Vukovic et al. 2008). In
addition, substances derived from lignin in POME can possibly inhibit embryonic development in marine organisms
(Pillai et al. 1997). There is great concern from the public on
the environmental effect of colored wastewater as compared
to harmful wastewater that is colorless (Gupta et al. 2006b).
Therefore, it is necessary that the color present in the effluent is removed before discharge into receiving water bodies.
The existing POME treatment technology such as ponding systems (combination of anaerobic and aerobic pond
processes) is inefficient for the removal of its dark brown
color. It should be highlighted that the POME is treated
without adding any chemicals or biological agents and is
dependent solely on the existence of indigenous microorganisms. Since color has recently emerged as a critical water
quality parameter under the Malaysian Environmental Quality Act 2009, the removal of colored compounds from
POME has become an important problem to be addressed.
Various studies such as membrane technology (Raja Ehsan
Shah and Kaka Singh 2004; Sulaiman and Ling 2004) and
activated sludgegranular-activated carbon (Zahrim et al.
2009) have been used to achieve the present discharge limit;
nevertheless, color removal in POME remains a major problem. Besides the inefficient removal of color, the application
of membrane technology also suffers from fouling due to
high suspended solids in POME, while granular-activated
carbon is costly and not economically feasible for industrial
application. Electrocoagulation caused the reduction of the
brown, opaque effluent of raw POME to a pale yellow
solution. Although effective in color removal, there is a
major operational issue in electrocoagulation (Agustin et
al. 2008). Electrode passivation (blockage of the surface of
the electrode) results in a drastic increase in maintenance
costs (Holt et al. 1999). A recent study conducted using
aerobic granular sludge in sequencing batch reactor for color
removal averaged at only 38 % removal with an initial
ADMI of 600 (Abdullah et al. 2011).
Adsorption is a promising technique for the removal of
impurities and color and has potential application for the

2913

treatment of real industrial wastewater. There are two types

of adsorption: One involving nonbiomass material such as
macrocomposite (Lim et al. 2011), bottom ash, and deoiled
soya for the removal of dye (Gupta et al. 2006a) and removal of dye (Gupta et al. 2007a), fluoride (Gupta et al. 2007b),
and hexavalent chromium (VI) (Gupta et al. 2010) by carbon slurry. The other type of adsorption involves the use of
biomass, either living or nonliving biomass, which is also
known as biosorption. Biosorption using biomass has been
found to be convenient, versatile, and economical for impurities removal and offer an alternative technology for the
removal of color in POME. Research on biomass adsorption
has been mostly used for the removal of heavy metal by
nonliving biomass such as cyanobacterium for the removal
of chromium (Gupta and Rastogi 2008d) and green algae for
the removal of hexavalent chromium (Gupta and Rastogi
2009; Gupta et al. 2001), lead (Gupta and Rastogi 2008a),
and copper (Gupta et al. 2006c). In addition, biosorption of
dye using nonliving biomass such as wheat husk (Gupta et
al. 2007c) has been also reported. Living biomass such as
growing fungal cells have also been used for the removal of
the dye Acid Brilliant Red B (Xin et al. 2012) as well as
cadmium and zinc (Liu et al. 2006). The use of growing
cells for biosorption of color leads to simpler nutrient requirement since POME can provide suitable buffering system and nutrients for the growth of fungi. In addition, the
system is simple and economical. Up till now, most of the
studies found in the literature focused on the biosorption of
color using dead cells of fungi (Patel and Suresh 2008; Aksu
and KarabayIr 2008). However, in this study, fungi were
grown in POME. The subsequent increase in biomass increased the adsorption of color and the removal of other
compounds in POME. This can further enhance the efficiency of the treatment process for the removal of chemical
oxygen demand (COD), total polyphenolic compounds, lignin content, and ammoniacal nitrogen content. Decolorization of colored wastewater such as pulp and paper
wastewater, molasses wastewater, and olive mill effluent
using fungi had been reported in the literature. To the best
of our knowledge, decolorization of POME using fungi has
yet to be reported.
The application of response surface methodology (RSM)
has been previously reported for the optimization of distillery wastewater decolorization using growing cells of Aspergillus fumigatus (Mohammad et al. 2006), decolorization of
dye using growing cells of Pseudomonas sp. (Du et al.
2010), laccase (Murugesan et al. 2007), and NaOHmodified rice husk (Chowdhury et al. 2012), and removal
of lignin in pulp mill wastewater (Wang et al. 2011). These
show the extensive and diverse applications of RSM, particularly in situations where various factors could affect the
efficiency of the process. Hence, this study aimed at optimizing the decolorization of POME by locally isolated A.

2914

fumigatus using RSM. The effects of the types and concentrations of carbon and nitrogen sources, pH, temperature,
and size of the inoculum to the decolorization activity were
identified.

Materials and methods

POME sampling and preparation
Treated POME, or better known as final POME, was
obtained from a local oil palm mill and stored at 4 C. It
was autoclaved at 121 C for 15 min to eliminate the
indigenous microbes. The autoclaved POME was then
centrifuged at 4,000 rpm for 15 min to eliminate suspended
solid materials. The POME was used in subsequent experiments (without filtration and dilution).
Fungal isolation, cultivation, and identification
Locally isolated fungi were obtained from diverse environmental samples such as POME, POME sludge, textile
sludge, soil, wood, grass, spoilt food, and pineapple wastes.
Samples were aseptically placed onto potato dextrose agar
containing 10 % v/v sterilized POME. Different fungal
strains were isolated into a single colony by repeated subculturings. A total of 24 fungal strains were maintained at
4 C on agar plate and were screened for their decolorization
potential. A small piece of agar block was cut out from the
agar plate of actively growing fungi and transferred into
POME (pH was adjusted to pH 5 using HCl). It was cultured
at ambient temperature (2628 C) under shaking condition
(150 rpm) supplemented with glucose (1 % w/v) and peptone (0.5 % w/v). Fungi that showed high decolorization
capability were further used for the study. For the preparation of the inoculum, the fungal strain that showed the best
decolorization was grown on agar plate and the spore was
harvested to make a spore suspension with 106/mL spore
number as described by Chidi et al. (2008).
Total DNA was extracted from fungal spores using the
Norgen DNA Isolation Kit. For the polymerase chain reactions (PCR), the primers ITS1 (5 TCC GTA GGT GAA CCT
TGC GG 3) and ITS4 (5 TCC TCC GCT TAT TGA TAT GC
3) were used to amplify the 18S rDNA fragment sequence.
PCR conditions were prepared as described by Korabecna
(2007) using the Bio-Rad MJ Mini Personal Thermal Cycler.
The aligned sequences were analyzed using the Basic Local
Alignment Search Tool (BLASTn) online analysis tool.

Environ Sci Pollut Res (2013) 20:29122923

enhance the decolorization efficiency of the isolated A.

fumigatus. A general factorial design (Stat Ease, Design
Expert software 6.0.4) is useful in the optimization of categorical factors and is able to identify the interaction of
carbon and nitrogen in influencing the decolorization process. Selected carbon sources such as glucose, sucrose,
fructose, carboxymethyl cellulose (CMC), and glycerol at
a concentration of 1 % w/v and nitrogen sources such as
yeast extract, peptone, urea, and ammonium sulfate at a
concentration of 0.5 % w/v were utilized in a series of
experiments. POME (pH 5) were inoculated with 10 % v/v
spores (106/mL) and incubated at ambient temperature for
the maximum predetermined time period of 5 days. A total
of 90 experimental runs were carried out and the average of
triplicate experiments of decolorization percentages was
recorded.
Glucose was chosen as the best carbon source and was
further optimized using a two-level factorial design together
with other numerical factors. Two-level factorial design was
used as a screening tool to determine important factors
affecting decolorization. The four independent variables that
may affect decolorization were coded at three levels between 1 and +1 for which the actual range is shown in
Table 1. A triplicate full-factorial design (total experimental
run is 48) was selected with 8 center points. All experiments
were set at different conditions as the experimental run and
incubated for 5 days at 150 rpm.
The central composite design (CCD) was used to find the
optimum response within the specified range of the factors.
It is built from the two-level factorial design with center
points and axial points which are able to provide more
precise results compared to the two-level factorial design.
Three independent variables, namely, pH, glucose concentration, and temperature, were included in face-centered
CCD (Table 2), while insignificant parameters (inoculum
size) from the result of the two-level factorial design were
kept at a minimum level throughout the studies. The actual
range of the independent variables is shown in Table 1. The
experiment was conducted in triplicates (total experimental
run is 42) with 6 center points. A final experiment was
conducted to validate the CCD model developed.
Table 1 Coded and uncoded values of the experimental variables for
two-level factorial design
Independent variables Symbols Coded level

pH

Optimization experimental design and data analysis

The first step in optimization was to identify supplementation of various sources of carbon and nitrogen into POME to

Glucose concentration B
(% w/v)
Temperature (C)
C
Inoculum size (% v/v) D

1 (low level) 0

+1 (high level)

0.5

1.0 1.5

30
2.5

35 40
7.5 12.5

Environ Sci Pollut Res (2013) 20:29122923

2915

Table 2 Coded and uncoded values of the experimental variables for

RSM
Independent variables Symbols Coded level
1 (low level) 0
pH
A
Glucose concentration B
(% w/v)
Temperature (C)
C

+1 (high level)

4.5
0.75

5 6.5
1.0 1.25

30

35 40

Analytical methods
To separate the fungal biomass and the liquid medium, the
whole fungi culture was centrifuged at 4,000 rpm for 15 min
at 4 C. The color (ADMI unit), COD (reactor digestion
method), and ammoniacal nitrogen (Nessler method) were
determined according to the HACH Method (2005) by
HACH DR 5000. The pH was measured using the Sartorius
PB-10 pH meter. Total polyphenolic compounds were quantified using a reaction with the FolinCiocalteu reagent
(Singleton et al. 1999). The total amount of polyphenolic
compound that remained in the culture medium was determined using gallic acid as standard. The lignin content of
the effluent was estimated using kraft lignin as standard
(Pearl and Benson 1990). All experiments were conducted
in triplicates.
Biodegradation and biosorption study
Fungal culture grown in POME was centrifuged at
4,000 rpm, 4 C for 15 min. The supernatant was discarded
and the pellet was mixed with an equal volume of NaOH
(0.1 M) solution. The sample was centrifuged at 4,000 rpm,
4 C for 15 min (Patel and Suresh 2008). The remaining
color in the supernatant was measured as described in the
Analytical methods section.
After 5 days of decolorization in POME and desorption
using 0.1 M NaOH, the mycelia of the A. fumigatus were
collected, washed several times with sterile distilled water,
and dried for 24 h at 70 C. The samples were gold-coated
using the Bio-Rad Polaron Division SEM Coating System.
Fourier transform infrared (FTIR) spectroscopy (Nicolet
iS5) was used to identify the functional groups present in the
samples. The sample/KBr mass ratio used for the preparation of the disks was 1:200 within the IR region of the
frequency 4004,000 cm1 at a scan speed of 16 cm/s.
Besides fungus after 5 days decolorization and desorption,
A. fumigatus was also cultured in mineral salts medium
(0.1 % w/v KH 2 PO 4 , 0.05 % w/v MgSO 4 7H 2 O, and
0.05 % w/v NaCl) supplemented with 1 % w/v glucose and
0.5 % w/v ammonium sulfate and analyzed for FTIR. The
samples were prepared by freeze-drying overnight.

The surface area of the fungus after 5 days decolorization

in POME was determined using single-point Brunauer, Emmet, and Teller surface area (Micromeritics Pulse ChemiSorb 2705). The gas mixture was composed of 30 mol%
nitrogen and 70 mol% helium.
Kinetic studies
The kinetics of color sorption by growing cultures of A.
fumigatus was analyzed using different kinetic models including pseudo-first order (Lagergren 1898) and pseudosecond order (Ho and Mckay 1998).
The conformity between experimental data and the
model-predicted values was expressed by the correlation
coefficient, r2. A relatively high r2 value indicates that the
model successfully describes the kinetics of adsorption of
color by A. fumigatus.

Results and discussion

Screening and molecular identification for POME
decolorizers
A total of 11 out of 24 fungi were successfully isolated to
decolorize POME, and the best fungus was further selected
for use in the subsequent experiments. In general, the decolorization capacity obtained ranged from 27 to 46 % (four
from Ananas comosus, one from dead branches and Musa
sp., and four from wild grass) and the highest was 63 %
(from POME sludge). The results of sequence alignment
based on BLAST analysis revealed that the best fungus for
decolorization was A. fumigatus. The sequence was submitted to the GenBank database and has been assigned the
accession number JF835995.
Screening results of nutrient supplement
Figure 1 shows the four carbon sources (glucose, sucrose,
fructose, and glycerol) that demonstrated the most significant
effect to decolorize POME up to 67 %. There was no significant difference between glucose and sucrose, as indicated by a
p value of more than 0.05 significance level (data not shown).
POME supplemented with fructose and glycerol gave a slightly low decolorization percentage, while POME supplemented
with CMC only gave a decolorization percentage of up to
27 %. These results were similar to the results of Jin et al.
(2007) who compared glucose, sucrose, and CMC. It was
found that CMC gave the lowest decolorization percentage
of dye industry effluent using A. fumigatus. Since glucose gave
the best POME decolorization (67 %), it was selected for use in
further experiments involving two-level factorial design to
determine optimal conditions for the decolorization of POME.

2916

Environ Sci Pollut Res (2013) 20:29122923

Fig. 1 Effect of additional carbon and nitrogen sources for POME decolorization by A. fumigatus

This study showed that an additional nitrogen source did

not significantly affect the decolorization of POME as compared to the addition of carbon sources. The addition of
peptone and ammonium sulfate showed the highest decolorization percentages and these were not significantly different from those with no nitrogen sources being added. The
addition of urea caused the decolorization of POME to be
significantly inhibited.
Two-level factorial and central composite design
The influence of four factors, pH (46), inoculum size
(2.512.5 % v/v), glucose concentration (0.51.5 % w/v),
and temperature (3040 C), on POME decolorization
were evaluated using a full-factorial design. Decolorization percentages of POME using A. fumigatus at different
conditions showed that the variation in decolorization
percentages ranged from 52 to 72 %. This indicated that
the selected factors had significant effects on decolorization. The analysis of variance (ANOVA) (data not
shown) showed that the model and curvature term was
statistically significant. The model lack-of-fit F value
of 1.05 implies that the lack of fit was not significant
relative to the pure error. Therefore, it was a good
predictor of the response. This study found that the size
of the inoculum (factor D) does not affect the decolorization of POME as a single factor (p<0.05). However,
this factor shows significant interaction with other factors
(AD, BD, CD, and ACD), though the difference in
decolorization between the high level (12.5 % v/v) and
low level (2.5 % v/v) of inoculum is only 2 % (data not
shown). This result differed from that observed by Erum
and Ahmed (2011) where an increase in the inoculum

size from 1 % v/v to 10 % v/v showed an adverse effect

on the dye removal efficiency using Aspergillus species.
A volume of 2.5 % v/v inoculum size was selected for
convenience and the cost of treatment. The data indicated that significant factors (p value<0.05) on POME
decolorization included pH, glucose concentration, and temperature. Through the statistical optimization studies, pH
(5.56.5), glucose concentration (0.250.75 % w/v), and temperature (3040 C) were selected for the RSM investigation.
The ANOVA of the RSM parameters (Table 3) indicated
that this quadratic model is significant with a p value
smaller than 0.05 and high adjusted R2 value of 0.9547.
Equation 1 shows the relationship between the decolorization of POME with pH, glucose, and temperature in
terms of coded factors.
Decolorization% 69:37  10:40A 10:70B
0:033C  11:89A2
 11:39B2  3:05C 2
 1:79AB  1:63AC
2:37BC

Since the interaction between factor C (temperature)

with factor A (pH) and factor B (glucose concentration)
is significant, factor C should therefore be included in
the model although by itself it is insignificant where the
p value is 0.9509. The optimization using CCD showed
that the three significant factors affecting the decolorization of POME were pH, glucose concentration, and
temperature. To investigate the interaction effect of two

Environ Sci Pollut Res (2013) 20:29122923

2917

Table 3 ANOVA for the RSM parameters

Term
Model
ApH
Bglucose concentration (w/v)
Ctemperature (C)
A2
B2
C2
AB
AC
BC
Residual
Lack of fit
Pure error

Sum of squares
12,273.85
3,244.80
3,434.70
0.033
1,117.20
1,025.17
73.66
77.04
63.38
135.38
329.81
80.48
249.33

df

Mean square

9
1
1
1
1
1
1
1

1,363.76
3,244.80
3,434.70
0.033
1,117.20
1,025.17
73.66
77.04

1
1
38
5
33

63.38
135.38
8.68
16.10
7.56

F value
157.13
373.86
395.74
3.841E003
128.72
118.12
8.49
8.88

Prob>F (p value)
<0.0001
<0.0001
<0.0001
0.9509
<0.0001
<0.0001
0.0060
0.0050

7.30
15.60

0.0102
0.0003

2.13

0.0863

R2 00.9738; adjusted R2 00.9547

factors on the percentage decolorization, the threedimensional (3D) plots were drawn.
Figure 2a presents the effect of pH and glucose at fixed
temperature (35 C). As there was an increase in glucose
concentration, the decolorization efficiency was up to the optimum level with a pH of 5.7. There was no significant difference
observed for the glucose concentration ranges from 0.57
until 0.75 % w/v. The optimum pH for POME decolorization using A. fumigatus was at pH 5.7, which was similar
to the result of Sharma et al. (2009) who reported the
optimum pH for decolorization of dye effluent using A.
fumigatus fresinus at pH 5.5. Decolorization was inhibited
when pH was increased from 5.7 to 6.5. This may be due
to the inhibition of fungal growth and thus decreased the
decolorization capacity. Figure 2b presents the interaction
effect of pH and temperature at fixed glucose concentration
(0.57 % w/v). As there was an increase in temperature, the
decolorization efficiency was up to the optimum level with
the pH of 5.7. The 3D plot indicated that decolorization
efficiency is more dependent on pH than on temperature.
Experimental results for the biosorption of humic acid using
fungi biosorbents reached the optimum at low pH (Zhou
1992). Another study reported that decolorization of dye
industry effluent by A. fumigatus reached the optimum at
pH 3 (Jin et al. 2007). Figure 2c presents the effect of
glucose and temperature at pH 5.7. Temperature has little
effect on decolorization compared to glucose concentration.
It can be concluded that a small change in pH will bring a
significant change in decolorization, followed by glucose
concentration and finally the temperature. The application
of A. fumigatus for the decolorization of POME seemed to
be a practical approach since it was able to decolorize
POME up to a maximum of 71 % after 5 days of

incubation at 35 C with shaking (150 rpm) at pH 5.7

supplemented with 2.5 % v/v inoculum and 0.57 % w/v
glucose.
Decolorization studies
Table 4 summarizes the removal efficiency of color (71 %),
COD (71 %), ammoniacal nitrogen (35 %), total polyphenolic compounds (50 %), and lignin (54 %) after 5 days of
incubation under optimized RSM conditions. The COD,
ammoniacal nitrogen, total polyphenolic compounds, and
lignin in sterile POME (pH 5.7) remained constant with an
increase of color between 4 and 6 % and a slight increase in
pH after 5 days incubation.
Figure 3 shows a similar trend in color, pH, COD,
total polyphenolic compounds, and lignin using A. fumigatus. There is a decrease in pH after 5 days incubation. This is probably due to the secretion of acidic
metabolites, consequently leading to an increase in the
acidity of the POME. For the fermentation of POME by
Aspergillus niger, citric acid, oxalic acid, and gluconic
acid were produced (Jamal et al. 2007). The release of
acidic metabolites decreases the pH of the culture and
affects the surface charge of the mycelium. At low pH,
high concentrations of protons lead to an increase in
adsorption as the repulsive forces between fungi with
humic acid and lignin were reduced (Zhou and Banks
1993). Besides, the ionization of humic acid and lignin
molecules decrease at low pH and self-aggregation takes
place (Belgacem and Gandini 2008) and thus easily
trapped by fungi mycelium. This explained why the
adsorption of humic acid and lignin by fungi occurs
best at low pH.

2918
Fig. 2 a 3D surface plots for
the decolorization of POME as
a function of pH and glucose
with temperature kept at 35 C.
b 3D surface plots for the
decolorization of POME as a
function of pH and temperature
with glucose concentration kept
at 0.57 %w/v. c 3D surface plots
for the decolorization of POME
as a function of glucose and
temperature with pH kept at 5.7

Environ Sci Pollut Res (2013) 20:29122923

Environ Sci Pollut Res (2013) 20:29122923

2919

Table 4 Reduction in effluent quality parameters using A. fumigatus

Parameter

Initial

Final

Percentage

Color (ADMI)
3,26041
93535
71
pH
5.710.02 2.720.05
COD (mg/L)
2,42991
70332
71
Ammoniacal nitrogen (mg/L)
1577
1024
35
Total polyphenolic compounds
30312
1516
50
(mg/L)
Lignin concentration (mg/L)
3382
1553
54

Figure 3b shows a similar trend of color and COD removal during the treatment period. The addition of glucose
was necessary for fungi growth and color removal during
Fig. 3 Profile of color, pH,
COD, and total polyphenolic
compounds/lignin
concentration versus time at the
optimal condition predicted by
RSM. a Plot of color (green
triangles) and pH (purple
multiplication sign) versus
time. b Plot of color (green
triangles) and COD (orange
squares) versus time. c Plot of
color and total polyphenolic
compounds (purple
multiplication sign)/lignin
concentration (orange squares)
versus time

the treatment. The consumption of glucose by A. fumigatus

caused an increase in biomass and thus increased the biosorption surface area. Besides, consumption of glucose and
other compounds in POME causes a decrease in pH which
consequently increases the color and COD removal.
Figure 3c shows the removal of total polyphenolic compounds, lignin, and color during the treatment process. The
correlation analysis (r2) for color and polyphenolic compounds is 0.9394, whereas for color and lignin is 0.9524.
In other words, lignin and total polyphenolic compounds are
directly proportional to color. Similar results were obtained
from the decolorization of debarking water which was contributed mainly by the lignin content (Kindsigo and Kallas
2009). To the best of our knowledge, there is no study
related to the removal of polyphenolic compounds in treated

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Environ Sci Pollut Res (2013) 20:29122923

POME. The only work reported in the literature was the

removal of polyphenolic compound in anaerobic POME
using Lactobacillus plantarum (Limkhuansuwan and Chaiprasert 2010). A. fumigatus showed polyphenol removal of
more than ten times as compared to L. plantarum.
Decolorization mechanism
For the growing cultures, desorption of color from fungus
pellet using 0.1 M NaOH produced about 892 % of color
removal from POME. From the results obtained, it may be
inferred that most of the color-causing compounds were
bound to the mycelium via adsorption; this typically
involves a combination of active and passive transport
mechanism starting with the diffusion of the adsorbed component to the surface of the cell (Bayramolu and Yakup
Arca 2007). The active transport mechanism generally
involves the use of energy generated by living cells, whereas
the passive transport mechanism is the diffusion of the
colored compounds and is mainly influenced by the affinity
between the biosorbent and sorbate (Volesky 2007). Desorption using NaOH indicates that electrostatic attraction was
the main the active force between the fungi and colored
compounds (Xin et al. 2010) and the resulting adsorption
is reversible in nature (Gupta et al. 2009).
Figure 4 shows that the fungus pellets after desorption
had a highly porous mycelium matrix and their large surface
areas were clean for colored compound uptake (Fig. 5). The
appearance of the hyphae of A. fumigatus implies a very
high capacity for uptake of colored compounds. A porous
structure is very important for colored molecules to be
adsorbed on the fungi. The surface area of A. fumigatus after
5 days decolorization in POME is 6.67 m2/g. This value is
higher than the immobilized A. niger for biosorption of dyes
that is in the range of 2.40 to 3.16 m2/g (Fu and Viraraghavan

Fig. 4 SEM image of A. fumigatus after desorption at 1,500

Fig. 5 SEM image of A. fumigatus after decolorization of POME at

1,500

2003). This shows that using living A. fumigatus gives a

higher surface area compared with the immobilized fungus
for biosorption of colored compounds. Besides, the scanning
electron microscopy (SEM) image in Fig. 5 shows that the
mycelium still kept a smooth morphology even after adsorption of color, and this was different from the study of Wang
and Hu (2008) on the adsorption of reactive dye by immobilized growing A. fumigatus beads. Wang and Hu (2008)
reported that the damaged part of the cell walls of A. fumigatus
may be due to the toxicity of the dye in the medium.
The FTIR spectra of A. fumigatus cultured in colorless
mineral salts medium after 5 days decolorization and desorption are given in Fig. 6. A similar and very strong peak was
observed for fungus before decolorization (3,445.27 cm1),
fungus after 5 days decolorization (3,435.62 cm1), and fungus after desorption (3,448.05 cm1). The broad overlapping
peak of the fungus after 5 days of decolorization might be
due to hydroxyl or amine groups present in the POME.
The appearance of weak bands (2,922.69, 2,922.49, and
2,923.47 cm1) in all of the samples could be assigned to
the CH sp3. The medium band for fungus before decolorization (1,634.99 cm1), fungus after 5 days decolorization (1,640.60 cm 1 ), and fungus after desorption
(1,635.00 cm1) was a consequence of the amide carbonyl
group (C0O amide) and the shifted peak, suggesting its
role in adsorption. The weak band for fungus before
decolorization (1,433.42 cm1), fungus after 5 days decolorization (1,430.86 cm1), and fungus after desorption
(1,384.82 cm1) could be attributed to the presence of additional nitro groups (N0O) in POME. The overlapped band for
fungus before decolorization (1,055.69 cm1), fungus after
5 days decolorization (1,056.23 cm1), and fungus after desorption (1,071.43 cm1) could be assigned to the CN
stretching vibration of the chitinchitosan and protein fractions (Gupta and Rastogi 2008b). The appearance of a band

Environ Sci Pollut Res (2013) 20:29122923

2921

respectively. The pseudo-first-order kinetics had the

highest r2 values, as shown in Fig. 7, with the rate
constant (k) of 0.1044. The pseudo-first-order kinetics
also had the highest precision between the experimental
qe and calculated qe compared to the pseudo-secondorder kinetics. Thus, the pseudo-first-order kinetics
model was taken as the best fit equation to describe
the sorption mechanism of color. The pseudo-first-order
kinetics considers the rate of adsorption to be proportional to the number of unoccupied sites. The applicability of the pseudo-first-order kinetics shows that the
adsorption rate depends on the ADMI value. Numerous
studies reported on the pseudo-first-order kinetics for
the sorption of dyes such as the sorption of acid dyes
onto chitosan (Wong et al. 2004) and sorption of hexavalent chromium using Acinetobacter junii (Paul et al.
2012). However, to the best of our knowledge, this is
the first report on pseudo-first-order kinetics for the
absorption of color using growing fungi where the biomass of the fungi changed with time.

Conclusions

Fig. 6 FTIR spectra for a fungus before decolorization, b fungus after

5 days decolorization, and c fungus after desorption using 0.1 M NaOH

The results of this study indicated that growing cultures

of A. fumigatus can be used for the decolorization of
POME to a maximum of 71 % in 5 days. Optimization
studies indicated that pH 5.7, incubation temperature of
35 C, and inoculum size of 2.5 % v/v with the addition
of glucose 0.57 % w/v were optimal for maximum
decolorization of POME. This is the first reported work
on the application A. fumigatus for the decolorization of
POME. These results suggested that the decolorization
process mediated by A. fumigatus has potential applications for treatment operations through the biosorption
and biodegradation processes. Further research should
be carried out in nonsterile wastewater and scale-up in
a bioreactor.

for the fungus before decolorization (554.99 cm1), fungus

after 5 days decolorization (463.20 cm1), and fungus after
desorption (666.55, 578.42, and 502.57 cm1) represents the
CNC scissoring that is only found in protein structures
(Akar et al. 2009). Significant changes in wave numbers for
the fungus after 5 days decolorization suggested that the
hydroxyl group, CH sp3, and the amide carbonyl group,
N0O and CN, could combine intensively with the colored
compound in POME (Gupta and Rastogi 2008c).
Kinetic studies
The correlation analysis values for pseudo-first-order
and pseudo-second-order kinetics are 0.9892 and 0.012,

Fig. 7 Pseudo-first-order kinetic modeling of the decolorization of

POME by A. fumigatus

2922
Acknowledgments The authors would like to express their appreciation to the Ministry of Sciences and Innovation Malaysia (National
Science Fellowship) and Universiti Teknologi Malaysia for the financial support. The authors also would like to thank the local company
Johor for sampling of the wastewater.

References
Abdullah N, Ujang Z, Yahya A (2011) Aerobic granular sludge formation for high strength agro-based wastewater treatment. Bioresour Technol 102(12):67786781
Agustin M, Sengpracha W, Phutdhawong W (2008) Electrocoagulation
of palm oil mill effluent. Int J Environ Res Public Health 5
(3):177180
Akar ST, Gorgulu A, Kaynak Z, Anilan B, Akar T (2009) Biosorption
of Reactive Blue 49 dye under batch and continuous mode using a
mixed biosorbent of macro-fungus Agaricus bisporus and Thuja
orientalis cones. Chem Eng J 148(1):2634
Aksu Z, KarabayIr G (2008) Comparison of biosorption properties of
different kinds of fungi for the removal of Gryfalan Black RL
metal-complex dye. Bioresour Technol 99(16):77307741
Bayramolu G, Yakup Arca M (2007) Biosorption of benzidine based
textile dyes Direct Blue 1 and Direct Red 128 using native and
heat-treated biomass of Trametes versicolor. J Hazard Mater 143
(12):135143
Belgacem MN, Gandini A (2008) Monomers, polymers and composites from renewable resources. Elsevier, Amsterdam
Chidi SB, Godana B, Ncube I, Rensburg EJV, Cronshaw A, Abotasi
EK (2008) Production, purification and characterization of
celullase-free xylanase from Aspergillus terreus UL 4209. Afr J
Biotechnol 7(21):39393948
Chowdhury S, Chakraborty S, Saha P (2012) Response surface optimization of a dynamic dye adsorption process: a case study of
crystal violet adsorption onto NaOH-modified rice husk. Environ
Sci Pollut Res Int. doi:10.1007/s11356-012-0989-7
DOE (1999) Industrial processes and the environment (handbook no.
3): crude palm oil industry. Department of Environment Malaysia,
Kuala Lumpur
Du L-N, Yang Y-Y, Li G, Wang S, Jia X-M, Zhao Y-H (2010)
Optimization of heavy metal-containing dye Acid Black 172
decolorization by Pseudomonas sp. DY1 using statistical designs.
Int Biodeter Biodegr 64(7):566573
Erum S, Ahmed S (2011) Comparison of dye decolorization efficiencies of indigenous fungal isolates. Afr J Biotechnol 10(17):3399
3411
Fu Y, Viraraghavan T (2003) Column studies for biosorption of dyes
from aqueous solutions on immobilised Aspergillus niger fungal
biomass. Water SA 29(4):465472
Gupta VK, Rastogi A (2008a) Biosorption of lead from aqueous
solutions by green algae Spirogyra species: kinetics and equilibrium studies. J Hazard Mater 152(1):407414
Gupta VK, Rastogi A (2008b) Biosorption of lead(II) from aqueous
solutions by non-living algal biomass Oedogonium sp. and Nostoc sp.a comparative study. Colloids Surf B Biointerfaces 64
(2):170178
Gupta VK, Rastogi A (2008c) Equilibrium and kinetic modelling of
cadmium(II) biosorption by nonliving algal biomass Oedogonium
sp. from aqueous phase. J Hazard Mater 153(12):759766
Gupta VK, Rastogi A (2008d) Sorption and desorption studies of
chromium(VI) from nonviable cyanobacterium Nostoc muscorum
biomass. J Hazard Mater 154(13):347354
Gupta VK, Rastogi A (2009) Biosorption of hexavalent chromium by
raw and acid-treated green alga Oedogonium hatei from aqueous
solutions. J Hazard Mater 163(1):396402

Environ Sci Pollut Res (2013) 20:29122923

Gupta VK, Shrivastava AK, Jain N (2001) Biosorption of chromium
(VI) from aqueous solutions by green algae Spirogyra species.
Water Res 35(17):40794085
Gupta VK, Mittal A, Gajbe V, Mittal J (2006a) Removal and recovery
of the hazardous azo dye acid orange 7 through adsorption over
waste materials: bottom ash and de-oiled soya. Ind Eng Chem Res
45(4):14461453. doi:10.1021/ie051111f
Gupta VK, Mittal A, Kurup L, Mittal J (2006b) Adsorption of a
hazardous dye, erythrosine, over hen feathers. J Colloid Interface
Sci 304(1):5257
Gupta VK, Rastogi A, Saini VK, Jain N (2006c) Biosorption of copper
(II) from aqueous solutions by Spirogyra species. J Colloid Interface Sci 296(1):5963
Gupta VK, Ali I, Saini VK (2007a) Adsorption studies on the removal
of Vertigo Blue 49 and Orange DNA13 from aqueous solutions
using carbon slurry developed from a waste material. J Colloid
Interface Sci 315(1):8793
Gupta VK, Ali I, Saini VK (2007b) Defluoridation of wastewaters
using waste carbon slurry. Water Res 41(15):33073316
Gupta VK, Jain R, Varshney S (2007c) Removal of Reactofix golden
yellow 3 RFN from aqueous solution using wheat huskan
agricultural waste. J Hazard Mater 142(12):443448
Gupta VK, Carrott PJM, Carrott MMLR, Suhas (2009) Low-cost
adsorbents: growing approach to wastewater treatmenta review.
Crit Rev Environ Sci Tech 39(10):783842
Gupta VK, Rastogi A, Nayak A (2010) Adsorption studies on the
removal of hexavalent chromium from aqueous solution using a
low cost fertilizer industry waste material. J Colloid Interface Sci
342(1):135141
Ho YS, McKay G (1998) Sorption of dye from aqueous solution by
peat. Chem Eng J 70:115124
Holt P, Barton G, Mitchell C (1999) Electrocoagulation as a wastewater treatment. Paper presented at the Third Annual Australian
Environmental Engineering Research Event, Castlemaine, Victoria, November, pp 2326
Jamal P, Alam MZ, Mohamad AB (2007) Microbial bioconversion of
palm oil mill effluent to citric acid with optimum process conditions. In: 3rd Kuala Lumpur International Conference on Biomedical Engineering 2006, pp 483487
Jin X-C, Liu G-Q, Xu Z-H, Tao W-Y (2007) Decolorization of a dye
industry effluent by Aspergillus fumigatus XC6. Appl Microbiol
Biotechnol 74(1):239243. doi:10.1007/s00253-006-0658-1
Kindsigo M, Kallas J (2009) Wet oxidation of debarking water:
changes in lignin content and biodegradability. Environ Chem
Lett 7(2):121126. doi:10.1007/s10311-008-0144-3
Korabecna M (2007) The variability in the fungal ribosomal DNA
(ITS1, ITS2, and 5.8S rRNA gene): its biological meaning and
application in medical mycology. In: Mendez-Vilas A (ed) Communicating current research and educational topics and trends in
applied microbiology. Formatex, Spain, pp 783787
Kushairi A, Parveez GKA (2009) Development of value-added products
in oil palm via genetic modification and non-genetic modification
routes. In: Accelerating Commercialization in Biotechnology, Kuala
Lumpur Convention Centre
Lagergren S (1898) About the theory of so-called adsorption of soluble
substances. Kungliga Svenska Vetenskapsakademiens Handlingar
24(4):139
Lim CK, Bay HH, Kee TC, Majid ZA, Ibrahim Z (2011) Decolourisation of reactive black 5 using Paenibacillus sp. immobilised onto
macrocomposite. J Bioremed Biodegrad S1:004. doi:10.4172/
2155-6199.S1-004
Limkhuansuwan V, Chaiprasert P (2010) Decolorization of molasses
melanoidins and palm oil mill effluent phenolic compounds by
fermentative lactic acid bacteria. J Environ Sci 22(8):12091217
Y-g L, Fan T, G-m Z, Li X, Tong Q, Ye F, Zhou M, W-h X, Y-e H
(2006) Removal of cadmium and zinc ions from aqueous solution

Environ Sci Pollut Res (2013) 20:29122923

by living Aspergillus niger. Trans Nonferrous Met Soc China 16
(3):681686
Mohammad P, Azarmidokht H, Fatollah M, Mahboubeh B (2006)
Application of response surface methodology for optimization
of important parameters in decolorizing treated distillery wastewater using Aspergillus fumigatus UB2 60. Int Biodeter Biodegr
57(4):195199
Mohan SV, Karthikeyan J (1997) Removal of lignin and tannin colour
from aqueous solution by adsorption onto activated charcoal.
Environ Pollut 97(12):183187
Murugesan K, Dhamija A, Nam I-H, Kim Y-M, Chang Y-S (2007)
Decolourization of reactive black 5 by laccase: optimization by
response surface methodology. Dyes Pigments 75(1):176184
Oswal N, Sarma PM, Zinjarde SS, Pant A (2002) Palm oil mill effluent
treatment by a tropical marine yeast. Bioresour Technol 85(1):3537
Patel R, Suresh S (2008) Kinetic and equilibrium studies on the biosorption of reactive black 5 dye by Aspergillus foetidus. Bioresour
Technol 99(1):5158
Paul ML, Samuel J, Chandrasekaran N, Mukherjee A (2012) Comparative kinetics, equilibrium, thermodynamic and mechanistic studies on biosorption of hexavalent chromium by live and heat killed
biomass of Acinetobacter junii VITSUKMW2, an indigenous
chromite mine isolate. Chem Eng J 187:104113
Pearl IA, Benson HK (1990) The determination of lignin in sulphide
pulping. PapTrade J 111(18):3536
Pillai MC, Blethrow H, Higashi RM, Vines CA, Cherr GN (1997)
Inhibition of the sea urchin sperm acrosome reaction by a ligninderived macromolecule. Aquat Toxicol 37(23):139156
Raja Ehsan Shah RSS, Kaka Singh PK (2004) Treatment of palm oil
mill effluent (POME) using membrane technology. In: Regional
Symposium on Membrane Science and Technology, Puteri Pan
Pacific Hotel, Johor Bharu
Sharma P, Singh L, Dilbaghi N (2009) Optimization of process variables for decolorization of Disperse Yellow 211 by Bacillus subtilis
using Box-Behnken design. J Hazard Mater 164(23):10241029

2923
Singleton VL, Orthofer R, Lamuela-Ravents RM (1999) Analysis of
total phenols and other oxidation substrates and antioxidants by
means of FolinCiocalteu reagent. In: Lester P (ed) Methods in
enzymology, vol. 299. Academic, San Diego, pp 152178
Sulaiman N, Ling CK (2004) Membrane ultrafiltration of treated palm
oil mill effluent (POME). J Teknol 41:113120
Volesky B (2007) Biosorption and me. Water Res 41(18):40174029
Vukovic M, Domanovac T, Briki F (2008) Removal of humic substances by biosorption. J Environ Sci 20(12):14231428
Wang B-E, Hu Y-Y (2008) Bioaccumulation versus adsorption of
reactive dye by immobilized growing Aspergillus fumigatus
beads. J Hazard Mater 157(1):17
Wang J-P, Chen Y-Z, Wang Y, Yuan S-J, Yu H-Q (2011) Optimization
of the coagulation-flocculation process for pulp mill wastewater
treatment using a combination of uniform design and response
surface methodology. Water Res 45(17):56335640
Wong YC, Szeto YS, Cheung WH, McKay G (2004) Pseudo-firstorder kinetic studies of the sorption of acid dyes onto chitosan. J
Appl Polymer Sci 92(3):16331645. doi:10.1002/app.13714
Xin B, Chen G, Zheng W (2010) Bioaccumulation of Cu-complex
reactive dye by growing pellets of Penicillium oxalicum and its
mechanism. Water Res 44(12):35653572
Xin B, Xia Y, Zhang Y, Aslam H, Liu C, Chen S (2012) A feasible
method for growing fungal pellets in a column reactor inoculated
with mycelium fragments and their application for dye bioaccumulation from aqueous solution. Bioresour Technol 105:100105
Zahrim AY, Rachel FM, Menaka S, Su SY, Melvin F, Chan ES (2009)
Decolorisation of anaerobic palm oil mill effluent via activated
sludge-granular activated carbon. World Appl Sci J 5:126129,
Special Issue for Environment
Zhou JL (1992) Biosorption and desorption of humic acid by microbial
biomass. Chemosphere 24(11):15731589
Zhou JL, Banks CJ (1993) Mechanism of humic acid colour removal
from natural waters by fungal biomass biosorption. Chemosphere
27(4):607620

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