2013 Optimization of Decolorization of Palm Oil Mill Effluent (POME) by Growing Cultures of Aspergillus Fumigatus Using Response Surface Methodology (1)
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Environmental Science and Pollution
Research
ISSN 0944-1344
Volume 20
Number 5
Environ Sci Pollut Res (2013)
20:2912-2923
DOI 10.1007/s11356-012-1193-5
Optimization of decolorization of palm oil mill effluent (POME) by growing cultures
of Aspergillus fumigatus using responsesurface methodology
Chin Hong Neoh, Adibah Yahya, Robiah
Adnan, Zaiton Abdul Majid & Zaharah
Ibrahim
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8/18/2019 2013 Optimization of Decolorization of Palm Oil Mill Effluent (POME) by Growing Cultures of Aspergillus Fumigatus …
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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 photosyn-thetic activities, produce carcinogenic by-products in drink-
ing 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. Char-
acterization of the fungus was performed using scanningelectron microscopy, Fourier transform infrared (FTIR)
spectroscopy, and Brunauer, Emmet, and Teller surface area
analysis. Optimum conditions using response surface meth-
ods 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 com-
pounds, 50 %; and lignin, 54 % after 5 days of treatment.
The decolorization process was contributed mainly by bio-
sorption involving pseudo-first-order kinetics. FTIR analy-
sis revealed that the presence of hydroxyl, C – H 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 sug-
gested 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 1999and 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 world’s 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
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
Environ Sci Pollut Res (2013) 20:2912 – 2923
DOI 10.1007/s11356-012-1193-5
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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 re-
leased 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 com-
pounds may chelate with metal ions and thus become di-
rectly toxic to aquatic biota (Mohan and Karthikeyan 1997).
Besides, the humic substances will react with chlorine in
drinking water treatment and produces carcinogenic by-
products such as trihalomethanes (Vukovic et al. 2008). In
addition, substances derived from lignin in POME can pos-
sibly 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 efflu-
ent is removed before discharge into receiving water bodies.
The existing POME treatment technology such as pond-
ing 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 microor-
ganisms. Since color has recently emerged as a critical water
quality parameter under the Malaysian Environmental Qual-
ity 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 sludge – granular-activated carbon (Zahrim et al.
2009) have been used to achieve the present discharge limit;
nevertheless, color removal in POME remains a major prob-
lem. 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
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 remov-
al of dye (Gupta et al. 2007a ), fluoride (Gupta et al. 2007b),
and hexavalent chromium (VI) (Gupta et al. 2010) by car-
bon slurry. The other type of adsorption involves the use of
biomass, either living or nonliving biomass, which is alsoknown as biosorption. Biosorption using biomass has been
found to be convenient, versatile, and economical for impu-
rities 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 asgrowing 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 re-
quirement since POME can provide suitable buffering sys-
tem 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 in-
creased the adsorption of color and the removal of other
compounds in POME. This can further enhance the efficien-
cy of the treatment process for the removal of chemical
oxygen demand (COD), total polyphenolic compounds, lig-
nin content, and ammoniacal nitrogen content. Decoloriza-
tion 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 distill-
ery wastewater decolorization using growing cells of Asper-
gillus fumigatus (Mohammad et al. 2006), decolorization of
dye using growing cells of Pseudomonas sp. (Du et al.
2010), laccase (Murugesan et al. 2007), and NaOH-
modified 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, par-
ticularly in situations where various factors could affect the
efficiency of the process. Hence, this study aimed at opti-
mizing the decolorization of POME by locally isolated A.
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fumigatus using RSM. The effects of the types and concen-
trations 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 experi-
ments (without filtration and dilution).
Fungal isolation, cultivation, and identification
Locally isolated fungi were obtained from diverse environ-
mental 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 sub-
culturings. 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 (26 – 28 °C) under shaking condition
(150 rpm) supplemented with glucose (1 % w/ v ) and pep-
tone (0.5 % w/ v ). Fungi that showed high decolorization
capability were further used for the study. For the prepara-
tion 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 reac-
tions (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.
Optimization experimental design and data analysis
The first step in optimization was to identify supplementa-
tion of various sources of carbon and nitrogen into POME to
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 cate-
gorical factors and is able to identify the interaction of
carbon and nitrogen in influencing the decolorization pro-
cess. Selected carbon sources such as glucose, sucrose,
fructose, carboxymethyl cellulose (CMC), and glycerol at
a concentration of 1 % w/ v and nitrogen sources such asyeast 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 factorsaffecting decolorization. The four independent variables that
may affect decolorization were coded at three levels be-
tween −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 concen-
tration, 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
−1 (low level) 0 +1 (high level)
pH A 4 5 6
Glucose concentration
(% w/ v )
B 0.5 1.0 1.5
Temperature (°C) C 30 35 40
Inoculum size (% v / v ) D 2.5 7.5 12.5
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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 quan-tified using a reaction with the Folin – Ciocalteu reagent
(Singleton et al. 1999). The total amount of polyphenolic
compound that remained in the culture medium was deter-
mined 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 prepara-
tion of the disks was 1:200 within the IR region of the
frequency 400 – 4,000 cm−1 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 KH2PO4, 0 . 0 5 % w/ v MgSO4·7H2O, 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, Em-
met, and Teller surface area (Micromeritics Pulse Chemi-
Sorb 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 in-
cluding pseudo-first order (Lagergren 1898) and pseudo-
second order (Ho and Mckay 1998).
The conformity between experimental data and the
model-predicted values was expressed by the correlation
coefficient, r 2. A relatively high r 2 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 de-
colorization 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 submit-
ted 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 signif-
icant 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 slight-
ly 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.
Table 2 Coded and uncoded values of the experimental variables for
RSM
Independent variables Symbols Coded level
−1 (low level) 0 +1 (high level)
pH A 4.5 5 6.5
Glucose concentration
(% w/ v )
B 0.75 1.0 1.25
Temperature (°C) C 30 35 40
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This study showed that an additional nitrogen source did
not significantly affect the decolorization of POME as com-
pared to the addition of carbon sources. The addition of
peptone and ammonium sulfate showed the highest decol-
orization percentages and these were not significantly dif-
ferent 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 (4 – 6), inoculum size
(2.5 – 12.5 % v / v ), glucose concentration (0.5 – 1.5 % w/ v ),
and temperature (30 – 40 °C), on POME decolorization
were evaluated using a full-factorial design. Decoloriza-
tion 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 decoloriza-
tion. 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 decolor-
ization of POME as a single factor ( p
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factors on the percentage decolorization, the three-
dimensional (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 opti-
mum 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 decoloriza-
tion 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 polyphe-
nolic 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. fumi-
gatus. There is a decrease in pH after 5 days incuba-
tion. 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
plac e (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.
Table 3 ANOVA for the RSM parameters
Term Sum of squares df Mean square F value Prob> F ( p value)
Model 12,273.85 9 1,363.76 157.13
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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 POMEas a function of glucose and
temperature with pH kept at 5.7
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Figure 3b shows a similar trend of color and COD re-
moval during the treatment period. The addition of glucose
was necessary for fungi growth and color removal during
the treatment. The consumption of glucose by A. fumigatus
caused an increase in biomass and thus increased the bio-
sorption 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 com-
pounds, lignin, and color during the treatment process. The
correlation analysis (r 2) for color and polyphenolic com- pounds 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 con-
tributed 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
Table 4 Reduction in effluent quality parameters using A. fumigatus
Parameter Initial Final Percentage
Color (ADMI) 3,260±41 935±35 71
pH 5.71± 0.02 2.72±0.05 –
COD (mg/L) 2,429±91 703±32 71
Ammoniacal nitrogen (mg/L) 157± 7 102± 4 35
Total polyphenolic compounds
(mg/L)
303±12 151±6 50
Lignin concentration (mg/L) 338± 2 155± 3 54
Fig. 3 Profile of color, pH,
COD, and total polyphenolic
compounds/lignin
concentration versus time at the
optimal condition predicted byRSM. 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
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POME. The only work reported in the literature was the
removal of polyphenolic compound in anaerobic POME
using Lactobacillus plantarum (Limkhuansuwan and Chai-
prasert 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 89± 2 % of color
removal from POME. From the results obtained, it may be
inferred that most of the color-causing compounds were
bou nd to the myc eli um via ads orp tion; thi s typ icall y
involves a combination of active and passive transport
mechanism starting with the diffusion of the adsorbed com-
ponent to the surface of the cell (Bayramoğlu and Yakup
Ar ıca 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). Desorp-
tion 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
2003). This shows that using living A. fumigatus gives a
higher surface area compared with the immobilized fungusfor 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 adsorp-
tion of color, and this was different from the study of Wang
and Hu (2008) on the adsorption of reactive dye by immobi-
lized 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 desorp-
tion are given in Fig. 6. A similar and very strong peak was
observed for fungus before decolorization (3,445.27 cm−1),
fungus after 5 days decolorization (3,435.62 cm−1), and fun-
gus after desorption (3,448.05 cm−1). 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 cm−1) in all of the samples could be assigned to
the C – H sp3. The medium band for fungus before decol-
orization (1,634.99 cm−1), fungus after 5 days decoloriza-
tion (1,640.60 cm−1), and fungus after desorption
(1,635.00 cm−1) 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 cm−1), fungus after 5 days decol-
orization (1,430.86 cm−1), and fungus after desorption
(1,384.82 cm−1) could be attributed to the presence of addi-
tional nitro groups (N0O) in POME. The overlapped band for
fungus before decolorization (1,055.69 cm−1), fungus after
5 days decolorization (1,056.23 cm−1
), and fungus after de-
sorption (1,071.43 cm−1) could be assigned to the – CN
stretching vibration of the chitin – chitosan and protein frac-
tions (Gupta and Rastogi 2008b). The appearance of a bandFig. 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
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for the fungus before decolorization (554.99 cm−1), fungus
after 5 days decolorization (463.20 cm−1), and fungus after
desorption (666.55, 578.42, and 502.57 cm−1) represents the
C – N – C 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, C – H sp3, and the amide carbonyl group,
N0O and C – N, 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,
respectively. The pseudo-first-order kinetics had the
highest r 2 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-second-
order 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 propor-
tional to the number of unoccupied sites. The applica-
bility 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 hex-
avalent 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 bio-
mass of the fungi changed with time.
Conclusions
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 applica-
tions for treatment operations through the biosorption
and biodegradation processes. Further research should
be carried out in nonsterile wastewater and scale-up in
a bioreactor.
Fig. 7 Pseudo-first-order kinetic modeling of the decolorization of
POME by A. fumigatus
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
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