International Journal of Chemical Engineering and Applications, Vol. 3, No.

3, June 2012

Batch Biodegradation of Phenol of Paper and Pulp

Effluent by Aspergillus Niger
Neha Sharma and Vikas. C. Gupta

large quantities of sludge [6]. No. of microorganisms can

AbstractBiodegradation of industrial phenol by a fungal utilize phenol under aerobic conditions as source of carbon &
isolate Aspergillus niger was studied in batch flask system with energy [7]-[9]. The biological treatment of industrial
synthetic & industrial effluent. Aspergillus niger, was efficiently wastewaters usually depends upon the oxidative activities of
immobilized on sodium alginate beads. The immobilized cells
were used in the batch culture flasks for paper & pulp industry
microorganisms. Filamentous fungi can be an important
as well as synthetic effluent phenol removal. All the flasks were source of phenol-degrading species as they grow frequently in
operated at temperature 25C at 125 rpm for five days in wood where phenolic structures are present. Nevertheless
continuous mode. The immobilized cells showed over all better filamentous fungi are not frequently used due to difficulties in
performance as compared to free cells. The highest cell growth their cultivation in liquid media and their slow growth rate in
and the amount of phenol degraded were observed on 5 day. comparison with most of the other microbial species.
average overall pH, Temp., Conductivity, B.O.D, C.O.D, T.S,
T.D.S, T.S.S, Chlorides and phenol were upto 7.5, 34.75C, 39.6 During this oxidation process, contaminants and pollutants
mohs/cm, 139.5 mg/l, 430.5 mg/l, 1490 mg/l, 900 mg/l, 590 mg/l, are broken down into end products such as carbon dioxide,
281.25 mg/l and 268 mg/l respectively. While culture with water, nitrates, sulphates and biomass (microorganisms). The
immobilized cell reached 110 mg/L whereas in free cell it is 119 most efficient Aspergillus Niger is capable of using phenol as
mg/L in industry effluent whereas in synthetic effluent culture the sole source of carbon and energy for cell growth and
with immobilized cell reach 28 mg/L whereas in free cell it is 150
metabolism degrade phenol via metapathway. That is the
mg/L with the same conditions. Reduction in phenol level proved
the biodegradation. Results from this study showed that benzene ring of phenol is dehydroxylated to form catechol
Aspergillus niger has potential to be used in biodegradation of derivative and the ring is then opened through
wastewater containing phenol. meta-oxidation.
Biodegradation is used to describe the complete
Index TermsAspergillus niger, batch culture, mineralization of the starting compound to simpler ones like
Biodegradation, immobilized cells, phenol. CO2, H2O, NO3 and other inorganic compounds. Microbial
degradation of phenol with different initial concentration
ranging from 50-2000mg/L have been actively studied using
I. INTRODUCTION
shake flask, fluidized- bed reactor, continuous stirred tank
Phenol and its derivatives is the basic structural unit in a bioreactor, multi stage bubble column reactor, air lift
wide variety of synthetic organic compounds [1]. Phenol and fermenter and two phase partitioning bioreactor methods
its higher homology are aromatic molecules containing [10]-[16] and these studies have shown that phenol can be
hydroxyl group attached to the benzene ring structure. The aerobically degraded by wide variety of fungi and bacteria
origin of phenol in the environment is both industrial and cultures such as Candida tropicalis [17] and Pseudomonas
natural. Phenol pollution is associated with pulp and paper putida [18].
mills, coal mines, refineries, wood preservation, plants & The efficiency of the phenol degradation could be further
various chemicals industries as well as their wastewaters. Due enhanced by the process of cell immobilization. Various
to their high inhibitory and antibacterial activity, phenols may methods have been described for the immobilization [19].
create problems in the operation of biological treatment plants. Alginates represent however several advantages such as high
They also add odour to drinking and food processing water porosity and chemical stability with a mild, fast, simple and
and have mutagenic and carcinogenic effects [2]-[5]. Phenol low cost immobilization method [20]. Under many conditions,
is also a priority pollutant and is included in the list of EPA immobilized cells have advantages over either free cells or
(1979). immobilized cells. The biodegradation of phenol by
Biological processes using microbial systems provide an immobilized cells had been investigated for several
alternative to the existing physical/ chemical technologies microorganisms but the use of immobilized Aspergillus niger
(expensive and commercially unattractive) because they are cells for phenol biodegradation is scanty. This study has the
more cost-effective, environment friendly and do not produce results obtained from the biodegradation of phenol by free
and immobilized cells of Aspergillus sp. Batch experiments
Manuscript received April 24, 2012; revised June 12, 2102. This work were carried out in order to obtain the maximum phenol
was supported by the Shobhit University, Meerut biodegradation rates by analyzing the influence of the
Neha Sharma was with the Shobhit University, Meerut, India, She is now immobilization in sodium- alginate gel beads on
working with the Department of Applied Sciences in Delhi Institute of Engg.
& Technology, Meerut (e-mail: nehasharma_bio@yahoo.co.in). biodegradation performance.
Vikas. C. Gupta is with Shobhit University, Meerut; India. He is working
with the Department of Biotechnology (e-mail: viky18@gmail.com).

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 International Journal of Chemical Engineering and Applications, Vol. 3, No. 3, June 2012

II. MATERIAL & METHODS were Pseudomonas alkaligenes, Bacillus pumilus, Bacillus
subtilis, Klebsielle sp, Proteus sp. The isolated fungal culture
A. Chemicals and Reagents was identified as Aspergillus niger using Lactophenol cotton
All the chemicals used were of analytical grade and the blue staining method.
chemicals were supplied by Qualigens Fine chemicals
(Mumbai, India).
B. Characterization of Industrial Effluent
The effluent was collected from Anand tissues ltd. (Meerut)
which is a paper industry. The effluent collected from industry,
was analyzed for different physico- chemical properties
(Table I) viz. Temperature, pH, Conductivity, B.O.D, C.O.D,
T.S, T.D.S, T.S.S, Chlorides, Phenol. The concentration of
each of the component was determined as per the procedure
outlined in APHA (2005). [3].

TABLE I: PHYSIC-CHEMICAL PARAMETERS OF

EFFLUENT.

Physico
S.No chemical Test Standard Fig. 1. Aspergillus niger a fungal strain growing on PDA agar medium.
parameters

1 Temp 34.75C _ E. Isolation, Growth and Processing of Cells

2 pH 7.5 - Aspergillus niger previously isolated from paper industry
effluent, and was capable of using phenol as carbon source
39.6 5
3 Conductivity [22]. Strain was maintained on potato dextrose agar (PDA) at
mohs/cm mohs/cm
4C. For mass culturing, liquid broth was used as a culture
4 B.O.D
139.5
30 mg/L medium which was having the following composition (g l-1 ):
mg/L
Dextrose (20 g l-1), Peptone (10 g l-1), NaCl (0.2 g l-1),
430.5
5 C.O.D
mg/L
250 mg/L CaCl2.2H2O (0.1 g l-1), KCl (0.1 g l-1), K2HPO4 (0.5 g l-1),
6 T.S
1490
1200mg/L
NaHCO3 (0.5 g l-1), MgSO4 (0.25g l-1) and FeSO4.7H2O
mg/L (0.005 g l-1). The liquid broth pH was adjusted to 4.5. The
7 T.D.S 900 mg/L 1000mg/L liquid broth was inoculated with a loop of culture grown on
8 TSS 590 mg/L 200 mg/L PDA medium and incubated on an orbital shaker (Orbitek, sci,
281.25
Genics Biotech Ltd) at 125 rpm and 25C for 5 days in 500 ml
9 Chlorides 1000 mg/L conical flasks. The biomass produced was collected by
mg/L
10 Phenol 268 mg/L 1 mg/L
filtration and washed twice with extra pure double distilled
water.

C. Synthetic Effluent
Sterile synthetic effluent composition (mg/l) was proposed
by Passos et al. (2009), [21]: KH2PO4; 200 MgSO4.7H2O;
100 NaCl; 25 Cacl2.2H2O; 3MnSO4.H2O; 500 NH4NO3.H2O;
500 Glucose; Phenol (250 or 500).
D. Isolation and Identification of Predominant
Microorganisms
The sample was serially diluted using sterile pipettes from
10-1 to 10-8 dilution. Five different types of bacteria were
predominant in the raw effluent and single type of fungi was
found to be present. For enumeration of bacteria nutrient agar
medium containing peptone (5 g/L), yeast extract (1.5 g/L),
Fig. 2. Aspergillus niger biomass produced
sodium chloride (5 g/L), agar (15 g/L) was used and for
enumeration of fungi Potato dextrose agar containing potato F. Fungal Spore Immobilization
(200 gm), dextrose (20 gm), agar (15 gm), distilled water Spores were immobilized in calcium alginate according to
(1000 ml) at pH 5.6 was used. To obtain pure culture, the Ellaiah et al. [23]. About 20 ml of sterile sodium alginate
cultures were repeatedly streaked on nutrient agar medium solution (3% W/V) and 5 ml of spore suspension (5 10 6
and incubated at 37C for 24 hrs. The isolated bacteria were spores/ml) were mixed fully and the slurry was dripped into
identified by colony morphology, gram staining, microscopic 0.2 M CaCl2.2H2O solution at room temperature. The beads
observation and confirmation test. The identified bacteria were maintained in CaCl2 solution for 1 hour at 4C and then

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 International Journal of Chemical Engineering and Applications, Vol. 3, No. 3, June 2012

the beds were washed three times with sterile distilled water. conditions in the batch cultivation process, which is crucial
for the objectives of our investigations. The fluctuations of the
number of fungal cells were higher at the 5 day. Growth of
G. Inoculum
Aspergillus niger in industrial wastewater has been shown in
The free and immobilized spores were inoculated in 250 ml Fig. 3. The free cell growth curve has a typical exponential
flasks containing 100 ml of synthetic medium and industry and stationary phase whereas the growth curve of the
effluent and were incubated at 25C for 5 days to spores immobilized cells of Aspergillus niger showed that the cell
germination. under immobilized conditions could make a better growth.
H. Phenol Biodegradation Experiment Immobilized cells in synthetic & industrial wastewater.
Experiments in triplicate were carried out. Flasks The Aspergillus niger cells exhibits diauxic type of growth
containing synthetic and industry effluent were inoculated (Fig. 4) in industrial wastewater. During incubation time of 5
with free and immobilized cultures. Temperature and days it is observed that there is slight increase in the biomass
agitation were set at 25C and 200 rpm on a rotary shaker. but the maximum growth is observed on the fifth day as shown
Control assay (without inoculum) were performed under the in Fig. 4. When the growth was studied on synthetic
same experimental conditions in order to verify abiotic losses. wastewater free cell show slight lag phase whereas
Samples were withdrawn at regular intervals for phenol immobilized cells has a typical exponential phase as shown in
determination. Fig. 4.
Since degradation proceed with biomass growth as shown
in the respective growth curves. It could be seen from Fig. 4.
that the phenol concentration (250 mg/L) was maximum
degraded on fifth day but in the industrial wastewater
maximum degradation was also observed on fifth day (Fig.4).
The control was observed constant throughout the study.

Fig. 3. Phenol biodegradation free by and immobilized cells of

Aspergillus niger

I. Phenol Determination
For Phenol determination, the Folin-Ciocalteau phenol
reagent was used, involving the successive addition of 1ml
sodium carbonate (200mg/l) and 0.5 ml Folin- Ciocalteau
phenol reagent to 10 ml sample. After 60 minutes at 20C, the
absorbance was measured at 725 nm against a distilled water
Fig. 4. Growth kinetics of free and immobilized cells in industrial and
and regent blank [24].
synthetic effluent.
J. Growth Determination
TABLE II: GROWTH KINETICS OF FREE CELLS IN INDUSTRIAL WASTEWATER
The growth determination was done by measuring the
Incubation SYNTHETIC EFFLUENT INDUSTRY EFFLUENT
absorbance at 600 nm using UV-Vis spectrophotometer
time (days) O.D at 600 O.D at 600 O.D at 600 O.D at 600
(Shimadzu).
nm(Free nm(Immobil nm(Free nm(Immobi
cells) ized cells) cells) lized cells)

III. RESULT AND DISCUSSION 0 0.4 0.52 0.84 0.9

A batch cultivation experiment was carried out using 1 1.22 1.42 2.76 2.84
phenol as limiting substrate for Aspergillus niger. Initial 2 1.48 1.82 2.9 3.09
phenol concentration in synthetic effluent was taken as 250 3 1.51 2.01 2.94 3.67
mg/L.
4 1.67 2.22 3.05 3.88
The extent of phenol concentration was investigated for
5 1.76 2.3 3.09 4.01
several batch residence times by intermittent sampling,
showing the biodegradation potential of Aspergillus niger in
degrading synthetic phenol waste. The initial phenol A batch cultivation experiment was carried out using
concentration observed in industrial effluent is 268 mg/L. phenol as single limiting substrate for Aspergillus niger.
The measured absorption at 600 nm gives us valuable Initial phenol concentration of 250 mg/L was used. The extent
information about the biomass accumulation (Table II). of phenol degradation using this initial phenol concentration
Dynamics of cell number has the major importance for was investigated for several batch residence times by
explanation of Aspergillus niger interactions with the intermittent sampling Fig. 3 shows the biodegradation
available substrate. The variation of the concentration of potential of Aspergillus niger in degrading synthetic phenol
microbial cells is lowest at the starting hour of the experiment. waste. Since degradation proceed with biomass growth as
This result confirms the existence of very similar initial shown in the respective growth curves. It could be seen from

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 International Journal of Chemical Engineering and Applications, Vol. 3, No. 3, June 2012

Fig. 5 that the phenol concentration (250 mg/L) was protective cover against toxicity of phenol by forming
maximum degraded on fifth day but in the industrial networks of the beads, a diffusion barrier for phenol is build
wastewater maximum degradation was also observed on fifth up which is not present in free cell culture [17]. The present
day Fig. 6 finding will be useful to treat the waste containing phenol to
After five days of incubation with Aspergillus niger convert the toxicant into nutrient, biomass and CO2 via
tremendous reduction in phenol concentration is observed. biodegradation through their intermediates. This technology
The culture with immobilized cell reached 110 mg/L whereas will be useful to the Paper industry which generates the waste
in free cell it is 119 mg/L. As shown in Table II phenol containing compounds such as phenol. The present
concentration in synthetic effluent initially provided was 250 technology will also be efficient and beneficial to treat the
mg/L, after five days of incubation the culture with waste generated by paper industry.
immobilized cell reach 28 mg/L whereas in free cell it is 150 Our work shows that Aspergillus niger. immobilized cells
mg/L. Immobilize cell of Aspergillus niger results in better in calcium alginate is promising for application in
performance than the free cell in batch process by reducing bio-degradation schemes in order to degrade phenol and
the adaptation and consequently the time for complete phenol possibly other related aromatic compounds at high
biodegradation. concentrations in industry generated wastewater which leads
to a reduction in time for complete phenol removal in relation
to free cells.

IV. CONCLUSION
In the present work the phenol degrading fungi Aspergillus
niger was isolated from industry effluent. Investigated culture
of Aspergillus niger are able to eliminate almost entirely the
introduced quantity of phenol as sole source of carbon and
energy for a period of 5 days. The present finding will be
useful to treat the waste containing phenol to convert the
toxicant into nutrient, biomass and CO2 via biodegradation
through their intermediates. This technology will be useful to
the Paper industry which generates the waste containing
Fig. 5. Comparative analysis of phenol biodegradation by free and compounds such as phenol. The present technology will also
immobilized cells in synthetic wastewater
be efficient and beneficial to treat the waste generated by
paper industry
Our work shows that Aspergillus niger. immobilized cells
in calcium alginate is promising for application in
bio-degradation schemes in order to degrade phenol and
possibly other related aromatic compounds at high
concentrations in industry generated wastewater which leads
to a reduction in time for complete phenol removal in relation
to free cells.
Better biodegradation rate of phenol was observed in
immobilized cells due to absence of internal and external
mass transfer resistance.
An immobilized cell is one of the approaches for
Fig. 6. Comparative analysis of phenol biodegradation by free and
incorporating fungal biomass into an engineering process.
immobilized cells in industrial wastewater The advantage of the process based on immobilized biomass
include enhancing microbial cell stability, allowing
TABLE III: COMPARATIVE ANALYSIS OF PHENOL BIODEGRADATION continuous process operation and avoiding the biomass
Sample Synthetic Industrial liquid separation requirement.
wastewater wastewater The present work open up new dimensions in research in
Phenol Free cell Immobiliz Free cell Immobili the areas of biological treatment of phenolic effluents and also
degradat (O.D at ed cell (O.D at zed cell into the mechanism of phenol biodegradation. The fungus
ion time 725 nm) (O.D at 725 nm) (O.D at used in the present study is an efficient one and can further be
(Days) 725 nm) 725 nm) exploited for industrial scale applications.
1 1.72 2.04 2.8 2.84
2 1.7 1.71 2.22 2.26
3 1.68 1.58 2.14 2.13 ACKNOWLEDGMENT
4 1.64 1.32 1.74 1.91
The authors are grateful to Dr Shobhit kumar,Chancellor,
5 1.46 0.22 1.21 1.18
Rajendra P. Agarwal, Vice Chancellor, Dr D.V
In encapsulated cell culture, the carrier material act as a Rai, ,Head,Department of Biotechnology, Shobhit University,

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Meerut (India) for providing encouragement and laboratory phenol degradation by Pseudomonas aeruginosa and Pseudomonas
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Neha Sharma received her B.Sc, MSc (Biotechnology)
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degree from C.C.S University,Meerut (U.P),India and
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M.Phil(Biotechnology) from Shobhit University ,Meerut
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(U.P),India. Now she is working as Asst Professor in the
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interest includes Environmental Biotechnology. She is
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Vikas Chnadra Gupta received the B.Tech.. degree
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from AIDU, Allahabad and MITS Gwalior, India
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with Department of Biotechnology, Faculty of
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