R. Ravikumar et al.
Int. J. Environ. Sci. Tech., 8 (1), 97-106, Winter 2011
ISSN: 1735-1472
©
IRSEN, CEERS, IAU
*Corresponding Author Email: ravi_cbe1@rediffmail.com
Tel.: +9942 9522 6255; Fax: +9942 9522 3775
Received 13 May 2010; revised 10 August 2010; accepted 20 November 2010; available online 1 December 2010
Single factorial experimental design for decolorizing anaerobically
treated distillery spent wash using cladosporium cladosporioides
1
*
R. Ravikumar;
1
N. S. Vasanthi;
2
K. Saravanan
1
Department of Biotechnology, Bannari Amman Institute of Technology, Sathyamangalam, Erode District - 638 401,
Tamilnadu, India
2
Department of Chemical Engineering, Kongu Engineering College, Perundurai, Erode District -638052, Tamilnadu,
India
ABSTARCT:
This study presents the standardization of nutrient concentration, pH and temperature required to
decolorize the anerobically treated distillery spent wash using the fungus Cladosporium cladosporioides. Experiments
were carried out to measure the decolorization of distillery spent wash effluent and it was found to be effective in acidic
environment. From the results it was observed that a maximum color reduction of 52.6 % and Chemichal Oxygen
Demand. removal of 62.5 % were achieved. The optimum conditions required for the growth of the fungus was found
to be 5 g/L of fructose, 3 g/L of peptone, 5 pH and 35 °C. It was also observed that during the process a maximum of
1.2 g of fungal growth was attained. Decolorizing ability of the fungus was confirmed using spectrophotometer and High
Performance Liquid Chromatography analysis. Single factorial experimental design was used to optimize the parameters.
Apart from decolorization it was observed that fungus also has the ability to degrade the spent wash efficiently. This
investigation could be an approach towards control of environmental pollution and health hazards of people in and
around the distillery unit.
Key words: Biodegradation; Biomass Chemical Oxygen demand; Fructose; Melanoidin
INTRODUCTION
The waste water from molasses based alcohol
distilleries is known as molasses spent wash. When
released in aquatic system, leads to reduction of
sunlight penetration in rivers, lakes and thereby
reducing the photosynthetic activity. Disposal on land
cause reduction in soil alkalinity, manganese
availability and inhibits seed germination (Agarwal, et
al., 1994; FitzGibbon et al., 1995; Ling et al., 2009). In
molasses based distilleries anaerobic treatment is an
accepted practice and various high rate anaerobic
reactor designs have been tried at pilot and full-scale
operation (Lata et al., 2002). However, anaerobic ally
treated effluent still contains high concentrations of
organic pollutants and as such cannot be discharged
directly (Nandy et al., 2002). Spent wash contains about
2 % of dark brown recalcitrant pigment called
melanoidin which has an empirical formula of C
17-18
H
26-
27
O
10
N and molecular weight between 5000 and 40,000
Da. Melanoidins is known as a natural browning
polymer produced by Maillard reaction between amino
and carbonyl groups of organic matters and is closely
related to humic substances in the natural environment.
(Aoshima et al., 1985; Fujita et al., 2000; Manishankar
et al., 2004; Samarghandi et al., 2007). Physicochemical
treatment with active activated carbon was
investigated to removal the color causing components
(mainly melanoidins) from biomethanated distillery
spent wash by (Satyawali, et al., 2008; Saetang and
Babel, 2009 ) but was in effective for application to
large scale as it requires a pretreatment with sulphuric
acid, phosphoric acid, nitric acid and hydrochloric acid
for the adsorbent .However, recently an increasing
attention has been directed towards utilization of
microbial activities for decolorisation of effluents (Pant
and Adholeya, 2007; Nwuche; Ugoji, 2008 and 2010).
Several fungi such as Phanerochaete chrysosporium
JAG-40, Aspergillus species, A.gaisen, P.pinophilum
and Emericella nidulans have been used for the
R. Ravikumar et al.
98
treatment of distillery spent wash (Dahiya et al., 2001;
Thakkar et al., 2006; Pant et al., 2007; Pazouki et al.,
2008; Cuthbertson et al., 2010; Singh et al., 2010).But
these organism were not able to maintain a consistency
in the result and hence not been implemented in the
distillery industries. In order to avoid the drawbacks,
an organism which can be isolated from the same
distillery site and which requires minimum nutrients
for its growth , potiential to decolorize , degrade
melanoidine present in spent wash is needed.
Optimization of process parameters required for the
particular organism for its growth plays a major role in
the degradation process.
It is evident from the report that Cladosporium
cladosporioides has high potential to decolorize two
model dye and also showed maximum decolorization
when tested with 1:2 metal complex acid blue 193 dye.
(Vijayakumar et al., 2006; Antonella Anastasi et al.,
2009; Malakootian M. et al., 2009;
Shah et al., 2009).
Hence in this report an effort was made to use the
fungus, Cladosprium cladosporioides which was
isolated from the spentwash disposal sites of distillery
unit to degrade and decolorize the anerobically treated
distillery spent wash. Since the use of statistics based
experimental design in the optimization of the
biotreatment processes has been well documented
(Annadurai et al., 2008; Madukasi et al., 2010). In this
paper sequential statistics based experimental designs
were applied with single factor to optimize the range of
medium concentration, initial pH and temperature for
obtaining maximum percentage decolorization. So far
no work was carried out using Cladosporium
cladosporioides for decolorizing Anerobically treated
distillery spent wash. This study was carried out at
Bioprocess research laboratory, Department of
Biotechnology, Bannari Amman Institute of
Technology, Sathyamangalam, India during the period
of February 2009 to November 2009.
MATERIALS AND METHODS
Anaerobically Treated Distillery Spent wash (ADSW)
The molasses spent wash after biomethanation from
anaerobic digester was collected aseptically from
distillery division of Bannari Amman Sugars Limited,
Periyapuliyur, Erode District, and Tamilnadu., India. The
spentwash was centrifuged at 4200 Xg for 15 min before
use to remove the suspended solids and stored at 4
°C.( Pazouki et al., 2008) The stored ADSW was filtered
through (What man No: 42) filter paper and was diluted
with deioinized distilled water (Millipore Direct – Q.3
UV). The analysis of different physico-chemical
parameters like Color, odour, Ph, Biochemical oxygen
demand (BOD), Chemical oxygen demand (COD), total
sugars, total dissolved solids (TDS), sulphates,
phosphorous and free chlorine were analysed for
employing standard methods for examination of water
and waste water. (APHA, 1995) and is shown in Table1.
Isolation and Screening of Cladosprium
cladosporioides
Soil sample was collected from the Anerobically
Treated Distillery Spent Wash (ADSW ) disposal sites
of Periyapuliyu. Several organisms were isolated from
the soil sample using serial dilution method and was
then screened for its decolorization ability of ADSW
using Giant Colony and Shake Flask method. (Dahiya
et al., 2001). During the screening process
Cladosprium cladosporioides showed largest zone
of decolorization (Ravikumar et al., 2010) and hence
was selected for this investigation to optimize the
process parameters using single factorial experiment.
Conidal spore structure of Cladosprium
cladosporioides was observed through Light
microscope photomicrograph and was shown in Fig. 1.
The selected strain was allowed to grow separately in
a agar plate containing PDA and was transferred to
slant culture for storing it to long duration. The Potato
dextrose agar medium used was prepared by taking
200 g of peeled and sliced potato with one litre distilled
water which was then subjected to steaming for 30
min. The extract was filtered and made up to a volume
of 1 liter. To this 20 g of dextrose, 0.1 g of yeast extract,
20 g of agar and 1 % diluted ADSW were added
(Chavan, et al., 2006; Mullai et al., 2007). Fungal
Table 1: Physico chemical characteristics ADSW before treatment
Parameters Magnitude
Color Greenish dark brown
Odour Burnt sugar
pH 7.20
Biochemical oxygen demand (BOD)
(mg/L)
5000 -6500
Chemical oxygen demand (COD)
(mg/L)
34,800
Total dissolved solids(TDS) (mg/L) 4500-4620
Sulphates (mg/L) 160
Potassium (mg/L) 850
free chlorine (mg/L) 800
R. Ravikumar et al.
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Int. J. Environ. Sci. Tech., 8 (1), 97-106, Winter 2011
inoculum for the batch process was prepared by
transferring a loopful of strain from the slant into a 250
mL conical flask containing 50 mL Potato dextrose broth
and was incubated in a orbatory shaker at 250 rpm at
30
o
C for 7 days until phase of spores formation was
observed. Reseeding was carried out every 25 days to
maintain the active population. In order to avoid the
extended growth of population 30 %HCl was added to
acidify the medium. Growth medium used for counting
spores alone was prepared with solution and method
(Jimenez et al., 2004). Acclimatization study was carried
out at room temperature with 15 % diluted ADSW as
stock solution which was the dilution made upto optical
density value of maximum percentage decolorization
obtained from the previous work carried out in treating
synthetic melanoidine distillery spent wash It was
performed by gradually exposing Cladosprium
cladosporioides to the higher concentration of ADSW
and was consecutively transferred into the nutrient
medium. During the process nutrient broth
concentration was decreased from 90 % (w/v) to 0 %
(w/v) and finally the organism was provided with spent
wash solution as sole source of nutrient. (Gopinath et
al., 2009).
Experimental
In any microbial decolorization process, medium
components of carbon, nitrogen source, pH and
temperature were considered to be important
Fig. 1: Light microscope photomicrograph of Cladosprium
cladosporioides sowing conidial spore structure
parameters affecting the process. Batch experiment was
conducted in a series of 500 mLconical flask containing
100 mL of 15 % diluted ADSW cultivation medium
supplemented with carbon and nitrogen source. 5 mL
of inoculum (8x10
6
spores,5
th
day) from 250 mL conical
flask was transferred into each flask and was incubated
in a rotary incubator shaker at 250 rpm for 10 days.
Effect of varying the concentration of Fructose
(5,10,15,20 g/L), peptone(1,3,5,7 g/L), pH(4,5,6,7,8) and
temperature (30,35,40,45
o
C) on the percentage
decolorization of the ADSW medium were studied by
single factorial experiment. In each experiment only
one factor, was changed with the other factors
remaining constant. Role of optimization was
confirmed based on the results of biodegradability
index improvement when the process condition was
optimized for tannery effluent. (Aboulhassan et al.,
2004). The initial conditions required for the
experiment was taken from the previous work carried
out in treating synthetic melanoidine distillery spent
wash. A 5mL of the aliquot was withdrawn for every 8
hour time interval and centrifuged for assaying
decolorization for 10 days. Experiment was repeated
with the optimized parameters and COD reduction with
residual color was observed. The observance was
measured as decrease in optical density of
supernatant of treated ADSW medium at 475 nm
(Krishna Prasad et al., 2009; Seyis, et al., 2009) on UV
Spectrophotometer (Perkin Elmer Model: Lambda 35).
R. Ravikumar et al.
100
Decolorizing anaerobically treated distilleryVpent wash using Cladosporium cladosporioides
pH determination was made by using Digital portable
pH meter (Model pt-10P, Sartorius). COD was measured
by using Open Reflux (APHA, 1995). The OD value
was measured and the percentage decolorization was
calculated using the formulae:
% Decolorization = (Initial OD – Final OD)*100/
(Initial OD)
HPLC analysis of treated ADSW
HPLC analysis was carried out at quality control
and assurance laboratory, Centre for
Pharmacognosy and Pharmaceutics FRLHT,
Bangalore, India. Decolorization of ADSW was
monitored by HPLC (Shimadzu). 10 mL of samples
were taken, and centrifuged, filtered through 0.45
µm membrane filter (Millipore). Filtered sample was
analysed using mobile phase consisting of
acetonitryl and methanol (45:55) (HPLC grade ) with
1mL glacial acid and 0.5 mL sodium acetate. (Chavan,
et al.,2006; Ramya, et al.,2007) The sample was
eluted using C-18; reverse phase column of 5 µm
SGE, 250 x 4.6mm SS. Resultant peak was analyzed
with UV –detector 475 nm. The flow rate of the mobile
phase was 1 mL/min.
RESULTS AND DISCUSSION
Effect of fructose concentration on color removal
The influence of fructose on percentage of
decolorization was studied by varying the fructose
concentration from 5 g/L to 20 g/L while other
parameters temperature 35
o
C, 4 pH and1 g/L of
peptone was maintained constant. From Fig. 1 it is
observed that during the initial period of 2 days
there is no significant change in color removal of
the spent wash and maximum of 42.6 %
decolorization was achieved at fructose
concentration of 5 g/L on the 7
th
day and no
significant change on the remaining days. It was
also observed that when the concentration was
increased above 5 g/L concentration the percentage
decolorization decreased. This is due to large mycelia
growth and lignolytic activity of laccase enzyme
produced. Laccase enzyme was tested using guaiacol
and found to be oxidized to form reddish brown zone
similar to the results obtained by (Viswanathan et
al., 2008).Effect of laccase in the degradation of DDT
showed the potential of the enzyme present in the
white rod fungi.(Zhao et al., 2009) Reports reveal
that anaerobically treated distillery spent wash
medium was a better laccase inducer medium than
the mineral media . Laccase production by
Aspergillus heteromorphus was studied using
anaerobically treated distillery spent wash
(ADSW). During the initial phase of growth
organism utilities easily available carbon source
added as medium and later on starts to degrade
spent wash components for carbon source.
Although spent wash contains large amount of
sugar, its easily metabolisable carbon source is
almost negligible. So addition of readily available
external carbon source like fructose is required for
its metabolism. It was reported that the percentage
decolorization of ADSW was highest when glucose
was fed as carbon source with flavodon flavus
isolated from a marine habitat (Raghukumar and
Rivonkar., 2001) In addition to that effect it also
enhanced the degradation of xenophobic
compounds. It was also observed from that
maximum color removal is favored for 5 g/L of
fructose concentration with the biomass increased
to 0.7 g/L till 8th day. Above this concentration
there was no significant change in the percentage
of color removal resulting an inhibiting effect to
the enzyme.
Effect of peptone concentration on color removal
The influence of peptone on percentage of
decolorization was studied by varying the peptone
concentration from 1 g/L to 7 g/L while other parameters
temperature 35
o
C, 4pH and 5 g/L of optimum fructose
concentration was maintained constant
Supplementation of various concentration of peptone
in addition to 5 g/L of fructose and with ADSW medium
was found to be less effective for decolorization and
microbial growth. It was reported that among the
nitrogen source supplemented along with the carbon
source, yeast extract and peptone gave highest growth
with decolorization for culture Phanerochaete
Chrysosporium (Kumar et al., 1998). It is observed from
the Fig. 2 that maximum of 45.6 % decolorization was
achieved with concentration of 1 g/L attaining 0.72 g/L
of biomass. At high concentration there was no
significance in decolourization due to excess
supplementation of nitrogen which inturn leads to
inhibition of the source to fungal growth. Similar
R. Ravikumar et al.
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Int. J. Environ. Sci. Tech., 8 (1), 97-106, Winter 2011
Fig. 2: Effect of fructose concentration on % decolorization at 35
o
C, 4 pH and 1g/L of peptone
Fig. 3: Effect of Peptone concentration on % decolorization at 35
o
C ,4 pH, at 5 g/L of fructose
Decolorization (%)
3 g/L
Time (days)
1 g/L
5 g/L 7 g/L
3 4 5 6 7 8 9 10
50
45
40
35
30
25
20
15
10
5
0
3 4 5 6 7 8 9 10
60
50
40
30
20
10
0
Decolorization (%)
Time (days)
5 g/L
10 g/L
20 g/L
15 g/L
effect was observed when low concentration of
peptone was used as nitrogen source for
decolorizing melanoidine pigment present in spent
wash using Phanerochaete Chrysosporium (Dahiya.
et al., 2001). Laccase production was high on the 7
th
day and thereafter was inhibited. In contrary to the
report by (Viswanathan et al., 2008) that nitrogen
source of peptone was the critical factor for efficient
laccase production .But in this investigation it was
observed that the production was high only at low
concentration of nitrogen source compared to carbon
source.
