Tiwari et al.

BMC Biotechnology 2012, 12:30

http://www.biomedcentral.com/1472-6750/12/30

RESEARCH ARTICLE Open Access

Decolorization of a recalcitrant organic

compound (Melanoidin) by a novel
thermotolerant yeast, Candida tropicalis RG-9
Soni Tiwari1, Rajeeva Gaur1* and Ranjan Singh2

Abstract
Background: Sugarcane distilleries use molasses for ethanol production and generate large volume of effluent
containing high biological oxygen demand (BOD) and chemical oxygen demand (COD) along with melanoidin
pigment. Melanoidin is a recalcitrant compound that causes several toxic effects on living system, therefore, may be
treated before disposal. The aim of this study was to isolate a potential thermotolerant melanoidin decolorizing
yeast from natural resources, and optimized different physico-chemical and nutritional parameters.
Results: Total 24 yeasts were isolated from the soil samples of near by distillery site, in which isolate Y-9 showed
maximum decolorization and identified as Candida tropicalis by Microbial Type Culture Collection (MTCC)
Chandigarh, India. The decolorization yield was expressed as the decrease in the absorbance at 475 nm against
initial absorbance at the same wavelength. Uninoculated medium served as control. Yeast showed maximum
decolorization (75%) at 45°C using 0.2%, glucose; 0.2%, peptone; 0.05%, MgSO4; 0.01%, KH2PO4; pH-5.5 within 24 h
of incubation under static condition. Decolorizing ability of yeast was also confirmed by high performance liquid
chromatography (HPLC) analysis.
Conclusion: The yeast strain efficiently decolorized melanoidin pigment of distillery effluent at higher temperature
than the other earlier reported strains of yeast, therefore, this strain could also be used at industrial level for
melanoidin decolorization as it tolerated a wide range of temperature and pH with very small amount of carbon
and nitrogen sources.
Keywords: Melanoidin, Decolorization, Thermotolerant, Candida tropicalis, Static condition

Background is formed by the reaction between amino acid and

Existing population bang globally urges rise of industrial carbohydrate called “Maillard reaction” [1,3]. These
sectors resulting in pollution of water, air and soil. The highly colored components hinder sunlight penetration
release of pollutants into the environment from various in rivers, lakes or lagoons which inturn decrease both
industries causes hazard to living organisms resulting in photosynthetic activity and dissolved oxygen concentra-
a greater environmental stress. One such industry of fast tion causing harm to aquatic life. Disposal of spentwash
development is the distillery industry. There are more on land is equally hazardous causing a reduction in
than 295 distilleries in India, producing approximately soil pH, inhibition of seed germination and potable
2.7 billion liters of alcohol and releasing 40 billion liters water [4].
of spentwash (distillery effluent) annually [1]. The colored compound in spentwash has antioxidant
Dark brown color of distillery spentwash is mainly due properties and become toxic to all living system includ-
to the presence of organic compound known as mela- ing microorganisms, therefore, must be treated before
noidin [2]. Melanoidin is main content of spentwash and disposal into environment [5,6]; melanoidin can be
removed by several common physico-chemical methods.
* Correspondence: [email protected] Still, these methods require high reagent dosages and
1
Department of Microbiology (Centre of Excellence), Dr. Ram Manohar Lohia
Avadh University, Faizabad 224001Uttar Pradesh, India
generate large amount of sludge [7,8]. Biological meth-
Full list of author information is available at the end of the article ods present an incredible alternate for decolorization/
© 2012 Tiwari et al.; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative
Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and
reproduction in any medium, provided the original work is properly cited.
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degradation of spentwash due to their low cost, environ-

mental friendly and publicly acceptable treatment and
cost-competitive alternative to chemical decomposition
processes [8,9].
A number of biological processes such as bioadsorp-
tion and biodegradation have been reported having pro-
spective application in color removal from spentwash
[10-16]. A wide variety of aerobic microorganisms cap-
able of decolorizing spentwash include bacteria, fungi,
cyanobacteria and yeasts. Some bacterial strains isolated
from sewage and acclimatized on increasing concentra- Figure 1 Selection of efficient culture medium for melanoidin
tions of distillery waste, which were able to reduce decolorization. Decolorization yield ( ), Biomass production
chemical oxygen demand (COD) by 80% in 4–5 days ( ). The inoculated flasks were contained three different
medium at 37°C temperature for 24–48 h at static condition. Error
without any aeration and the major products left after bars presented are mean values of ± standard deviation of triplicates
the degradation process were biomass, carbon dioxide of three independent experiments.
and volatile acids [17]. Raghukumar and Rivonkar [18]
isolated a marine fungus, Flavodon flavus, which was
more effective in decolorizing raw molasses spentwash peptone; 0.05%, MgSO4; 0.05%, K2HPO4 with 3.5 OD ef-
than was the molasses wastewater collected either after fluent) when compared to medium A and C. Medium B
anaerobic treatment or after aerobic treatment. Tondee was found most suitable because this medium could
and Sirianutapiboon [19] isolated Issatchenkia provide more organic form of nitrogen source than
orientalis yeast from fruit sample which showed 60% others. Therefore, nitrogen requirement by the isolate
melanoidin decolorization at 30 °C in 7 days under was higher for better decolorization, this could probably
aerobic condition. by improving metabolic activities for enzyme secretion
In the present investigation, an attempt was made to or the biomass could be promoted. However, medium B
isolate such strain from natural ecosystem which has was selected for optimization of physico-chemical and
ability to grow at higher temperature with minimum ex- nutritional parameters for melanoidin decolorization by
pense of simple sugar and higher percentage of melanoi- yeast strain Y-9 (Figure 1).
din decolorization ability.

Results and discussion Effect of different temperature on melanoidin

Isolation, screening and identification of the isolates decolorization
A total of 24 yeast isolates capable of dye decolorization The influence of temperature regime on melanoidin
were isolated on the GPYE agar medium from the soil of decolorization and biomass production was studied by
distillery near by the Masudha distillery Faizabad, India. varying the temperature from 25°C to 50°C while other
The isolates showing higher clear zone around the col-
ony on GPYE agar were selected for further study (pH
5.5, 24–48 h and 45 °C). The clear zone diameter of
more than 1 cm around the colony was considered as ef-
fective isolates for decolorization (data not shown).
For further study, isolates were inoculated in 50 ml of
medium and incubated at 35°C and 45°C for 24–48 h for
selection of thermotolerant melanoidin decolorizing yeast
individually. Among yeast isolates, higher decolorization
(67%) was shown by yeast isolate Y-9 identified by
MTCC Chandigarh as Candida tropicalis RG-9. How-
ever, this isolate of yeast was separately optimized for
higher decolorization at different medium with varying
contents of carbon, nitrogen sources and their different Figure 2 Effect of different temperature on melanoidin
concentrations. decolorization. Decolorization yield ( ), Biomass production
The effect of medium composition on decolorization ( ). The inoculated flasks were incubated at different
by yeast is clear as mentioned in Figure 1. Yeast strain temperature (°C) for 24–48 h at static condition in medium. Error
bars presented are mean values of ± standard deviation of triplicates
showed higher melanoidin decolorization (67%) in
of three independent experiments.
medium B (0.5%, glucose; 0.2%, yeast extract; 0.3%,
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parameters were maintained constant. From Figure 2 it the decolorization. On contrary Tondee and Sirianuta-
was observed that melanoidin decolorization by yeast piboon. [19] reported 60% decolorization by Issatchen-
strain Y-9 was active at all temperatures employed with kia orientalis, but after 7 days of incubation. In another
maximum decolorization at 40°C to 50°C. It exhibited study Sirianuntapiboon et al. [20] had been reported a
72% decolorization with 5.0 g l−1 biomass production. maximum 68% decolorization using Citeromyces sp. WR-
The remarkable decolorization (72%) in the temperature 43-6 after 7 days of incubation. Therefore, decolorization
range of 40–50°C reveals thermotolerant as well as and growth efficiency of our strain Y-9 is certainly better
mesophilic nature of the yeast strain. Our strain showed than that reported by other researchers.
better decolorization potential at higher temperature
than Sirianuntapiboon et al. [20] who reported a max- Effect of different pH on melanoidin decolorization
imum of 68% spentwash decolorization at 30°C by The influence of pH on melanoidin decolorization and
Citeromyces sp. WR-43-6. Similarly, Tondee and biomass production was studied at varying pH from 4.0
Sirianutapiboon. [19] reported that Issatchenkia orientalis to 7.0 at their optimal temperature and incubation
showing maximum 60% spentwash decolorization at 30 °C. period. Maximum 72% decolorization was achieved at
Further, increase in temperature could not affect the bio- pH 5.5 with 4.95 g l−1 biomass production (Figure 4).
mass production as well as decolorization efficiency by Strain Y-9 exhibited its optimal decolorization at pH 5.5
yeast strain Y-9. According to Cetin and Donmez [21], high and any deviation from optimum level of pH reduced
temperature may cause loss in cell viability or deactivation the melanoidin decolorization activity. The decrease in
of the enzymes responsible for decolorization resulted into decolorization activity was drastic towards more acidic
suppressed decolorizing activity. Therefore, the melanoi- pH leading to no activity at pH 3.0–4.0. Melanoidin
din decolorization and biomass production efficiency of decolorization from other yeast strain was also reported
our strain Y-9 was certainly better than reported by other by several researchers having maximum decolorization
researchers. Thus, it may be suggested that the optimal activity in 5.0–6.0 optimum pH range [19,20]. Several
temperature for melanoidin decolorization depends on the workers have studied that enzymes produced by micro-
variation of microbial strains and their genetic diversity as organism during the decolorization were effective only
they have been isolated from a very wide range of climatic in acidic conditions [22]. An increase in color at higher
conditions. pH was due to the polymerization of melanoidin and
higher rate nutrient utilization [23,24]. Similar results
Effect of different time course on melanoidin have been reported when soil samples were used as
decolorization inoculum instead of isolated organisms [23,25,26].
Time course of melanoidin decolorization was studied Melanoidin decolorization got reduced at above and
alongwith biomass production of yeast strain Y-9. Max- below of this pH due to inhibition of enzyme production
imum decolorization (72%) was achieved in 24 h of incu- as well as activity. All enzymes are protein in nature,
bation with 4.95 g l−1 biomass production (Figure 3). therefore, some proteins denatured at higher or lower pH
Further increase in the incubation period did not increase value. Each microorganism has a specific pH for their

Figure 3 Effect of different incubation periods on melanoidin Figure 4 Effect of different pH on melanoidin decolorization.
decolorization. Decolorization yield ( ), Biomass production Decolorization yield ( ), Biomass production ( ). The
( ). The inoculated flasks were incubated at different incubation inoculated flasks were incubated at different pH at 45°C for 24 h
period at 45°C under static condition in medium. Error bars under static condition in medium. Error bars presented are mean
presented are mean values of ± standard deviation of triplicates of values of ± standard deviation of triplicates of three independent
three independent experiments. experiments.
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growth and enzyme activity in the surrounding environ- Effect of different concentration of glucose on melanoidin
ment. Therefore, the physiological function of yeast varies decolorization
from strain to strain. In another set of the experiment, the effect of various
concentration of glucose (0.1, 0.2, 0.3, 0.4, 0.5 and 0.6%,
Effect of different carbon sources on melanoidin w/v) on melanoidin decolorization and biomass produc-
decolorization tion was also investigated and the results are depicted in
In another approach, the effect of various carbon Figure 6. Melanoidin decolorization by yeast strain Y-9
sources (0.5%, w/v) on melanoidin decolorization and is extraordinarily stable in the presence of all glucose
biomass production was also investigated for 24 h of in- concentrations under study. It was observed that glucose
cubation, and the results are depicted in Figure 5. Mela- concentration above 0.3% (w/v), decreased the melanoi-
noidin decolorization by yeast strain Y-9 is din decolorization. From Figure 6 it was observed that
extraordinarily stable in the presence of all carbon maximum 75% decolorization with 5.0 g l−1 biomass pro-
sources under study. It was observed that except lactose, duction was achieved at 0.2% (w/v) glucose concentra-
presence of other carbon sources enhanced the melanoi- tion, above this concentration decolorization reduced
din decolorization when compared to control (without and biomass was slightly increased. This effect can be
carbon source). With control (without carbon source), explained as during initial phase of growth organism uti-
sucrose, glucose, fructose and starch, yeast strain Y-9 lizes easily available carbon sources added to the medium
showed 70, 72, 74, 73, and 72% decolorization, respect- and then starts to degrade spentwash components for car-
ively. The presence of lactose marginally reduced bon source [11]. Tondee and Sirianutapiboon. [19] have
decolorization. From Figure 5 it was observed that also reported that Issatchenkia orientalis utilized 2.5%
higher decolorization (74%) and biomass (5.0 g l−1) was glucose for maximum decolorization (60%) and above this
reported by glucose when compared to control (without concentration of glucose there was decrease in the
sugar), while fructose favored the decolorization. Mela- decolorization. Similarlly, Sirianuntapiboon et al. [20] have
noidin decolorization from other yeast are maximum in reported that Citeromyces sp. WR-43-6 showed 68%
the presence of glucose have reported by others decolorization in the presence of 2.0% glucose concentra-
researchers also [19,20]. Watanabe et al. [27] have tion. Ohmomo et al. [10] have reported that glucose was
reported the enzymatic degradation of melanoidin by the best carbon source, which utilized by Aspergillus
Coriolus sp. No. 20 having an intracellular enzyme, fumigatus G-2-6 for maximum degradation of melanoidin
which required active oxygen molecules and sugars (sor- and further increase in glucose concentration resulted in an
bose as well as glucose) in reaction mixture, was later increase in mycelial biomass but no further increase in
identified as sorbose oxidase which oxidize glucose into decolorization yield. It is, therefore, evident from our study
gluconic acid. It is, therefore, evident from our study that melanoidin decolorization by yeast strain Y-9 is
that melanoidin decolorization by yeast strain Y-9 is re- remarkably higher in the presence of 0.2% (w/v) glucose
markably stable in the presence of broad range of carbon within 24 h of incubation when compared to other
sources employed in this study. researchers.

Figure 5 Effect of different carbon sources on melanoidin Figure 6 Effect of different glucose concentration on
decolorization. Decolorization yield ( ), Biomass production melanoidin decolorization. Decolorization yield ( ), Biomass
( ). The control flask does not contain any carbon sources. Test production ( ). The control flask does not contain glucose. Test
flasks contained different carbon sources in the medium at a level of flasks contained different concentration of glucose in the medium at
0.5% (w/v). Inoculated flasks were incubated at 45°C for 24 h. Error a level of 0.6% (w/v). Inoculated flasks were incubated at 45°C for
bars presented are mean values of ± standard deviation of triplicates 24 h. Error bars presented are mean values of ± standard deviation
of three independent experiments. of triplicates of three independent experiments.
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Effect of different nitrogen sources on melanoidin w/v) on melanoidin decolorization and biomass produc-
decolorization tion was also investigated and the results are depicted in
The influence of various organic and inorganic nitrogen Figure 8. Melanoidin decolorization by yeast strain Y-9
sources (0.5%, w/v) on melanoidin decolorizatin and bio- is extremely stable in the presence of all peptone con-
mass production was also investigated for 24 h of incu- centrations under study. It was observed that peptone
bation, and the results are depicted in Figure 7. concentration above 0.3% (w/v), decreased the melanoi-
Melanoidin decolorization by yeast strain Y-9 is extraor- din decolorization. From Figure 8 it was observed that
dinarily stable in the presence of all nitrogen sources maximum 75% decolorization with 5.0 g l−1 biomass
under study. It was observed that except sodium nitrate production was achieved at 0.2% (w/v) peptone
and beef extract, presence of other nitrogen sources concentration, above this concentration reduced
enhanced the melanoidin decolorization when compared decolorization. Similarly Ravikumar et al. [26] have also
to control (without nitrogen source). From Figure 7 it reported that cladosporium cladosporioides showed
observed that yeast strain showed higher 75% maximum decolorization at low concentration of pep-
decolorization with 4.95 g l−1 biomass production in the tone (1.0 g l−1) because at high concentration there was
presence of peptone, it was practically high compared to no significance in decolorization due to surplus supple-
the extent of decolorization reported by other workers mentation of nitrogen which inhibition the growth.
[8]. Sirianuntapiboon et al. [28] have reported that yeast Similar effect was observed when low concentration of
extract and peptone inducing decolorizing activity in peptone was used as nitrogen source by Phanerochaete
acetogenic bacterium strain No. BP103. But in case of Chrysosporium for decolorization of melanoidin pig-
Issatchenkia orientalis and Citeromyces sp. WR-43-6, ment present in spentwash [5]. It is, therefore, evident
maximum decolorization was reported in the presence from our study that melanoidin decolorization by yeast
of 0.1% NH4Cl and 0.1% sodium nitrate [19,20]. Kirk et strain Y-9 is remarkably higher in the presence of 0.2%
al. [29] have reported that enzymatic systems catalyze (w/v) peptone within 24 h of incubation. This culture uti-
degradation of lignin and lignin-like materials during the lized little amount of peptone for higher melanoidin
secondary phase of the metabolic growth in the presence decolorization compared to other researchers ever reported.
of peptone. Synthesis and secretion of lignin peroxidase
or ligninase (LiP) and manganese-dependent peroxidase HPLC analysis
(MnP) are triggered by nutrient limitations such as car- The HPLC analysis report representing the area, height,
bon and nitrogen sources. retention time, before and after the treatment of spent-
wash (Figure 9a and b) which confirms the biodegradation
Effect of different concentration of peptone on of melanoidin, main compound/pigment responsible for
melanoidin decolorization dark brown color of distillery effluent. A major peak
In another set of the experiment, the effect of various appeared at a retention time of 2.60 min in treated sample
concentration of peptone (0.1, 0.2, 0.3, 0.4, 0.5 and 0.6%, which was less compared to untreated and clearly indi-
cates the ability of the yeast to decolorize/degrade the spent-
wash. The reduction in physico-chemical characteristics

Figure 7 Effect of different nitrogen sources on melanoidin Figure 8 Effect of different peptone concentration on
decolorization. Decolorization yield ( ), Biomass production melanoidin decolorization. Decolorization yield ( ), Biomass
( ). The control flask does not contain any nitrogen sources. production ( ). The control flask does not contain peptone.
Test flasks contained different nitrogen sources in the medium at a Test flasks contained different concentration of peptone in the
level of 0.5% (w/v). Inoculated flasks were incubated at 45°C for 24 medium at a level of 0.6% (w/v). Inoculated flasks were incubated at
h. Error bars presented are mean values of ± standard deviation of 45°C for 24 h. Error bars presented are mean values of ± standard
triplicates of three independent experiments. deviation of triplicates of three independent experiments.
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Figure 9 HPLC analysis for monitoring the spentwash decolorization. 9(A) HPLC analysis for distillery spentwash before treatment showing
a maximum peak with 2.712 retention time, 3916140 area and 276697 height. 9(B) HPLC analysis for distillery spentwash after treatment showing
a maximum peak with 2.601 retention time, 1253879 area and 84452 height.

may be due to degradation of melanoidin in the presence of Isolation, screening and identification of melanoidin-
carbon and nitrogen sources through metabolism. decolorizing yeast
Melanoidin decolorizing yeast isolated from soil sample
collected from distillery was grown on GPYE agar
Conclusion medium for 24 to 48 h incubation. Culture medium
The thermotolerant Candida tropicalis has ability to consisted of 0.2%, K2HPO4; 0.1%, KH2PO4; 0.01%,
decolorized complex melanoidin compound at wide MgSO4.12H2O; 0.5%, glucose and 0.1%, yeast extract
range of temperature and pH in presence of little
amount of carbon and nitrogen sources within a short Table 1 Physico-chemical properties of distillery effluent
incubation period of 24 h. This strain has ability to re- (spentwash)
duce environment pollution by decolorizing melanoidin Parameters Value of distillery effluent
pigment with cost effective and eco-friendly nature. Color Dark brown
Odour Like molasses

Materials and methods Temperature °C 82

Distillery spent wash (DSW) pH 4.2
The molasses spent wash was collected aseptically from Total dissolved solid (mg l−1) 81733
Masuadha sugarcane distillery, faizabad U.P., India. The Total suspended solid (mg l−1) 5933
spentwash was centrifuged at 10,000 rpm for 15 min be-
Dissolved oxygen (mg l−1) 0
fore use to remove the suspended solids and stored at 4°C. −1
Biological oxygen demand (mg l ) 46666
The stored distillery spentwash was filtered through
(Whatman No: 1) filter paper and was diluted with dis- Chemical oxygen demand (mg l−1) 104130
tilled water [25]. The analysis of different physico-chem- Total nitrogen (mg l−1) 1635
ical parameters like color, odor, pH, biological oxygen Phosphorus (mg l−1) 163
demand (BOD), chemical oxygen demand (COD), total Potassium (mg l ) −1
8766
sugars, total dissolved solids (TDS), sulphates, phosphor-
Sodium (mg l−1) 211
ous and calcium were analyzed for employing standard −1
Calcium (mg l ) 1816
methods for examination of water and wastewater [30]
and is shown in Table 1. Sulphate (mg l−1) 1738
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with 3.5 OD effluent and the initial pH was adjusted to potential of the isolate. Three types of media with differ-
5.5. In order to isolate molasses decolorizing yeast, 1.0 g ent composition were used to evaluate.
of soil was serially dilution upto 10−5 to 10−6 and placed
in Petri plates along with the GPYE agar medium. The Medium A: - Distillery effluent without carbon and
plates were subsequently incubated for 24–48 h at nitrogen supplemented medium with 3.5 OD.
35 ± 2°C and 45 ± 2°C for thermotolerant yeast. After Medium B:- 0.5%, glucose; 0.2%, yeast extract; 0.3%,
24–48 h of incubation, decolorization efficiency was peptone; 0.05%, MgSO4; 0.05%, K2HPO4 with 3.5 OD
recorded visually. The isolates showing more decolorization effluent.
of the melanoidin were selected for further studies, main- Medium C: - 0.6%, glucose; 0.5%, peptone; 0.05%,
tained on the same medium at 4°C in slants, and sub- MnSO4; 0.05%, K2HPO4 with 3.5 OD effluent
cultured after two weeks. These cultures were identified respectively.
at genus and species level by Institute of Microbial
Technology (IMTECH) MTCC Chandigarh, India. Selection of Physico-chemical and nutritional parameters
for melanoidin decolorization
Inoculum preparation Optimization of experimental conditions
Mother culture was prepared by inoculating one full The various process parameters influencing melanoidin
loop of 24 h grown culture on basal agar plate in 50 ml decolorization and biomass production by fermentation
basal broth, and incubated at 37°C for 24 h to achieve were optimized individually and independently of the
active exponential phase consisting of 50x106 cfu ml−1 others, therefore, the optimized conditions were subse-
population. Appropriate volume (0.5%, v/v) of this cell quently used in all the experiments in sequential order.
suspension was used to inoculate the test flasks. For optimization, the basal medium contained glucose
0.5%; peptone 0.2%; yeast extract 0.3%; K2HPO4 0.05%
Decolorization assay and MgSO4 0.05% with 3.5 OD spentwash at pH −5.5
The melanoidin decolorizing yeast was inoculated in the was used for inoculation with 0.5% (v/v) of yeast culture
GPYE broth medium and after incubation; broth was having 50x106 cfu ml-1 and then incubated for different
centrifuged at 10,000 rpm for 10 min. The supernatant periods viz. 8, 16, 24, 32, 40 and 48 h at different
of the centrifuged sample was read at absorbance max- temperature viz. 25, 30, 35, 40, 45 and 50°C. For mela-
imum (Amax) of the melanoidin i.e. 475 nm using noidin decolorization all the experiments were carried
spectrophotometer [31]. The decolorization yield was out under static. Initial pH also plays an important role
expressed as the decrease in the absorbance at 475 nm in melanoidin decolorization and biomass production,
against initial absorbance at the same wavelength. Unin- so pH of medium was adjusted to 4.0, 4.5, 5.0, 5.5, 6.0,
oculated medium served as control. The entire assays 6.5 and 7.0 using either 1 N HCl or 1 N NaOH. For the
were performed in triplicate and compared with control. optimal melanoidin decolorization and biomass produc-
The decolorization efficiency of the isolate was expressed tion, the strains may require additional carbon and ni-
as per following equation: trogen sources with varying concentrations in its growth
media. Therefore, the growth medium was supplemen-
Decolorization ð%Þ ¼ I  F=I ted with the carbon sources viz. glucose, fructose, su-
crose, maltose, lactose and starch (at the level of 0.5%,
Where, I = Initial absorbance (Control) and F = Ab- w/v) and nitrogen sources viz. ammonium sulphate,
sorbance of decolorized medium broth. yeast extract, peptone, beef extract, malt extract, sodium
nitrate, and sodium nitrite (at the level of 0.5%, w/v).
Biomass determination Thereafter, optimized carbon and nitrogen sources were
Yeast cells in broth were collected by centrifugation further optimized at different concentration (0.1 to 0.6%,
(10,000 rpm for 10 min at 4°C), washed with distilled w/v). The fermentation medium was sterilized at 121°C
water, and dried in an oven at 80°C until getting a con- for 15 min and incubation was done at 45°C with all the
stant dried weight reported in the form of dry cell mass other conditions at the optimal levels determined previously.
(g l−1).
HPLC analysis of spentwash
Selection of efficient medium for melanoidin Decolorization of melanoidin (spentwash) was moni-
decolorization tored by HPLC (Shimadzu). 10 ml of samples were
An experiment was conducted to select a suitable taken, and centrifuged, filtered through 0.45 μm mem-
medium for efficient decolorization by the yeast strain. brane filter (Millipore). Filtered sample was analyzed
The medium having various combinations of glucose, using mobile phase consisting acetonitryl and methanol
peptone and effluent was used to evaluate decolorization (45:55) (HPLC grade) with 1 ml glacial acid and 0.5 ml
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Competing interests
the white-rot fungus Flavodon flavus, isolated from a marine habitat.
The author(s) declare that they have no competing interests.
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Authors’ contributions
activity in yeast strain. Int Conf Environ 2006, 99:5511–5519.
ST carried out the research work and drafted the manuscript. RS was
20. Sirianuntapiboon S, Zohsalam P, Ohmomo S: Decolorization of molasses
involved in revising the manuscript critically for important intellectual
wastewater by Citeromyces sp. WR-43-6. Process Biochem 2004, 39:917–924.
content. RG has designed the experiment, contributed substantially to
21. Cetin D, Donmez G: Decolorization of reactive dyes by mixed cultures isolated
analysis and interpretation of data and have given final approval of the
from textile effluent under anaerobic conditions. Enzym Microbial Technol 2006,
version to be published. All authors read and approved the final manuscript.
38:926–930.
22. Seyis I, Subasing T: Screeming of different fungi for decolorization of
Acknowledgement molasses. Brazilian J Microbiol 2009, 40:61–65.
Financial assistance to the author (Soni Tiwari, Rajeeva Gaur and Ranjan 23. Adikane HV, Dange MN, Selvakumari K: Optimization of anaerobically
Singh) by Council of Science and Technology, U.P., in the form of Major digested distillery molasses spent wash decolorization using soil as
Research Project is thankfully acknowledged. inoculum in the absence of additional carbon and nitrogen source.
Biores Technol 2006, 97:2131–2135.
Author details 24. Jiranuntipon S, Chareonpornwattana S, Damronglerd S, Albasi C, Delia ML:
1
Department of Microbiology (Centre of Excellence), Dr. Ram Manohar Lohia Decolorization of synthetic Melanoidins-Containing Wastewater by a
Avadh University, Faizabad 224001Uttar Pradesh, India. 2Amity Institute of Bacterial Consortium. Ind Microbiol Biotechnol 2008, 35:1313–1321.
Microbial Biotechnology, Amity University, NoidaG. B. NagarUttar Pradesh, 25. Pazouki M, Shayegan J, Afshari A: Screening of microorganisms for
India. decolorization of treated distillery wastewater. Iran J Sci Technol 2008,
32:53–60.
Received: 6 March 2012 Accepted: 22 May 2012 26. Ravikumar R, Vasanthi NS, Saravanan K: Single factorial experimental
Published: 18 June 2012 design for decolorizing anaerobically treated distillery spent wash using
cladosporium cladosporioides. Int J Environ Sci Tech 2011, 8:97–106.
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