- Research article
- Open Access
Decolorization of a recalcitrant organic compound (Melanoidin) by a novel thermotolerant yeast, Candida tropicalis RG-9
© Tiwari et al.; licensee BioMed Central Ltd. 2012
- Received: 6 March 2012
- Accepted: 22 May 2012
- Published: 18 June 2012
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.
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.
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.
- Candida tropicalis
- Static condition
Existing population bang globally urges rise of industrial sectors resulting in pollution of water, air and soil. The release of pollutants into the environment from various industries causes hazard to living organisms resulting in a greater environmental stress. One such industry of fast development is the distillery industry. There are more than 295 distilleries in India, producing approximately 2.7 billion liters of alcohol and releasing 40 billion liters of spentwash (distillery effluent) annually .
Dark brown color of distillery spentwash is mainly due to the presence of organic compound known as melanoidin . Melanoidin is main content of spentwash and is formed by the reaction between amino acid and carbohydrate called “Maillard reaction” [1, 3]. These highly colored components hinder sunlight penetration in rivers, lakes or lagoons which inturn decrease both photosynthetic activity and dissolved oxygen concentration causing harm to aquatic life. Disposal of spentwash on land is equally hazardous causing a reduction in soil pH, inhibition of seed germination and potable water .
The colored compound in spentwash has antioxidant properties and become toxic to all living system including microorganisms, therefore, must be treated before disposal into environment [5, 6]; melanoidin can be removed by several common physico-chemical methods. Still, these methods require high reagent dosages and generate large amount of sludge [7, 8]. Biological methods present an incredible alternate for decolorization/degradation of spentwash due to their low cost, environmental friendly and publicly acceptable treatment and cost-competitive alternative to chemical decomposition processes [8, 9].
A number of biological processes such as bioadsorption and biodegradation have been reported having prospective application in color removal from spentwash [10–16]. A wide variety of aerobic microorganisms capable of decolorizing spentwash include bacteria, fungi, cyanobacteria and yeasts. Some bacterial strains isolated from sewage and acclimatized on increasing concentrations of distillery waste, which were able to reduce chemical oxygen demand (COD) by 80% in 4–5 days without any aeration and the major products left after the degradation process were biomass, carbon dioxide and volatile acids . Raghukumar and Rivonkar  isolated a marine fungus, Flavodon flavus, which was more effective in decolorizing raw molasses spentwash than was the molasses wastewater collected either after anaerobic treatment or after aerobic treatment. Tondee and Sirianutapiboon  isolated Issatchenkia orientalis yeast from fruit sample which showed 60% melanoidin decolorization at 30 °C in 7 days under aerobic condition.
In the present investigation, an attempt was made to isolate such strain from natural ecosystem which has ability to grow at higher temperature with minimum expense of simple sugar and higher percentage of melanoidin decolorization ability.
Isolation, screening and identification of the isolates
A total of 24 yeast isolates capable of dye decolorization were isolated on the GPYE agar medium from the soil of distillery near by the Masudha distillery Faizabad, India. The isolates showing higher clear zone around the colony 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 effective 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. However, this isolate of yeast was separately optimized for higher decolorization at different medium with varying contents of carbon, nitrogen sources and their different concentrations.
Effect of different temperature on melanoidin decolorization
Effect of different time course on melanoidin decolorization
Effect of different pH on melanoidin decolorization
Effect of different carbon sources on melanoidin decolorization
Effect of different concentration of glucose on melanoidin decolorization
Effect of different nitrogen sources on melanoidin decolorization
Effect of different concentration of peptone on melanoidin decolorization
The thermotolerant Candida tropicalis has ability to decolorized complex melanoidin compound at wide range of temperature and pH in presence of little amount of carbon and nitrogen sources within a short incubation period of 24 h. This strain has ability to reduce environment pollution by decolorizing melanoidin pigment with cost effective and eco-friendly nature.
Distillery spent wash (DSW)
Physico-chemical properties of distillery effluent (spentwash)
Value of distillery effluent
Total dissolved solid (mg l−1)
Total suspended solid (mg l−1)
Dissolved oxygen (mg l−1)
Biological oxygen demand (mg l−1)
Chemical oxygen demand (mg l−1)
Total nitrogen (mg l−1)
Phosphorus (mg l−1)
Potassium (mg l−1)
Sodium (mg l−1)
Calcium (mg l−1)
Sulphate (mg l−1)
Isolation, screening and identification of melanoidin-decolorizing yeast
Melanoidin decolorizing yeast isolated from soil sample collected from distillery was grown on GPYE agar medium for 24 to 48 h incubation. Culture medium consisted of 0.2%, K2HPO4; 0.1%, KH2PO4; 0.01%, MgSO4.12H2O; 0.5%, glucose and 0.1%, yeast extract with 3.5 OD effluent and the initial pH was adjusted to 5.5. In order to isolate molasses decolorizing yeast, 1.0 g of soil was serially dilution upto 10−5 to 10−6 and placed in Petri plates along with the GPYE agar medium. The plates were subsequently incubated for 24–48 h at 35 ± 2°C and 45 ± 2°C for thermotolerant yeast. After 24–48 h of incubation, decolorization efficiency was recorded visually. The isolates showing more decolorization of the melanoidin were selected for further studies, maintained on the same medium at 4°C in slants, and sub-cultured after two weeks. These cultures were identified at genus and species level by Institute of Microbial Technology (IMTECH) MTCC Chandigarh, India.
Mother culture was prepared by inoculating one full loop of 24 h grown culture on basal agar plate in 50 ml basal broth, and incubated at 37°C for 24 h to achieve active exponential phase consisting of 50x106 cfu ml−1 population. Appropriate volume (0.5%, v/v) of this cell suspension was used to inoculate the test flasks.
Where, I = Initial absorbance (Control) and F = Absorbance of decolorized medium broth.
Yeast cells in broth were collected by centrifugation (10,000 rpm for 10 min at 4°C), washed with distilled water, and dried in an oven at 80°C until getting a constant dried weight reported in the form of dry cell mass (g l−1).
Selection of efficient medium for melanoidin decolorization
An experiment was conducted to select a suitable medium for efficient decolorization by the yeast strain. The medium having various combinations of glucose, peptone and effluent was used to evaluate decolorization potential of the isolate. Three types of media with different composition were used to evaluate.
Medium A: - Distillery effluent without carbon and nitrogen supplemented medium with 3.5 OD.
Medium B:- 0.5%, glucose; 0.2%, yeast extract; 0.3%, peptone; 0.05%, MgSO4; 0.05%, K2HPO4 with 3.5 OD effluent.
Medium C: - 0.6%, glucose; 0.5%, peptone; 0.05%, MnSO4; 0.05%, K2HPO4 with 3.5 OD effluent respectively.
Selection of Physico-chemical and nutritional parameters for melanoidin decolorization
Optimization of experimental conditions
The various process parameters influencing melanoidin decolorization and biomass production by fermentation were optimized individually and independently of the others, therefore, the optimized conditions were subsequently used in all the experiments in sequential order. For optimization, the basal medium contained glucose 0.5%; peptone 0.2%; yeast extract 0.3%; K2HPO4 0.05% and MgSO4 0.05% with 3.5 OD spentwash at pH −5.5 was used for inoculation with 0.5% (v/v) of yeast culture having 50x106 cfu ml-1 and then incubated for different periods viz. 8, 16, 24, 32, 40 and 48 h at different temperature viz. 25, 30, 35, 40, 45 and 50°C. For melanoidin decolorization all the experiments were carried out under static. Initial pH also plays an important role in melanoidin decolorization and biomass production, so pH of medium was adjusted to 4.0, 4.5, 5.0, 5.5, 6.0, 6.5 and 7.0 using either 1 N HCl or 1 N NaOH. For the optimal melanoidin decolorization and biomass production, the strains may require additional carbon and nitrogen sources with varying concentrations in its growth media. Therefore, the growth medium was supplemented with the carbon sources viz. glucose, fructose, sucrose, maltose, lactose and starch (at the level of 0.5%, w/v) and nitrogen sources viz. ammonium sulphate, yeast extract, peptone, beef extract, malt extract, sodium nitrate, and sodium nitrite (at the level of 0.5%, w/v). Thereafter, optimized carbon and nitrogen sources were further optimized at different concentration (0.1 to 0.6%, w/v). The fermentation medium was sterilized at 121°C for 15 min and incubation was done at 45°C with all the other conditions at the optimal levels determined previously.
HPLC analysis of spentwash
Decolorization of melanoidin (spentwash) was monitored by HPLC (Shimadzu). 10 ml of samples were taken, and centrifuged, filtered through 0.45 μm membrane filter (Millipore). Filtered sample was analyzed using mobile phase consisting acetonitryl and methanol (45:55) (HPLC grade) with 1 ml glacial acid and 0.5 ml sodium acetate [26, 32]. The sample was eluted using C-18; reverse phase column of 5 μm SGE, 250 x 4.6 mm SS. Resultant peak was analyzed with UV–detector 475 nm. The flow rate of the mobile phase was 1 ml min−1.
All experiments were carried out in triplicates and the results are presented as the mean of three independent observations. Standard deviation for each experimental result was calculated using Microsoft Excel.
Financial assistance to the author (Soni Tiwari, Rajeeva Gaur and Ranjan Singh) by Council of Science and Technology, U.P., in the form of Major Research Project is thankfully acknowledged.
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