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.
The effect of medium composition on decolorization by yeast is clear as mentioned in Figure 1. Yeast strain showed higher melanoidin decolorization (67%) in medium B (0.5%, glucose; 0.2%, yeast extract; 0.3%, peptone; 0.05%, MgSO4; 0.05%, K2HPO4 with 3.5 OD effluent) when compared to medium A and C. Medium B was found most suitable because this medium could provide more organic form of nitrogen source than others. Therefore, nitrogen requirement by the isolate was higher for better decolorization, this could probably by improving metabolic activities for enzyme secretion or the biomass could be promoted. However, medium B was selected for optimization of physico-chemical and nutritional parameters for melanoidin decolorization by yeast strain Y-9 (Figure 1).
Effect of different temperature on melanoidin decolorization
The influence of temperature regime on melanoidin decolorization and biomass production was studied by varying the temperature from 25°C to 50°C while other parameters were maintained constant. From Figure 2 it was observed that melanoidin decolorization by yeast strain Y-9 was active at all temperatures employed with maximum decolorization at 40°C to 50°C. It exhibited 72% decolorization with 5.0 g l−1 biomass production. The remarkable decolorization (72%) in the temperature range of 40–50°C reveals thermotolerant as well as mesophilic nature of the yeast strain. Our strain showed better decolorization potential at higher temperature than Sirianuntapiboon et al. [20] who reported a maximum of 68% spentwash decolorization at 30°C by Citeromyces sp. WR-43-6. Similarly, Tondee and Sirianutapiboon. [19] reported that Issatchenkia orientalis showing maximum 60% spentwash decolorization at 30 °C. Further, increase in temperature could not affect the biomass production as well as decolorization efficiency by yeast strain Y-9. According to Cetin and Donmez [21], high temperature may cause loss in cell viability or deactivation of the enzymes responsible for decolorization resulted into suppressed decolorizing activity. Therefore, the melanoidin decolorization and biomass production efficiency of our strain Y-9 was certainly better than reported by other researchers. Thus, it may be suggested that the optimal temperature for melanoidin decolorization depends on the variation of microbial strains and their genetic diversity as they have been isolated from a very wide range of climatic conditions.
Effect of different time course on melanoidin decolorization
Time course of melanoidin decolorization was studied alongwith biomass production of yeast strain Y-9. Maximum decolorization (72%) was achieved in 24 h of incubation with 4.95 g l−1 biomass production (Figure 3). Further increase in the incubation period did not increase the decolorization. On contrary Tondee and Sirianutapiboon. [19] reported 60% decolorization by Issatchenkia orientalis, but after 7 days of incubation. In another study Sirianuntapiboon et al. [20] had been reported a maximum 68% decolorization using Citeromyces sp. WR-43-6 after 7 days of incubation. Therefore, decolorization and growth efficiency of our strain Y-9 is certainly better than that reported by other researchers.
Effect of different pH on melanoidin decolorization
The influence of pH on melanoidin decolorization and biomass production was studied at varying pH from 4.0 to 7.0 at their optimal temperature and incubation period. Maximum 72% decolorization was achieved at pH 5.5 with 4.95 g l−1 biomass production (Figure 4). Strain Y-9 exhibited its optimal decolorization at pH 5.5 and any deviation from optimum level of pH reduced the melanoidin decolorization activity. The decrease in decolorization activity was drastic towards more acidic pH leading to no activity at pH 3.0–4.0. Melanoidin decolorization from other yeast strain was also reported by several researchers having maximum decolorization activity in 5.0–6.0 optimum pH range [19, 20]. Several workers have studied that enzymes produced by microorganism during the decolorization were effective only in acidic conditions [22]. An increase in color at higher pH was due to the polymerization of melanoidin and higher rate nutrient utilization [23, 24]. Similar results have been reported when soil samples were used as inoculum instead of isolated organisms [23, 25, 26]. Melanoidin decolorization got reduced at above and below of this pH due to inhibition of enzyme production as well as activity. All enzymes are protein in nature, therefore, some proteins denatured at higher or lower pH value. Each microorganism has a specific pH for their growth and enzyme activity in the surrounding environment. Therefore, the physiological function of yeast varies from strain to strain.
Effect of different carbon sources on melanoidin decolorization
In another approach, the effect of various carbon sources (0.5%, w/v) on melanoidin decolorization and biomass production was also investigated for 24 h of incubation, and the results are depicted in Figure 5. Melanoidin decolorization by yeast strain Y-9 is extraordinarily stable in the presence of all carbon sources under study. It was observed that except lactose, presence of other carbon sources enhanced the melanoidin decolorization when compared to control (without carbon source). With control (without carbon source), sucrose, glucose, fructose and starch, yeast strain Y-9 showed 70, 72, 74, 73, and 72% decolorization, respectively. The presence of lactose marginally reduced decolorization. From Figure 5 it was observed that higher decolorization (74%) and biomass (5.0 g l−1) was reported by glucose when compared to control (without sugar), while fructose favored the decolorization. Melanoidin decolorization from other yeast are maximum in the presence of glucose have reported by others researchers also [19, 20]. Watanabe et al. [27] have reported the enzymatic degradation of melanoidin by Coriolus sp. No. 20 having an intracellular enzyme, which required active oxygen molecules and sugars (sorbose as well as glucose) in reaction mixture, was later identified as sorbose oxidase which oxidize glucose into gluconic acid. It is, therefore, evident from our study that melanoidin decolorization by yeast strain Y-9 is remarkably stable in the presence of broad range of carbon sources employed in this study.
Effect of different concentration of glucose on melanoidin decolorization
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%, w/v) on melanoidin decolorization and biomass production was also investigated and the results are depicted in Figure 6. Melanoidin decolorization by yeast strain Y-9 is extraordinarily stable in the presence of all glucose concentrations under study. It was observed that glucose concentration above 0.3% (w/v), decreased the melanoidin decolorization. From Figure 6 it was observed that maximum 75% decolorization with 5.0 g l−1 biomass production was achieved at 0.2% (w/v) glucose concentration, above this concentration decolorization reduced and biomass was slightly increased. This effect can be explained as during initial phase of growth organism utilizes easily available carbon sources added to the medium and then starts to degrade spentwash components for carbon source [11]. Tondee and Sirianutapiboon. [19] have also reported that Issatchenkia orientalis utilized 2.5% glucose for maximum decolorization (60%) and above this concentration of glucose there was decrease in the decolorization. Similarlly, Sirianuntapiboon et al. [20] have reported that Citeromyces sp. WR-43-6 showed 68% decolorization in the presence of 2.0% glucose concentration. Ohmomo et al. [10] have reported that glucose was the best carbon source, which utilized by Aspergillus fumigatus G-2-6 for maximum degradation of melanoidin and further increase in glucose concentration resulted in an increase in mycelial biomass but no further increase in decolorization yield. It is, therefore, evident from our study that melanoidin decolorization by yeast strain Y-9 is remarkably higher in the presence of 0.2% (w/v) glucose within 24 h of incubation when compared to other researchers.
Effect of different nitrogen sources on melanoidin decolorization
The influence of various organic and inorganic nitrogen sources (0.5%, w/v) on melanoidin decolorizatin and biomass production was also investigated for 24 h of incubation, and the results are depicted in Figure 7. Melanoidin decolorization by yeast strain Y-9 is extraordinarily stable in the presence of all nitrogen sources under study. It was observed that except sodium nitrate and beef extract, presence of other nitrogen sources enhanced the melanoidin decolorization when compared to control (without nitrogen source). From Figure 7 it observed that yeast strain showed higher 75% decolorization with 4.95 g l−1 biomass production in the presence of peptone, it was practically high compared to the extent of decolorization reported by other workers [8]. Sirianuntapiboon et al. [28] have reported that yeast extract and peptone inducing decolorizing activity in acetogenic bacterium strain No. BP103. But in case of Issatchenkia orientalis and Citeromyces sp. WR-43-6, maximum decolorization was reported in the presence of 0.1% NH4Cl and 0.1% sodium nitrate [19, 20]. Kirk et al. [29] have reported that enzymatic systems catalyze degradation of lignin and lignin-like materials during the secondary phase of the metabolic growth in the presence of peptone. Synthesis and secretion of lignin peroxidase or ligninase (LiP) and manganese-dependent peroxidase (MnP) are triggered by nutrient limitations such as carbon and nitrogen sources.
Effect of different concentration of peptone on melanoidin decolorization
In another set of the experiment, the effect of various concentration of peptone (0.1, 0.2, 0.3, 0.4, 0.5 and 0.6%, w/v) on melanoidin decolorization and biomass production was also investigated and the results are depicted in Figure 8. Melanoidin decolorization by yeast strain Y-9 is extremely stable in the presence of all peptone concentrations under study. It was observed that peptone concentration above 0.3% (w/v), decreased the melanoidin decolorization. From Figure 8 it was observed that maximum 75% decolorization with 5.0 g l−1 biomass production was achieved at 0.2% (w/v) peptone concentration, above this concentration reduced decolorization. Similarly Ravikumar et al. [26] have also reported that cladosporium cladosporioides showed maximum decolorization at low concentration of peptone (1.0 g l−1) because at high concentration there was no significance in decolorization due to surplus supplementation of nitrogen which inhibition the growth. Similar effect was observed when low concentration of peptone was used as nitrogen source by Phanerochaete Chrysosporium for decolorization of melanoidin pigment present in spentwash [5]. It is, therefore, evident from our study that melanoidin decolorization by yeast strain Y-9 is remarkably higher in the presence of 0.2% (w/v) peptone within 24 h of incubation. This culture utilized little amount of peptone for higher melanoidin decolorization compared to other researchers ever reported.
HPLC analysis
The HPLC analysis report representing the area, height, retention time, before and after the treatment of spentwash (Figure 9a and b) which confirms the biodegradation of melanoidin, main compound/pigment responsible for dark brown color of distillery effluent. A major peak appeared at a retention time of 2.60 min in treated sample which was less compared to untreated and clearly indicates the ability of the yeast to decolorize/degrade the spentwash. The reduction in physico-chemical characteristics may be due to degradation of melanoidin in the presence of carbon and nitrogen sources through metabolism.