INTRODUCTION:

Molasses is a byproduct of the sugar factory. After fermentation of molasses, alcohol is produced. After distillation of alcohol, the effluent is formed. It is called as spent wash (SW). It is colored material and hazardous to the environment. Disposal of spent wash is a major problem due its colour and ingredients. Some industries have closed due to this problem. Some industries dispose of the effluent (SW) in the river which causes trouble to residents near the rivers. Some sugar factories make ponds of SW, which smells bad. Efforts are going on so many years by differents groups of researchers to solve this problem but still satisfactory solution has not achieved. Therefore, it is necessary to biodegrade it. Spent wash biodegradation study is reported by many researchers [Ravikumar et. al. (2015)¹, Naik et al. (2010)², Singh et al. (2007)³, Kumar et al. (1998)⁴].

In this study, we have used microbial cultures for biodegradation of spent wash. Decolorization and sugar utilization of SW have reported in this paper.

MATERIAL AND METHOD:

Chemicals

Malt extract, yeast extract, beef extract, peptone and agar purchased from Hi-media, Mumbai. Glucose, NaCl, sodium hydroxide etc. were purchased from S.D. Fine Chemicals, Mumbai.

Cultures:

Cultures were brought from NCIM, Pune. Bacterial cultures were maintained on Nutrient agar (Peptone 1%, NaCl 0.5, Beef extract 1 %. pH 7.5, 2.0 % agar). Yeast cultures were maintained on MGYP agar (malt extract 0.3, glucose 1, yeast extract 0.3, peptone 0.5% pH 6.4-6.8, agar 2%)

Growth:

10 ml NB/MGYP medium inoculated from slant. Incubation carried at 28°C, 220 rpm for 24h. It was added to 50 ml NB/MGYP medium and incubated at 28°C, 220 rpm for 24h. Cells were separated by centrifugation at 8000 rpm for 10 minutes. Cells used for biodegradation.

Analysis:

Sugar was analyzed by Miller (1959)⁵ method. Colour of spent wash was estimated at 475nm [Kumar et al. (1998)⁴].

Experimental:

Biodegradation of Spent Wash (SW) pH 4.5

80% Spent wash (SW) and 2.5% cells used for biodegradation. Biodegradation was carried out at 220 rpm, 28⁰C for 24h. 30 cultures (17 bacterial and 13 yeast) used for screening. Results have shown in Table 1.

Table 1: Biodegradation of Spent wash at pH 4.5

Cultures	NCIM no.	Decolorization (%)
Alcaligenes sp.	5085	9.61
Candida brumptii	3402	7.15
Candida magnoliae	3470	10.62
Candida utilis	3055	6.49
E.coli	2067	9.98
Pacchysolan tannophilus	3504	2.30
Pacchysolan tannophilus	3508	3.00
Pseudomonus oleovoran	2867	3.99
P.putida	5050	5.53
Pseudomonus sp.	2223	12.40
P.syringae	5102	15.76
P.testosteroni	5098	17.69
P.testosteroni	5099	0.38
Sacchromyces cerevisiae	3077	16.52

Precipitation of spent wash by various additives:

Spent wash treated with different additives (CaCl₂. H2O, NaHCO₃, CaCO₃, kieselguhr and Ca(OH)₂ ). It is observed that Ca(OH)₂ was more effective among the additives used. SW is treated with 1.5% Ca(OH)₂ . The precipitate was found. It is centrifuged. The brown coloured solution was obtained. Results are shown in figure 1. After removal of precipitate, pH of supernatant adjusted to 8.0 or 6.0 and used for screening of cultures.

Figure 1: Effect of calcium hydroxide concentration of precipitation of SW.

Screening of cultures at pH 8.0:

2.5% cells and 80% SW (Ca(OH)₂ treated, pH 8.0) was kept at 28°C, 220rpm for 24h. 23 cultures (12 Bacterial and 11 yeast) were used for biodegradation of SW at pH 8 (table 2).

Table 2: Biodegradation of Spent wash at pH 8.0

Cultures	NCIM no.	Decolorization (%)
Alcaligenes sp.	5085	0
Arthrobacter sp.	2263	4.37
Candida brumptii	3402	0
Candida shethiae	3500	1.87
Candida magnolaie	3470	25.33
Candida utilis	3055	30
E.coli	2067	11.86
Kluyveromyces marxianus	3232	30
Pacchysolan tannophilus	3445	3.75
Pacchysolan tannophilus	3504	0
Pacchysolan tannophilus	3508	18.66
Pseudomonus fluorescens	2653	5
Pseudomonus fluorescens	5101	6.25
Pseudomonus oleovoran	2867	18
P.putida	5050	25.98
Pseudomonus sp.	2223	36.25
Pseudomonus sp.	2668	9.37
P.syringae	5102	36.72
P.testosteroni	5098	18.64
P.testosteroni	5099	17.51
Sacchromyces carlsbergensis	3455	24.37
Sacchromyces cerevisiae	3161	28.12
Sacchromyces cerevisiae	3077	8.66

Screening of cultures at pH 6.0:

4 cultures selected from the study at pH 8 and used for biodegradation of SW at pH 6. 2.5% cells and 80% SW (Ca(OH)₂ treated, pH 6.0) was kept at 28°C, 220rpm for 24h. Results are shown in table 3.

RESULTS AND DISCUSSION:

The biodegradation of spent wash has studied by many researchers^1-4. Ravikumar (2015)¹ have reported 72.3% decolorization of spent wash by using Cladosporium cladosporioides in 5 days. Some literature survey has cited in Table 4. In this study, 30 cultures (17 bacterial and 13 yeast) were used for screening of spent wash (SW) biodegradation at pH 4.5. 16 cultures have not shown decolorization (Arthrobacter sp. NCIM 2263; Candida shethiae 3500; Kluyveromyces marxianus 3232; Pacchysolan tannophilus 3445,3502; Pseudomonus fluorescens 2653, 5101; Pseudomonus pictorum 2077; Pseudomonus putida 2872; Pseudomonus sp. 2847, 2304, 2667, 2668; Sacchromyces carlsbergensis 3455; Sacchromyces cerevisiae NCIM 3161, 3215). P. testosteronii NCIM 5098, S. cerevisiae NCIM 3077 have shown 17.69, 16.52 % decolorization respectively. P. testosteronii NCIM 5098 has shown maximum 17.69 % decolorization at pH 4.5 (Table 1). Black colour was still there. Therefore, it was decided to give treatment to SW and then use for screening. NaHCO₃, CaCO₃, Kieselguhr, etc. were used for precipitation of SW.

Table 3: Biodegradation of Spent wash at pH 6.0

Cultures	NCIM no.	Decolorization (%) : pH 6.0	RS-sugar% at pH 6
Candida utilis	3055	26.78	67.86
Kluyveromyces marxianus	3232	14.59	68.76
Pseudomonas sp.	2223	27.56	0
Pseudomonas syringae	5102	3.78	67.86

Table 4: Literature survey

Cultures	SW (%)	Decolorization (%)	Time	References
Mixed consortia	80	58-59	72h	Nikam et al. (2014)⁷
Bacterial strain K₅	20	55	12 h	Mane and Rokade (2013)⁸
Fungal isolate K₁	70	65.49	7days	Naik et al. (2010)²
Bacillus sp. isolate	10	81.10	9 days	Singh et al. (2007)³
Pseudomonas sp. isolate	10	56	72h	Chavan et al. (2006)⁹
Coriolus versicolor	6.25	71.5	10 days	Kumar et al. (1998)⁴

It is observed Ca(OH)₂ is more effective for precipitation. As concentration of calcium hydroxide increased, precipitation was increased (figure 1). 1.5% concentration is good. The increase in stirring time has not increased precipitation much. Rendleman (1987)⁶ has studied complexation of calcium with melanoidin on a diet. Melanoidin is present in spent wash. Melanoidin in the spent wash can form the complex with calcium and precipitated. Also other ingredients which can form calcium salts are precipitated. This removes the barrier for decolorization. 23 cultures (12 bacterial and 11 yeast) were used for biodegradation of SW at pH 8. P. syringae NCIM 5102 have shown 36.72% decolorization and 68.85 % sugar utilization (Table 2). From 23 cultures we have selected four cultures (on the basis of decolorization) for further work. Candida utilis NCIM 3055 and P. sp. 2223 are good cultures at pH 6.0. Candida utilis NCIM 3055 has shown sugar consumption, but Pseudomonas sp. 2223 has not shown (Table 3). Therefore, Candida utilis NCIM 3055 is the best culture. Brownish colour of the effluent was changed by this culture satisfactorily. Literature survey is shown in table 4.

CONCLUSION:

Among 30 cultures, P. testosteronii NCIM 5098, S. cerevisiae NCIM 3077 have shown 17.69, 16.52 % decolorization of SW respectively. P. testosteronii NCIM 5098 has shown maximum 17.69 % decolorization at pH 4.5. 1.5% Ca(OH)₂ is useful for precipitation of SW. Study of biodegradation at pH 8.0 was carried out using 23 cultures. P. syringae NCIM 5102 have shown 36.72% decolorization pH 8.0. Candida utilis NCIM 3055 and Pseudomonas sp. 2223 are good cultures at pH 6.0. Candida utilis NCIM 3055 has shown sugar consumption, but Pseudomonas sp. 2223 has not shown. Therefore, Candida utilis NCIM 3055 is the best culture at pH six which can decolorize spent wash to the minimum level.

REFERENCES:

1. Ravikumar R. Effect of transport phenomena of Cladosporium cladosporioides on decolorization and chemical oxygen demand of distillery spent. Int. J. Environ. Sci. Technol. 12; 2015: 1581–1590.

2. Naik N, Jagadeesh KS and Noolvi MN. Enhanced degradation of melanoidin and caramel in biomethanated distillery spentwash by microorganisms isolated from mangroves. Iranica Journal of Energy & Environment. 1 (4); 2010: 347-351.

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4. Kumar V, Wati L, Nigam P, Banat IM, Yadav BS, Singh D and Marchant R. Decolorization and biodegradation of anaerobically digested sugarcane molasses spent wash effluent from biomethanation plants by white-rot fungi. Process Biochemistry. 33(1); 1998: 83-88.

5. Miller GL. Use of dinitrosalicylic acid reagent for determination of reducing sugar. Analytical Chemistry. 31(3); 1959: 426-428.

6. Rendleman JA Jr. Complexation of calcium by melanoidin and its role in determining bioavailability. Journal of Food Science. 52(6); 1987: 1699-705.

7. Nikam SB, Saler RS and Bholay AD. Bioremediation of distillery spent wash using Pseudomonas aeruginosa, Aspergillus niger and mixed consortia. Journal of Environmental Research and Development. 9(1); 2014: 129-137.

8. Mane C and Rokade K. Physico-chemical analysis and microbial degradation of spent wash from sugar industries. Research Journal of Chemical Sciences. 3(8); 2013: 53-56.

9. Chavan MN, Kulkarni MV, Zope VP and Mahulikar PP. Microbial degradation of melanoidins in distillery spent wash by indigenous isolate. Indian Journal of Biotechnology. 5; 2006: 416-421.

Received on 17.02.2016 Modified on 04.04.2016

Asian J. Research Chem. 9(4):April 2016; Page 188-190

DOI: 10.5958/0974-4150.2016.00031.6