Statistical Optimization of Parameters for Fungal Decolourization of Digested Molasses Spentwash S. S. Singh1 and A. K.

Dikshit2 Research Scholar, Centre for Environmental Science and Engineering, IIT Bombay, Powai, Mumbai 400 076 (India); PH (+91-22) 2576 4865; FAX (+91-22) 2576 4650; email: ss_singh@iitb.ac.in Professor, Centre for Environmental Science and Engineering, IIT Bombay, Powai, Mumbai- 400 076 (India); PH (+91-22) 2576 7862; FAX (+91-22) 2576 4650; email: dikshit@iitb.ac.in ABSTRACT The spent wash is characterized by high chemical oxygen demand and dark brown colour. Cane molasses contain dark brown pigment called melanoidins that impart colour to the spentwash. In the recent years, interest is being directed towards utilizing pure microbial cultures for the decolourization and biodegradation of melanoidins. In this study, decolourization of anaerobically digested spentwash (DSW) was studied after pretreatment with Poly aluminium chloride using Aspergillus niger species IITB-V8 as an inoculum. The optimum values of different variables were determined by three sequential Plackett-Burman designs. The average decolourization efficiency for the three designs were 47.44%, 58.13% and 65.07%, which shows that the changes in the level of parameters studied from first to third design had a positive effect on decolourization efficiency. The optimum values of parameters for maximum decolourization were 5 g/L Glucose, 1 g/L KH2PO4, pH 5 and 0.15 g/L inoculum. INTRODUCTION Distilleries are one of the most polluting Industries in India. In India, the demand of ethanol is projected to increase because of Central Government notification directing 5% ethanol-blended petrol, as per Bureau of Indian Standards specifications, to be sold in the several States and Union Territories (MPNG, 2004). Most of the distilleries in India use sugarcane molasses for making alcohol. The main source of wastewater generation is the distillation step wherein large volumes of dark brown effluent (spentwash) are generated. Molasses spentwash has very high levels of BOD, COD, COD/BOD ratio as well as high potassium; phosphorus and sulfate content (Satyawali and Balakrishnan, 2007). Colour of spentwash is mainly due to melanoidin which is produced from the non-enzymatic browning reaction between aldose and amino compounds. Conventional wastewater treatment cannot breakdown these coloured pigments and therefore colour remains in the effluent. Many pure
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cultures of fungi were used for the decolourization of distillery effluent (Pant and Adholeya, 2007) Environmental conditions like nutrients and pH is important for decolourization. Colour elimination from Molasses spentwash using Aspergillus niger was studied and the nutrients used for this process were sucrose, NH4NO3, KH2PO4 and MgSO4.7H2O with an initial pH of 5 (Miranda et al., 1996). An Aspergillus species isolated from the soil was used for decolourization of anaerobically digested and aerobically treated distillery wastewater and 75% decolourization was achieved which reduced to 40% on using undiluted wastewater (Shayegan et al., 2005). In the study, decolourization of anaerobically digested and chemically pretreated spentwash was performed using Aspergillus niger. Sequential Plackett Burman design was applied for optimization of the most significant parameters enhancing decolourization. MATERIALS AND METHODS Wastewater. Anaerobically digested spentwash (DSW) was obtained from a cane molasses based SSK distillery, Nashik (India). DSW is pretreated with Polyaluminium chloride (coagulant), as it gives better decolourization without requiring dilution of wastewater (Chauhan, 2008). Wastewater was characterized based on the Standard Methods for Examination of Water and Wastewater (APHA, 1998). Microorganism and Inoculum. Aspergillus niger species IITB-V8 was isolated from the sludge for highest decolourization. This species is maintained on petri dish and slant, prepared in potato dextrose agar (PDA). Strain from the plates was transferred to saline solution containing 0.9% (w/v) NaCl and 0.1 % (w/v) Tween 80 and cultivated at 30oC in 250 mL shake flasks, containing 100 ml of culture media consisting 50 g/L glucose, 1 g/L KH2PO4, 2 g/L NaNO3, 0.5 g/L MgSO4.7H2O (pH adjusted to 4.5), in a rotary shaker at 150 rpm. Flask cultures. Optimization studies were performed in 250 mL shake flask with 100 mL of DSW after coagulation. The different concentration of nutrients added were glucose, NaNO3, KH2PO4 and MgSO4.7H2O. After sterilization in autoclave, flask was inoculated with fungal suspension. Flasks were, then, put inside the shaker at 150 rpm at 30oC. Samples were withdrawn every 24 hours for observing maximum decolourization. Colour measurement. 5 ml samples were taken from shake flasks and centrifuged at 10,000 rpm for 10 minutes. The supernatant after centrifugation was diluted 100 times and used for color reduction measurement. The absorbance was measured at 475 nm using Thermo Spectronic visible spectrophotometer (Model Helios Epsilon). The decolourization yield was expressed as the percentage decrease in absorbance at 475 nm related to the initial absorbance at the same wavelength.

PlackettBurman experimental design. The different important parameters affecting decolourization activity were calculated using PlackettBurman experimental design (Plackett and Burman, 1944). The optimum values of different variables can be determined by PlackettBurman sequential designs. Three sequential Placket-Burman designs were used to determine the optimum values. In First, the Plackett-Burman design for 11 parameters was used. In Second and third, the Plackett-Burman design for 7 parameters was used In the first design eleven variables comprising of six independent variables and five dummy variables were used in 12 runs of duplicate set of experiments(Table 1 and 2). In the second design (Table 3) and third design (Table 5) six main variables were used to run eight set of duplicate experiments and one dummy variable was used(Table 4). The rows in Table 3 represent the 12 different experiments and each column represents a different variable. For each independent variable, a high (+) or low (-) level was assigned. The effect of each variable was determined with the following equation:
E ( xi ) = M i+ M i N

(1)

Where E (xi) is the concentration effect of the tested variable, Mi+ and Mi- are the decolourization efficiencies from trials where the variable (xi) measured was present at high and low levels respectively, and N is the number of run or trials. Table 1. First experimental design for decolourization. Variable Parameter High level(+) Glucose(g/L) 15 X1 Dummy X2 Dummy X3 NaNO3(g/L) 3 X4 2 KH2PO4(g/L) X5 2 MgSO4.7H2O(g/L) X6 Dummy X7 pH 7 X8 Inoculum(g/L) 0.3 X9 Dummy X10 Dummy X11 Low level(-) 10 0 0 0 5 0.2 -

Table 2. The Plackett-Burman design for 11 parameters (First design). X2 X3 X4 X5 X6 X7 X8 X9 X10 Run X1 + + + + + + 1 + + + + + 2 + + + + + + 3 + + + + + + 4 + + + + + + 5 + + + + + 6 + + + + + 7 + + + + + 8 + + + + + + 9 + + + + + 10 + + + + + 11 12

X11 + + + + + + -

Table 3. Second experimental design for decolourization. Variable Parameter High level(+) Low level(-) Glucose(g/L) 10 5 X1 NaNO3(g/L) 2 0 X2 KH2PO4(g/L) 2 1 X3 1 0 MgSO4.7H2O(g/L) X4 Dummy X5 pH 5 4 X6 Inoculum(g/L) 0.2 0.1 X7

Table 4. The Plackett-Burman design for 7 parameters (Second and third design). X2 X3 X4 X5 X6 X7 Run X1 + + + + 1 + + + + 2 + + + + 3 + + + + 4 + + + + 5 + + + + 6 + + + + 7 8

Table 5. Third experimental design for decolourization. Variable Parameter High level(+) Low level(-) Glucose(g/L) 5 3 X1 NaNO3(g/L) 1 0 X2 1 0.5 KH2PO4(g/L) X3 MgSO4.7H2O(g/L) 0.5 0 X4 Dummy X5 pH 5.5 4.5 X6 Inoculum(g/L) 0.15 0.05 X7 RESULTS AND DISCUSSION Characterization of Digested spent wash after coagulation. In this study, digested spent wash after coagulation with Polyaluminum chloride (PAC), is used. The physicochemical characteristics of this wastewater are given in Table 6. Table 6. Physicochemical characteristics of digested spent wash after coagulation. S.No. Parameter Value 1 Colour (Absolute absorbance at 475 nm) 4.5 2 pH 4.82 3 Total Solids (mg/L) 25100 4 Total suspended Solids (mg/L) 568 5 Total dissolved solids (mg/L) 24532 6 BOD (5 days, 20oC) 4200 7 COD (mg/L) 11200 8 NH3-N (mg/L) 2400 Optimization of parameters by PlackettBurman experimental design. The first design was carried out using the Plackett-Burman design for 11 parameters as in Table 2. The second and third design was performed using the Plackett-Burman design for 7 parameters as in Table 4. The high and low levels of parameters for first, second and third designs are taken as in Table 1, 3 and 5. The decolourization efficiency found in each run for the first, second and third designs are shown in Table 7. The steady state decolourization was observed after 3 days. The effect of each parameter in three designs is found out using eq. (1) and given in Table 8.

Table 7. Decolourization efficiency (%) after 3 days in the first, second and third design. Run 1 2 3 4 5 6 7 8 9 10 11 12 First 61.0 60.7 31.8 41.7 47.2 49.8 45.2 33.6 50.1 36.9 60.7 50.6 Second 52.4 55.9 58.6 63.3 55.7 59.2 60.7 59.3 Third 63.2 65.1 62.7 67.5 65.6 64.5 68.7 63.3 -

Table 8. Effect of parameters for the first, second and third design. S.No. 1 2 3 4 5 6 Parameter Glucose (g/L) NaNO3 (g/L) KH2PO4 (g/L) MgSO4.7H2O (g/L) pH Inoculum(g/L) Effect, E(xi) First Second -0.375 -0.2625 -0.458 -0.4125 5.3 -0.0625 -0.09 -2.48 -8.04 1.4375 -0.26 -0.4875 Third 0.9 -0.3 1.05 -0.925 1.05 0.125

The effect of KH2PO4 and pH is significant in the first design as its value is 5.3 and -8.04 (Table 8).This means that from 0 to 2 g/L of KH2PO4, decolourization increased by 5.30 units and when pH was increased from 5 to 7, decolourization decreased by 8.04 units. The second sequential Plackett-Burman design was carried out considering the sign and magnitude of parameter effect in first design. As there is no considerable effect due to glucose, NaNO3, MgSO4.7H2O and inoculum at levels considered in first design, the high and low level of glucose and inoculum was decreased and high level of NaNO3 and MgSO4.7H2O was decreased in the second design (Table 3). The low level of KH2PO4 is increased to 1g/L to see the effect at that level. In the second design, there is very low negative effect in moving from low to high level in case of Glucose, NaNO3, KH2PO4 and inoculum; significantly negative effect in MgSO4.7H2O and positive effect on pH. So in the third design (Table 5) the high and low level of glucose, KH2PO4 and inoculum is decreased; high level of NaNO3 and MgSO4.7H2O is decreased and low and high level of pH is increased. In the third design, the effect of glucose, KH2PO4, pH and inoculum increases from low to high level. The average decolourization efficiency for the three designs is

given in Table 9. It can be seen that changes in the level of parameters studied from first to third design had a positive effect on decolourization efficiency. Table 9. Mean decolourization efficiency (%) for each design at 72 hrs. S.No. Design Mean decolourization efficiency (%) 1 First 47.44 2 Second 58.13 3 Third 65.07 The optimum level of parameters after three designs are decided by comparing the parameter effect in three designs and given in Table 10. Table 10. Optimum level of parameters after three designs. Parameter Optimum condition Design Glucose (g/L) 5 Third KH2PO4 (g/L) 1 Third pH 5 Second Inoculum (g/L) 0.15 Third CONCLUSION The optimization of nutrients and other parameters is very important for enhancing the decolourization. Plackett Burman design is an important tool to optimize the parameters.The optimum values of different variables were determined by three sequential Plackett-Burman designs. The maximum decolourization in all the three designs was obtained at 3 days. The optimum values of parameters for maximum decolourization were 5 g/L Glucose, 1 g/L KH2PO4, pH 5 and 0.15 g/L inoculum. REFERENCES APHA (1998) Standard Methods for the Examination of Water and Wastewater, American Public Health Association, New York. Chauhan, M.S. (2008). Integrated physico-chemical and fungal treatment for decolourisation of anaerobically digested molasses spentwash. PhD Thesis, CESE, IIT Bombay, Mumbai, India. Miranda, M.P., Benito, G.G., Cristobal, N.S. and Nieto, C.H. (1996). Colour elimination from molasses wastewater by Aspergillus niger. Biores. Technol., 57 (3), 229235. MPNG (Ministry of Petroleum & Natural Gas) (2004). Notification, http://petroleum.nic.in/gaznew.pdf> (July 21, 2009). Pant, D. and Adholeya, A. (2007). Biological approaches for treatment of distillery wastewater: a review. Biores. Technol., 98, 23212334. Plackett, R.L., Burman, J.P. (1944). The design of optimum multifactorial experiments. Biometrica, 33, 305325. Satyawali, Y., Balakrishnan, M. (2007). Wastewater treatment in molasses-based alcohol distilleries for COD and color removal: a review. J. Environ.

Manage., 86, 481497. Shayegan, J., Pazouki, M. and Afshari, A. (2005). Continuous decolourization of anaerobically digested distillery wastewater. Process Biochem., 40, 1323 1329.