Media Optimization for the Decolorization of Distillery ...ijiet.com/wp-content/uploads/2014/12/2..pdfDec 02, 2014 · the spent wash was acidic in nature i.e. 4.1 (the raw spent

International Journal of Innovations in Engineering and Technology (IJIET)

Vol. 4 Issue 1 August 2014 8 ISSN: 2319 – 1058

Media Optimization for the Decolorization of

Distillery Spent Wash by Biological Treatment

using Pseudomonas Fluorescence

M.Ananda Boopathy

Department of Biotechnology

Mepco Schelnk Engineering College, Sivakasi, Tamilnadu

S.N.S.Senthilkumar

Department of Biotechnology

Mepco Schlenk Engineering College, Sivakasi, Tamil Nadu

Abstract -The wastewater released from distilleries and fermentation industries are the major source of soil and aquatic

pollution due to presence of water-soluble recalcitrant colouring compounds called melanoidins. Biological

decolourisation of of these compounds found more advantages compared to chemical treatments. Hence in this study, the

bacterium Pseudomonas fluorescens showing higher decolorization efficiency on distillery spent wash was screened.

Physico-chemical properties like colour, pH, Biological Oxygen Demand (BOD), Chemical Oxygen Demand (COD), etc.,

were analyzed for the substrate. Effect of carbon and nitrogen supplementation on media was studied followed by media

optimization for different carbon, nitrogen and salt concentrations were carried out using Response Surface

Methodology. The central composite design (CCD), consisting of a three-factor-four-level pattern with 17 design points,

was then conducted in this optimum vicinity to locate the true optimum concentrations which are as follows: Glucose

(0.8%), Peptone (0.8%) and KH2Po4 (0.05%). Hence it is found that higher decolorization of distillery spent wash could

be achieved at low carbon and nitrogen concentrations. Thus this bacterium can be utilized for melanoidin decolorization

of distillery effluent at industrial scale.

Keywords: decolourization, Pseudomonas fluorescens, distillery spent wash, media optimization.

I. INTRODUCTION

Distilleries and fermentation industries produces dark brown color spent wash with a high Biological Oxygen

Demand (BOD), Chemical Oxygen Demand (COD), low pH and toxic substances such as phenols. The main

problem in treating Distillery Spent Wash (DSW) is its color, which contains nearly 2% (w/w) of a dark brown

recalcitrant pigment, melanoidin. Melanoidin is known as a natural browning polymer, produced by the “Maillard

reaction” between amino and carbonyl groups of organic matters and is closely related to humic substances in the

natural environment. Naturally melanoidins are widely distributed in food drinks and widely discharged in huge

amount by various agro-based industries especially from cane molasses based distilleries and fermentation industries

as environmental pollutants. The structure of melanoidins is still not completely understood but it is assumed that it

does not have a definite structure as its elemental composition and chemical structures largely depend on the

nature and molar concentration of parent reacting compounds and reaction conditions as pH, temperature,

heating time and solvent system used. Carbon isotope ratios support the stoichiometric ratios for the combination

of sugars with amino acids, which are based on the elemental composition data of melanoidins. Hence for its safe

disposal into the environment, pretreatment is required.

Degradation and decolourisation of these wastewaters by chemical methods, flocculation treatment and

physicochemical treatment such as ozonation and activated carbon adsorption have been accomplished, but these

methods are not economically feasible on large scale due to cost limitation whereas biological decolourisation by

using bacteria such as Pseudomonas fluorescens, Pseudomonas putida, Bacillus sp. , Alkaligenes sp., Lactobacillus

sp., have been successfully achieved and thus can be applied as bioremediation techniques. However, the biological

decolourisation of wastewaters containing melanoidins depends on pH, temperature, concentration of nutrients,

oxygen and inoculums size while the enzymatic system responsible for the degradation of melanoidins consists

mainly sugar oxidases and peroxidases as sarbos oxidase, glucose oxidase, manganese dependent and independent

peroxidases (MnP and MIP) (Watanabe et al., 1982; Ohmomo et al., 1985a; Aoshima et al., 1985).



Biological methods present an incredible alternate for decolorizaiton/degradation and bioremediation of spent wash

due to their low cost,environmental friendly and publicly acceptable treatment and cost competitive alternative to

chemical decomposition processes.

II. MATERIALS AND METHODS

Microorganisms and Culture Medium

Fifteen strains were initially taken. The strains were identified on the basis of its decolorizing capacity. The screened

strain was maintained in LB broth at 4oC and was used for further studies

Preparation of seed culture

Seed culture was prepared by inoculating organisms in nutrient broth at 32oC and overnight night cultures were used

for inoculating the decolorization medium.

Distillery spentwash (DSW)

The spentwash was collected aseptically from Dharani sugars and Chemicals pvt.ltd, Tirunelveli, India. The

spentwash was centrifuged at 10,000 rpm for 15 min before use to remove the suspended solids and stored at 4°C.

The stored distillery spentwash was filtered through (Whatman No: 1) filter paper and was diluted with distilled

water. The analysis of different physico-chemical parameters like color, odor, pH, biological oxygen demand

(BOD), chemical oxygen demand (COD) were analyzed by employing standard methods.

Determination of BOD (Biological oxygen demand)

BOD was determined as follows; 0.1, 1.0, 2.0, 4.0, 6.0 ml of samples (Spent wash) were taken and made up to

300ml with distilled water. To this,15 drops of alkaline iodide solution was added followed by addition of

conc.H2SO4 of same volume. 15 drops of starch solution was added to 203ml of above solution which was titrated

against Sodium thiosulphate solution taken in burette. The endpoint is the disappearance of blue colour.

Determination of COD (chemical oxygen demand)

COD was determined as follows; 50 ml of diluted sample was taken. To this 1g of mercuric sulphate and 5 ml

of sulphuricacid-silver sulphate reagent was added followed by addition of 25ml of std. potassium dichromate

solution and 30ml of sulphuric acid-silver sulphate reagent and boiled for 2hours.After cooling , 2-5drops of

ferroin indicator was added and titrated against std. Ferrous ammonium sulphate.The endpoint is the color change

from yellowish green to reddish brown.

Decolorization assay

The strains were inoculated in the LB broth containing distillery spent wash. After incubation, the broth was

centrifuged at 10,000 rpm for 10 min. The supernatant was read at absorbance maximum (Amax) of the melanoidin

i.e. 475 nm.The decolorization yield percentage was expressed as the decrease in the absorbance at 475 nm against

initial absorbance at the same wavelength. Uninoculated medium was served as control. The entire assay was

performed in triplicates and compared with control. The decolorization efficiency of the isolate was expressed as per

following equation:

Decolorization (%) = I – F/I

Where, I = Initial absorbance (Control) and F = Absorbance of decolorized medium broth.

Biomass determination

Biomass is determined by using the standard formula 1OD = 0.37g/l of dry cellular weight.

III. SELECTION OF PHYSICO-CHEMICAL AND NUTRITIONAL PARAMETERS FOR MELANOIDIN DECOLORIZATION:

Effect of pH, temperature and time on the decolourisation activity

Optimum pH for the decolourisation activity was determined by inoculating the microbial culture in spent wash

medium in the pH range of 3.5-9.0 and incubated at room temperature. Effect of temperature on the decolourisation

activity was determined by inoculating the microbial culture in the same medium over a temperature range of 30-

80oC.Effect of time for efficient decolorisation was determined by incubating the medium for 24h,48h,72h and the

results were analyzed.



Optimization of experimental conditions

The various process parameters influencing melanoidin decolorization and biomass production 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 glucose0.5%; peptone 0.1 %;

KH2Po4 0.05% with 3.5 OD spentwash was used for inoculation with 2% (v/v) of bacterial culture. 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 different

carbon sources viz. glucose, sucrose, cellulose and nitrogen sources viz. yeast extract, peptone, beef extract.

Thereafter, optimized carbon and nitrogen sources were further optimized at different concentration (0.2 to 1%,

w/v). The medium was sterilized at 121°Cfor 15 min and incubation was done at 30°C with all the other conditions

at the optimal levels.

Statistical analysis

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 MS Excel. All the graphs were

plotted using Origin pro.

IV. RESULTS AND DISCUSSION

Physico-chemical analysis of the effluent was carried out. The colour of spent wash was found dark brown. The

colour of spent wash is suspected due to presence of a derivative of caramelized sugar formed during the distillation,

termed melanoidin (9).Odour of the distillery effluent was offensive. Odourous compounds from distillery waste

water mainly consist of volatile fatty acids such as butyric and valeric acids that have a high odour index. Distillery

has distinct organic compositions. Various anaerobic bacteria ferment these compounds and generate products such

as volatile fatty acids for example glycerol is fermented into butyric acid by clostridium butyricum (17).The pH of

the spent wash was acidic in nature i.e. 4.1 (the raw spent wash is acidic in nature and the pH values of distillery

wastewaters range from 3.5 to 5.0) (18, 19). BOD and COD were found to be 38130 mg/l and 95564 mg/l

respectively. For assay determination, wavelength scan of distillery spent wash was done (Fig.1). The sample

showed maximum absorbance at 475nm.

Out of fifteen species screened, Pseudomonas fluorescens showed maximum efficiency of decolorization and hence

this bacterium is used further for media optimization studies.

Maximum decolourisation effect was found within the pH range of 6.5 to 7.0, the preferred range for the growth of

isolate. Temperature range of 30-35 oC was found to be suitable for the activity. Under optimum pH and

temperature, decolourisation efficiency was notable at 72h for the screened species.

The effect of various sugars and nitrogen as externally added carbon and nitrogen sources in the spent wash

medium, on decolourisation activity was studied. Maximum melanoidin decolorization was observed in glucose as

well as sucrose as carbon source at the level of 0.8%.This effect can be explained that during initial phase of growth,

organism utilizes easily available carbon sources added to the medium and then starts to degrade spentwash that is

complex carbon source(Kumar et al., 1997). Ohmomo et al. (1987) reported that glucose was the best carbon source,

which utilized by Aspergillus fumigates G-2-6 for maximum degradation of melanoidins and further increase in

glucose concentration, increased the mycelial biomass but no change in decolorization level. Watanabe et al.

(1982)have reported that 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 the reaction mixture,

was later identified as sorbose oxidase which oxidize glucose into gluconic acid (Miyata et al.,2000; D’souza

et al., 2006). The decline in melanoidin decolorization encountered with high sugar concentration in the medium is

probably due to inhibition effect to the enzyme like lignolytic activity of laccase enzyme and oxidation activity of

the peroxidase (Raghukumar and Rivonkar, 2001;Guimaraes et al.,2005; Pant et al., 2008; Jiranuntipon et al., 2008;

Zhao et al.,2010; Ravikumar et al., 2011). Different nitrogen sources were optimized for melanoidin decolorization.

Among different nitrogen sources (organic and inorganic), the highest melanoidin decolorization was reported with

peptone at the level of 0.8%. Similarly various nitrogen sources were optimized by different workers for melanoidin

decolorization, but peptone was the most effective for color removal (Ohmomo et al., 1988; Miyata et al.,2000;

Sirianuntapiboon et al.,2004a; 2004b; Ravikumar et al., 2011). Kirk et al. (1978) reported that enzymatic systems

catalyse degradation of lignin and lignin-like compound 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. At high concentration,

there was no significant decolorization due to surplus supplementation of nitrogen which inhibited the growth.



Similar effect was observed when low concentration of peptone was used as nitrogen source for decolorization of

melanoidin pigment present in the spent wash.

Response surface methodology uses statistical models. In practice, both the models and the parameter values are

unknown, and subject to uncertainty on top of ignorance. Of course, an estimated optimum point need not be

optimum in reality, because of the errors of the estimates and of the inadequacies of the model.

In our study, Response Surface Methodology (RSM) was used to study the effects of carbon, nitrogen, salts, on

Decolorization. Interactions between each variable were calculated by experimental designs. The factorial design

was performed to study the decolorization and the effects of glucose concentration, Sucrose, cellulose, peptone,

yeast extract,beef extract, Potassium di hydrogen Phosphate (KH2PO4-- ).

The Model F-value of 11.34 implies the model is significant. There is only a

0.04% chance that a "Model F-Value" this large could occur due to noise.Values of "Prob > F" less than 0.0500

indicate model terms are significant. In this case C, BC, A2, B2, C2 are significant model terms.Values greater than

0.1000 indicate the model terms are not significant.

The "Lack of Fit F-value" of 3.33 implies the Lack of Fit is not significant relative to the pureerror. There is a

10.66% chance that a "Lack of Fit F-value" this large could occur due to noise. Non-significant lack of fit is good.

"Adeq Precision" measures the signal to noise ratio. A ratio greater than 4 is desirable. Your ratio of 10.037

indicates an adequate signal. This model can be used to navigate the design space.

From this study, it was found that Melanoidin present in distillery spent wash can be degraded and hence the spent

wash can be decolorized by using the bacteria Pseudomonas fluorescens. Thus this bacterium can be used in

biological treatment of spent wash and this method could be used to treat distillery spent wash and hence can

prevent environmental pollution.

V. DETERMINATION OF CENTRAL LEVELS:

Effect of Different Carbon sources on Decolorisation

Figure 1: Effect of cellulose on decolorisation and biomass

Figure 2: Effect of glucose on decolorisation and biomass



Figure 3: Effect of sucrose on decolorization and biomass

Effect of Different Nitrogen Sources on decolorization and biomass

Figure 4: Effect of Yeast extract on decolorization and biomas

Figure 5: Effect of Peptone on decolorization and biomass



Figure 6: Effect of Beef extract on decolorization and biomass

Experimental run from Design expert

Table 1: Experimental runs

Factor 1 Factor 2 Factor 3 Response 1

Std Run A:Glucose B:Peptone C:KH2Po4 Decolorization

14 1 0.8 0.8 0.075 74

15 2 0.8 0.8

0.075 74

2 3 1 0.6 0.075 61

3 4 0.6 1 0.075 70

12 5 0.8 1 0.1 60

13 6 0.8 0.8 0.075 74

8 7 1 0.8 0.1 65

6 8 1 0.8 0.05 69

5 9 0.6 0.8 0.05 67

16 10 0.8 0.8 0.075 74

10 11 0.8 1 0.05 71

17 12 0.8 0.8 0.075 74

11 13 0.8 0.6 0.1 75

4 14 1 1 0.075 68

7 15 0.6 0.8 0.1 62

1 16 0.6 0.6 0.075 66

9 17 0.8 0.6 0.05 64

VI. ANOVA RESPONSE AND

VALUES



Std DEV Mean C.V

%

PRESS R-

Squared

Adj R-

squared

Pred R

squared

Adeq

Recession

372.96 4925.65 7.57 8.775E+006 0.9108 0.8304 0.4370 10.037

Effect of Interaction between nutrients

Glucose and Peptone Glucose and KH2Po4

Interaction between Peptone and KH2PO4

The advantage of using RSM is that the prediction of limiting nutrient which is given by the perturbation graph.

Here “A” i.e. glucose is the limiting nutrient



Perturbation graph for prediction of limiting nutrient

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Media Optimization for the Decolorization of Distillery ...ijiet.com/wp-content/uploads/2014/12/2..pdfDec 02, 2014 · the spent wash was acidic in nature i.e. 4.1 (the raw spent