Journal of Applied Sciences Research, 6(12): 1966-1974, 2010 2010, INSInet Publication

Biosorption of Molasses Pigments by Sludge Agriculture Residues and Fungal

1

Amber S. Gad and 2H. El Sayaad

Chemistry of Natural and Microbial Products Dept. National research center, Dokki 2 Middle Eastern Isotopes Regional Center for Arab countries,.Dokki, Egypt.

Abstract: Free and viable cells of Phanerochaete chrysosporium effectively decolorize heat sterilized molasses waste water with 62.956 % when supplemented with (% w/v): glucose,1; peptone,1.25; K H2PO4, 0.25,and MgSO4 .7H20, 0.125, pH 5 after 8 days of incubation at 30"C and 200 rpm.0.5% (w/v) chemically untreated wood chips,corn cob shred and sugarcane bagasse were also effective in molasses waste water color removal.Molasses waste water showed decreased COD and BOD when treated with Phanreovhaete chrysosporium under optimum conditions. Key words: Molasses waste water, De-colorization, Phanerochaete chrysosporium, corn cob shred, sugar cane bagasse, Wood chips. INTRODUCTION Molasses has a high commercial value due to its use as a carbon source for fermentation industries, biofertilizer and feed for domestic animals[1]. However, the use of molasses as a raw material for fermentation industries is associated with the presence of large amounts of colored substances which remain in the fermentation residues after recovery of the products. The main colored substance, melanoidin, can hardly be decomposed by usual biological treatment processes and accounts for the high COD value, which is a major problem for pollution control[2]. Melanoidin pigment are the products of millard reaction between sugars and amino compounds produced in heating[3]. Due to the hard nature of melanoidins, conventional waste water treatment process are unable to remove the color from the molasses spent wash (MSW) which has then the potential to block out light from contaminated waterways thus preventing oxygenation especially many dyes are made from known carcinogens dye waste water have to be removed[4]. However, microbial and physical de-colorization by agro-industrial wastes were tried; wheat straw, corn cob shred and wood chips or even seeds as Moringa oleifera[5,6]. Many studies have been observed to find low cost absorption which includes peat, benomite, steel plant slag, fly ash, china clay, maize cob, wood shavings and silica out on a pilot plants. Processes such as chemical precipitation, ozonization, flocculation, chemical adsorption or carbon adsorption are used for the removal of the colored substances. However, color removal by the above processes still has disadvantages due to the high operation cost, high consumption of chemicals, fluctuation of the color removal efficiency and the high volume of solid waste produced[7,8]. Generally,fungi are known for their ability to remove different pollutants either by excreting a protein to chelate it, excreting an enzyme that can decompose or degrade the pollutants;[9]. The aim of this study is the use of the white-rot fungus Phanerochaete chrysosporium and some adsorbents to decolorize molasses waste water also to investigate the effect of immobilization, cell viability, medium composition,temp, contact time, sterilization, and other cultivation conditions on the de-colorization process besides comparing some chemical characters for molasses with and without biological treatment. MATERIALS AND METHODS Microorganisms: Phanerochaete chrysosporium: ATCC 24725 was obtained from Cairo MERCIN, Ein Shams University. Rhizopus oryzae obtained from Dr. M.Usama Noaman; Biochemistry Lab, NRC., Egypt. Both strains were used for color removal of a previously fermented molasses. Manitenance Media: Phanerochaete chrysosporium and Rhizopus oryzae were grown aerobically for 7 days and maintained at 4C on Malt extract medium(g/L): malt extract, 20; peptone,1, dextrose, 20 and agar 15;[10]. Molasses Waste Water Basal Medium: The molasses waste water was centrifuged to remove suspended matter, diluted with distilled water (v/v) without any complementation,otherwise stated and was used as a basal medium initial pH 5, sterilized either by heat or

Corresponding Author: Amber S. Gad., Chemistry of Natural and Microbial Products Dept. National research center, Dokki

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J. Appl. Sci. Res., 6(12): 1966-1974, 2010

gamma irradiation for 15 min at 121" C .The rest of the chemical components of the medium were sterilized separately and added. Organisms were tested for decolorization in medium contains (g/l): glucose, 25; peptone,12.5; KH2PO4,2.5;MgSO4 .7H2O,1.25, (Fahy et al.,[7] with molasses waste water, where indicated the glucose concentration or the nitrogen source were altered. Original molasses stock was obtained from Delta sugar company, Kafr El Sheik, Egypt, and stored at4"C until required. Chemical composition of stock beet molasses was estimated[11]. Preparation of the Inoculum: Each strain was precultivated three days at 30C in fifty ml of malt extract medium contained in 250 ml Erlenmeyer flasks. The mycelium was recovered by centrifugation for 20 min at 6000 g, and further washed with sterile water. Vortex dispersion of the mycelium was carried out discontinuously in a sterile tube for 5 min in the presence of 5ml water and glass beads. The homogenate was centrifuged at 6000 g for 15 min and washed twice. The recovered pellet was re-suspended in 10 ml of sterile diluted water to be used as inoculums for further experiments[10]. A separate set of un-inoculated flasks was maintained in parallel as control. Culture Conditions: Fifty ml of the diluted molasses waste basal medium was properly, contained in 250 ml Erlenmeyer flask, autoclaved at 121C for 15 min then inoculated with 2 ml of the fungal inoculums (5x107 conidia) and incubated at 30"C on rotary shaker at 150 rpm. A corresponding amount of P. chrysosporium inoculums were used for cultivation with alginate immobilized cells;[10]. Determination of Mycelium Dry Weight: The molasses medium was filtered on weighed Whatman No.1 filter paper, biomass were dried at70"C for 24 h to get a constant weight. P. Chrysosporium Immobilization; [ 1 0 ] : Cell immobilization was carried out by restriction of the cells in a calcium alginate matrix. Two mls of the dispersed mycelium (~5x107 conidia), were mixed with 2% (w/v) solution of sodium alginate, and then homogenized in a sterile syringe. This solution was delivered to a cold 0.1M CaCl2 solution as droplets, the resulting beads were allowed to harden for 2 minutes and then the CaCl2 solution was removed. The formed beads were consolidated at 30C during 15 min. Thereafter the alginate beads were washed with physiological water (0.9% NaCl) in order to eliminate calcium chloride traces and free cells. In 250 ml Erlenmeyer flasks,equal numbers of the immobilized

cells and control beads were added to fifty ml molasses waste water medium. In control experiments, calcium alginate beads were prepared as described without the addition of living inoculum and were further subjected to the same treatment as immobilized cells beads. Determination of Beads Dry Weight: The growth of the immobilized cells was measured after filtration on Whatman No.1 filter paper. The number of randomly chosen beads; fifty beads were taken and washed thoroughly with distilled water and then dried at 110"C to a constant weight for 24h. The weight of entrapped cells was measured by calculation of the difference between the weight of immobilized beads with and without cultivation. i.e. an average pre-determined pellet dry weight was subtracted from the weight of pellet plus mycelium. P. chrysosporium Heat Killed Culture: Fifty ml molasses waste water basal media with biomass were autoclaved at 121C for 15 min.;[12] cultures were withdrawn after incubation for 6 days at 150rpm and 30"C, filtered, weighed, and clear centrifuged filtrate O.D. was measured at 475 nm. Results were estimated due to difference in decolonization on using heat killed and live culture in the 6th day. Effect of Glucose Concentration: The different glucose concentrations were sterilized separately and added to 50 ml molasses water in 250 ml Erlenmeyer flasks to attain a concentration of 0.5-2.5 %(w/v), inoculated, incubated at 150 rpm, and 30C for 6 days. The media were filtered and absorbance was measured at 475 nm;[12]. Effect of Nitrogen Source: 1.25%(w/v) yeast extract, peptone and malt extract were added to 50 ml diluted molasses waste water medium in 250ml flask inoculated, incubated at 150 rpm, and 30C for 6 days. The media were filtered and absorbance was measured at 475 nm;[12]. Effect of Temperature: Flasks with optimal glucose concentration and 1.25 % (w/v) suitable nitrogen source were inoculated and incubated at 150 rpm and different temp, 25-40"Cf or 6days,harvested and the absorbance was measured at 475nm. Effect of Contact Time: The molasses waste water media were dispensed in 50 ml portions, heat sterilized at 121"C. for 15 min,inoculated and incubated at 30"C, and 150 rpm for different time intervals (0-10 days). Cultures were harvested and the absorbance was measured at 475 nm.

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Effect of Nutrients: Nutrients were added; Fahy et al[7], sterilized, inoculated and incubated for 8 days,harvested and the absorbance was measured at 475nm,[7]. n: Effect of Agitatio: The optimum supplements concentration, optimal time and temperature were adjusted while agitation was varied. The used rpm were,100-250. Cultures were withdrawn after 8 days, filtered, and the absorbance was measured, O.D. was measured at 475 nm. Gamma Irradiation of Molasses: The process of irradiation was carried out at Middle Eastern Isotopes Regional Center for Arab countries. Dokki, Egypt. Portions of fifty ml diluted molasses waste water sterilized with 0.612 Gy/min gamma irradiation according to[13]. After inoculation, incubation at 200 rpm and 30"C for 8 days, the mycelia were filtered, weighed and the absorbance was measured at 475 nm;[12]. Chemical Analysis of Molasses Waste Water: Chemical Oxygen Demand (COD) and Biological Oxygen Demand (BOD), Nitrates, Ammonia of the molasses waste water after and before microbial treatment,[14]. Total sugars:was measured by the method of[15]. Total nitrogen:was measured according to.[16]. PH was estimated by pH meter model M62 (standard pH meter, Denmark) equipped with a glass electrode. Agro- Industrial Waste as Adsorbents: Wood Chips: Sieved local wood chips was used to decolorize molasses waste water;[17]. Corn cob shred was obtained from corn plant and stored in a freezer until used.; Starch and glucose company, Mustrud. Sugarcane bagasse were produced locally, washed thoroughly to remove dust,pulverized to mesh size using a blender and, air dried with occasional mixing.Agro-industrial wastes were used without pretreatment with 2.5g /50ml molasses waste water;[6]. Fifty ml molasses waste water medium was prepared as described, and the adsorbents were added. Sterilization, incubation conditions are the same as in P. chrysosporium inoculated flasks. Decolorization Assay: After being centrifuged at 6000 x g for 15 min, 2.0 ml of the clear cultured supernatant from experimental (treated) and control(untreated) culture were used to find the difference in absorbance maximum at 475 nm using UV-visible spectrophotometer[18].

Removal (%) = Initial ab s o r b a nce-Final absorbance/Initial absorbance x100 RESULTS AND DISCUSSION Screening the Potentiality of Fungi for Molasses Biotreatment: As it could be seen from Table (1), P. chrysosporium is more effective in removing 30.099 (%) of color after 6 days of incubation comparing to 7.223 (%) color removal by Rhizopus oryzae. In addition to P. chyrsosporium, A.niger, Rhizopus oryzae; Nagarathnamma and Bajpai[19] Flavodon flavus; Raghukumar et al[12] proved their efficiency in color removal. Fu&Viraghavan[20] and Coulibaly et al[21] stated that, the decolorizing ability of P. chrysosporium is related to extra cellular lignin peroxidase and manganese peroxidase produced in molasses waste water (MWW) in the course of de-colorization. Bio-treatment of Molasses Waste Water by Free and Immobilized: P.chrysosporium: Free cells are highly effective than immobilized as it gave 25.42 (%) removals compared to 14.23 (%) of the immobilized cells,this may be due to aerobic nature if the fungus. as indicated in Table (2), results are in the contrary to Raghukumar et al[12] who stated that Flavodon flavus, which is highly potent in de-colorization of MSW on immobilizing it in polyurethane foam, yielding values similar to its free mycelium. Effect of Viability of P. chrysosporium Cells: Table (3) compared the color removal ability between freshly grown vegetative cells and heat inactivated cells 30.87 (%) de-colorization by viable compared to 20.6 (%) by autoclaved cells after 6 days. This may be due to, the transportation across the cell membrane yields intracellular accumulations, which is dependent on the cell's metabolism. This means that this kind of bio-sorption may take place only with viable cells. It is often associated with an active defense system of the microorganism. While during non-metabolism dependent bio-sorption, substance uptake is by physico-chemical interaction between the compound and the functional groups present on the microbial cell surface. This is based on physical adsorption, ion exchange and chemical sorption, which is not dependent on the cells' metabolism. Cell walls of microbial bioma s s , ma i n l y c omposed of polysaccharides, proteins and lipids have abundant metal binding groups such as carboxyl, sulphate, phosphate and amino groups. This type of biosorption, i.e., non-metabolism dependent is relatively rapid and can be reversible[22].

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Table 1: Screening the potentiality of fungi for molasses bio-treatment. culture Final absorbance (O.D. at 475nm) (%)Color removal Dry weight (g/l) P.chrysosporium 0.987 30.099 6.97 -------------------------------------------------------------------------------------------------------------------------------------------------------------------------------Rhizopus oryzae 1.31 7.223 5.42 Initial absorbance O.D.1.412 Table 2: The effect of P. chrysosporium free and immobilized cells in de-colorization of molasses. Mode of cultivation Final absorbance (O.D. at 475nm) (%)Color removal Dry weight (g/l) P. chrysosporium free cells 1.053 25.42 8.11 -------------------------------------------------------------------------------------------------------------------------------------------------------------------------------Immobilized cells 1.211 14.23 7.12 Initial absorbance.O.D. 1.412. Table 3: Use of heat inactivated cell P. chrysosporium in molasses de-colorization. Treatment Final absorbance (O.D. at475) (%)Colour removal Dry weight(g/l) Live cells 0.976 30.87 10.87 -------------------------------------------------------------------------------------------------------------------------------------------------------------------------------Autoclaved cells 1.121 20.6 7.12 Initial absorbance O.D. 1.412.

Effect of Glucose Concentrations on Molasses Biotreatment by P. chrysosporium: Due to depletion of carbohydrate in the reused molasses; 11.34 mg/ml, it is a trial to supply glucose as carbon source for decolorization process. The mycelial weight was shown to increase with increase in glucose to 8.66 g/l at 2.5(%) glucose concentration. However, there is no relation between the mycelia weigh and color removal, this is in agreement with;[23]. As table 4 indicated, 1 (%) glucose had the most effect on color removal when supplemented to the molasses basal medium resulted in dry weight of 6.43 (g/l) as it result in 43.27 % color removal after 6 days,so it will be used in next decolorization experiments. Watanabe et al.[24] showed a relation between glucose concentration and color removal as it reached its maximum with 2.5 % (w/v) glucose. Ghosh et al[25] stated that glucose concentration was critical for de-colorization, and improved color removal efficiency was obtained by periodic replenishment of glucose. Effect of Nitrogen Source on Molasses Bio-treatment by P. chrysosporium: Molasses seemed to be poor in nitrogen, so external nitrogen compound may be used[11]. As, molasses waste water contains 0.58 % (w/v) nitrogen.so, enrichment with nitrogen will be somewhat limited. Data presented in table 5, showed that different nitrogen sources at 1.25% (w/v) with1% (w/v) glucose were somewhat effective in decolorization process; yeast extract decolorizes with 29.55(%) efficiency, peptone with 35.05 (%) and malt extract with 15.65 (%) .i.e. peptone is the most effective in color removal after 6 days and will be used in the next experiments.; Kirk et al.[26] stated that P. chrysosporium enzymatic systems catalyse degradation of lignin and lignin-like materials during the secondary phase of the metabolic growth. Synthesis and secretion of lignin peroxidase or ligninase (LiP) and manganese-dependent peroxidase (MnP) are

triggered by nutrient limitations such as source of carbon and/or nitrogen); the addition of these nitrogen sources increased the mycelial dry weight. Ohmomo et al.[27] used 0.05% peptone in highest decolorization with Aspergillus oryzae also,Sirianuntapiboon et al.[18] assured that highest activity of removal was obtained with addition of a nitrogen source. Effect of Temperature: As it could be estimated from table 6,300C was the optimum for maximum decolorization by P. chrysosporium. after 6 days with glucose and peptone addition; 40. 297 (%). Miranda et al.[28] used the same temperature 30 NC for decolorization by A.niger. Ohmomo et al.[27] established a direct relationship between increase in temp and increase in melanoidin removal was 40NC and 45NC for free and immobilized cells respectively. The increase in temperature may have caused the impairment to decolorization mechanism`. Also, Gomaa et al[29] stated that higher temp may lead to disorption processes on using A.niger. Dependence of Colorization on Contact Time: Table 7 indicated that, the maximum removal (46.883) % was after 8 days incubation with increase in dry weight to 9.45 g/l. After 10 days the mycelium dry weight was 10.41 g/l was with the least de-colorization (11.432) % in the contrary to results obtained by Aoshima et al.[23] who stated that the increase in the mycelial dry weight correlated to the pigment removal. Gomaa et al[29] stated that the time needed for de-colorization varied from strain to another and it increased the mycelia weight but not the de-colorization process. Effect of Nutrients Addition on Removal of Melanoidin Pigment in Molasses Waste Water by P. chrysosporium: As could be seen from Fig. (1), The overall addition (%w/v) of KH2PO4,0.25; MgSO4.7 H2O, 0.125; with glucose, 1; and peptone, 1.25, to molasses

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Table 4: Effect of glucose enrichment on molasses bio-treatment by P. chrysosporium. Glucose concentration(%w/v) Final absorbance (O.D. at 475nm) (%)Colour removaL Dry weight (g/l) 0.5 0. 952 32.57 5.9 -------------------------------------------------------------------------------------------------------------------------------------------------------------------------------1 0.8 01 43.27 6.43 -------------------------------------------------------------------------------------------------------------------------------------------------------------------------------1.5 0.9 46 33 7.57 -------------------------------------------------------------------------------------------------------------------------------------------------------------------------------2 0.96 32.91 7.91 -------------------------------------------------------------------------------------------------------------------------------------------------------------------------------2.5 0.974 31.01 8.66 Initial absorbance O.D. 1.412. Table 5: Effect of different nitrogen sources on bio-treatment of molasses by P.chrysosporium. Nitrogen source(1.25%w/v) Final absorbance(O.D. 475nm) (% )Colour removal Dry weight (g/l), Yeast extract 1.001 29.55 10.11 -------------------------------------------------------------------------------------------------------------------------------------------------------------------------------Peptone 0. 917 35.05 9.98 -------------------------------------------------------------------------------------------------------------------------------------------------------------------------------Malt extract 1.191 15.65 9.01 Initial absorbance O.D. 1.412. Table 6: Effect of temperature on molasses bio-treatment of molasses P. chrysosporium. Degree(EC) Final absorbance (O.D. at475) ( %)Colour removal Dry weight (g/l) 25 0.945 33.073 10.316 -------------------------------------------------------------------------------------------------------------------------------------------------------------------------------30 0.843 40.297 10.715 -------------------------------------------------------------------------------------------------------------------------------------------------------------------------------35 0.862 38.951 10.662 -------------------------------------------------------------------------------------------------------------------------------------------------------------------------------40 0.893 36.756. 10.971 Initial absorbance O.D. 1.412. Table 7: Effect of contact time on molasses bio-treatment by P.chrysosporium. Time in days Final absorbance ( O.D. at475nm) (%)Colour removal Dry weight (g/l) 0 1.412 0 1.27 -------------------------------------------------------------------------------------------------------------------------------------------------------------------------------2 1.16 17.84 3.52 -------------------------------------------------------------------------------------------------------------------------------------------------------------------------------4 0.99 29.886 6.11 -------------------------------------------------------------------------------------------------------------------------------------------------------------------------------6 0.8 43.342 7.33 -------------------------------------------------------------------------------------------------------------------------------------------------------------------------------8 0.75 46.883 9.45 -------------------------------------------------------------------------------------------------------------------------------------------------------------------------------10 1.25 11.432 10.41 Initial absorbance O.D. 1.412.

waste water resulted in obvious increase in color removal as indicated in fig(1). As 12.16 (g/l) fungus biomass resulted in 60 % color removal with all the above nutrients after 8 days at pH5,as initial acidic pH has a critical effect on color removal due to polymerization of melanoidins. Fig 1 showed that molasses used without supplements results in less decolorization as 10.4 % color removal with 8.6 g/l biomass.The same results obtained with Fahy et al.[7] This means that molasses waste water composition was not enough to activate the biological color removal This may lead to additional of extra chemicals in the system, de-colorization run in parallel with depletion of nutrients in the medium thus affect growth. It should be mentioned that, some micro-organisms that are shown to degrade melanoidine, are not suitable for

treating waste distilleries as there is a depletion of oxygen which is necessary for degradation of melanoidines. Effect of Agitation: Table 8 indicated that 61.756 % color removal was obtained with 200 rpm with 11.232 g/l dry weight after 8 days and all nutrients addition, a result comes in agreement with Miranda et al[28] who used 200 rpm while Ohmomo et al.[27] stated that an agitation speed of 125 rpm was sufficient for maximum decolonization. Color removal under aeration is higher than under limited aeration as P.chrysosporium is aerobic. Effect of Sterilization on Molasses Waste Water Decolorization: It could be seen from table 9, when

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Fig. 1: Effect of nutrients addition on decolorization of molasses by P.chrysosporium. molasses waste water was heat sterilized and enriched, P. chrysosporium results in 62.546 % color removal when incubated for 8 days at 200rpm and 30NC, compared to 60.130 % by gamma irradiation under the same conditions.,this comes in agreement with Miranda et al[28] who stated that heat sterilization cause better de-colorization, as they used heat sterilization at 120NC for 20 min and claimed that the high temp cause structural changes in compound present in molasses waste water giving chance for an increase in decolorization by A.niger. Sterilization is essential for color removal so as not to leave space for contaminating microorganisms that can inhibit the growth or compete for the nutrients in the media; Goma et al[29]. However, Dahyia et al.[30] used non sterile condition s to remove 76 % of colour by Pseudomonas fluoresenceas immobilized on porous cellulose carrier., while Benito et al[31] proved that sterilization was not necessary in melanoidin color removal. De-colourization by Agriculture Residues;nigam et Al. (2000): Phytoremediation, the use of plants to remove environmental pollutants, shows great promise as an approach to hazardous waste elimination and /or hydrogen peroxide evolution which is responsible for colour removal .The de-colorization may be by physical adsorption or enzymatic; as Watanabe et al[24] stated that the enzyme catalyzing melanoidin de-colorization for Coriolus sp. was proved to be by L-sorbose oxidase and glucose oxidase. Melanoidin was decolorized by active oxygen and H2O2 produced by the reaction of these oxidases .Gomaa et al[29] assume that the increase in molasses colour removal by A. niger is probably due to glucose oxidase produced by the mycelia that oxidize glucose to gluconic acid. Also, they stated that, one of the important characteristic of biosorbents whether it can be regenerated to be used by several sorption desorption cycle with similar efficiency as the fungal biomass could be eluted by some organic solvent. As it could be seen from table 10, Under optimum condition, P.chrysosporium resulted in 62.956(%) removal and is advanced over the other untreated culture residues, this may be due to the fungus enzymatic system. Comparison Between Treated and Untreated Molasses: The organic content of domestic agriculture and industrial wastes causes the largest pollution problems as it is metabolized by microorganisms present in the receiving water causes the largest pollution thereby causing a rapid depletion of oxygen and elimination of natural aquatic flora and fauna,the high NO3, NH4 and low pH were considered toxic to aquatic life. Also, the dark color affects penetration of light thus harm flora. Gonzales et al[32] obtained 61.7(%) reduction in COD after 7 days of fungal treatment by Trametes sp of distillery wastewater generated by ethanol using sugar cane molasses. Several methods have been devised for the treatment and removal of heavy metals as well.;[33]. Spent wash disposal into environment is hazardous and has high pollution potential. According to the Egyptian law of 48/1982 for maximum constituents limits in

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Table 8: Effect of agitation speed on molasses bio-treatment by P. chrysosporium.. (rpm) Final absorbance (O.D. at475) (%) Colour removal Dry weight(g/l) 100 0.85 39.8 10.283 -------------------------------------------------------------------------------------------------------------------------------------------------------------------------------150 0.61 56.79 11.661 -------------------------------------------------------------------------------------------------------------------------------------------------------------------------------200 0.54 61.75 11..232 -------------------------------------------------------------------------------------------------------------------------------------------------------------------------------250 0.77 45.467 12.452 Initial absorbance O.D. 1.412. Table 9: Effect of sterilization on molasses bio-treatment by P. chrysosporium. Mode of sterilization Final absorbance (O.D. at475) Colour removal (%) Dry weight(g/l) Heat sterilization 0.528 62.546 9.12 -------------------------------------------------------------------------------------------------------------------------------------------------------------------------------Gamma irradiation 0.565 60.13 11.98 Initial O.D 1.412. Table 10: Mode of bio-treatment by culture wastes and P. chrysosporium. Mode of adsorption Final (O.D. at475) (%)Colour removal P. chrysosporium 0. 523 62.956 -------------------------------------------------------------------------------------------------------------------------------------------------------------------------------Wood chips 0.56 60.33 -------------------------------------------------------------------------------------------------------------------------------------------------------------------------------Corn cob 0.621 56.019 -------------------------------------------------------------------------------------------------------------------------------------------------------------------------------Sugar cane bagasse 0.954 32.436 Initial absorbance O.D. 1.412. Table 11: Comparison of some environmental parameter for molasses waste. Factor Molasses waste Molasses treated by 48/1982 Permissible 44 /2000 Permissible water(mg/l) P. chrysosporium limits (mgO2/ml) limits (mgO2/ml) BOD 600 522 600 20-30 -------------------------------------------------------------------------------------------------------------------------------------------------------------------------------COD 8267 5557 1100 30-40 -------------------------------------------------------------------------------------------------------------------------------------------------------------------------------NO3 2.2 1.11 30 -------------------------------------------------------------------------------------------------------------------------------------------------------------------------------NH4 0.48 0.42 0.5

liquid effluent discharged into river Nile and 93 /1962 law which is modified to 44/2000 for liquid waste in the public sewage wastes. Table 11 indicated the efficiency of treating molasses waste water by fungal sludge before its disposal into the environment, by decreasing BOD to 522,COD to 5557 also, NO3 to 1.11 and NH4 to 0.42(mg/l). BOD, COD, chemical elements and color substance vary hourly,seasonally, in addition to molasses itself as colorants rather than melanoidins due to phenolic compounds are present. Beet molasses contains much melanin;Vassanasak and Pawinee[34]. However, Jiranuntipon, et al[35] stated that bioprocess of melanoidins removal stability and maintenance of removal activity depends upon growth, culture conditions, and nutrient supplements ACKNOWLEDGMENT

The authors are indebted to Prof. Dr.S. Abo El Ala, Water Pollution,Research Dept., NRC for her guide in this research. REFERENCES 1. Brown, Jr., V. Arthur, Karrasch, J. Richard, 1980. Molasses-containing animal feed having resilient structure, US4212896. Landas-Jocelyn, D.G., Elizabeth C. Bugante, Del Rosario, J. Ernesto, 2000. Characterization of Melanoidin Decolorization by Local Isolates of Aspergillus niger . Philippine Journal of Biotechnology, 11(1). Raghukumar, C. and G. Rivonkar, 2001. Decolorization of molasses spent wash by the white-rot fungus Flavodon flavus, isolated from a marine habitat. Appl. Microbiol. and Biotechnol., 55: 510-514.

2.

3.

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4.

5.

6.

7.

8.

9.

10.

11.

12.

13.

14.

15.

Banat, I.M., P. Nigam, D. Singh and R. Marchant, 1996. Microbial decolourization of textile dye containing effluent, A review. Biores. Technol., 58: 217-227. Nigam, P., G. Amour, L.M. Banat, D. Singh and R. Marchant, 2000. Physical removal of textile dyes from effluent and Solid state fermentation of dye-adsorbed agricultural residues. Biores. Technol., 72(32): 219-236. Prasad, R.K., 2009. Color removal from distillery spent wash through coagulation using Moringa oleifera seeds: Use of optimum response surface methodology. J. of Hazardous Materials, 165(1-3): 804-811. Fahy, V., F.J. Fitz Gibbon, G. McMullan, D. Singh and R. Marchant, 1997. Decolorization of molasses spent wash by Phanerochaete chrysosporium. Biotech. Lett., 19(1): 97-99. Kahraman, S. and O. Yesilada, 2003. Decolorization and bioremediation of molasses by white rot fungus in a semi solid condition. J. Biosc. Bioeng., 89: 145-150. Gadd, G.M., 1988. Accumulation of metals by microorganisms and algae in Biotecgnology, 6., Special microbial processes. Edited by HJ. Rrehm Weinheim. German Fedral Rebublic VCH.Verlags Gselleschsft MBH. Alaoui, S.M., M. Merzouki, M.J. Penninckx and M. Belemlih, 2008. Relationship between cultivation mode of white rot fungi and their efficiency for olive oil mill wastewater treatment. Elect. J. of Biotech. 11(4). Gad, A.S., 2003. Modification of molasses for kojic acid production. by A.parasiticus. N. Egy. J. Microbiol., 5: 14-26. Raghukumar, C., C. Mahandass, K. Shipa and M. Shailaja, 2004. Simultananeous detoxification and decolorization of molasses spent wash by immobilized culture of rot fungus Flavadon flavus isolated from marine habitat. Enz. Microb., 35: 197-202. Christensen, E.A., N.W. Holm and F.A. Juul, 1967. Radio-sterilization of medical devices and supplies. In Radio-sterilization of medical products STI/Pub/157. Vienna, IAEA. A.P.H.A., 2001. Standard methods for the examination of water and waste water (20th ed.). American Public Health Association, Washington. Dubois, M., D.A. Gilles, J.K. Hamilton, P.A. Rebers and F. Smith, 1956. Colorimetric method for the determination of sugars and related substances. Anal. Chem., 28(3): 350-356.

16. A.O.A.C., 1975. Official methods of analysis of association of official anal.chemist.published by Association of Official Anal. Chem. Washington. 17. Malakootian, M., A. Almasi and H. Hossaini, 2008. Pb and Co removal from paint industries effluent using wood ash. Int. J. Env.. Sci. Tevch., 5(2): 217-222. 18. Sirianuntapiboon, S., P. Somchai, S. Ohmomo and P. Attampasampunna, 1988. Screening of filamentous fungi having the ability to decolorize molasses pigment. Agric Biol. Chem., 52: 387-92. 19. Nagarathnamma, R. and P. Bajpai, 1999. Decolorization and detoxification of extractionstage effluent from chlorine bleaching of Kraft pulp by Rhizopus oryzae. Appl. Environ. Microbiol., 65: 1078-82. 20. Fu, Y. and T. Viraraghavan, 2001. Fungal decolorization of dye waste waters. A review. Biores.Technol., 79: 251-261. 21. Coulibaly, L., G. Gourene and A.N. Spiros, 2003. Utilization of fungi for biotreatment of raw wastewaters Afric.J. of Biotech., 2(12): 620-630. 22. Kuyucak, N. and B. Vole sky, 1988. Biosorbents for recovery of metals from industrial solutions. Biotechnol. Lett., 10(2): 137-142. 23. Aoshima, I., Y. Tozawa, S. Ohmomo and K. Uden, 1985. Production of decolorizing activity for molasses pigment by Coriolus versicolor Ps4a. Agric. Biol. Chem., 49: 2041-2045. 24. Watanabe, Y., R. Sugi, Y. Tanaka, S. Hayashida, 1982. Enzymatic decolorization of melanoidin by Coriolus sp.no. 20. Agric. Biol. Chem., 46: 16231630. 25. Ghosh, M., A. Ganguli and A.K. Tripathi, 2009. Decolorization of anaerobic-ally digested (MSW) by Pseudomonas putida. Appl. Biochem and Microbiol., 45(1): 68-73. 26. Kirk, T.K., E. Schultz, W.J. Connors, L.F. Lorenz, J.G. Zeikus, 1978. Influence of culture parameter of lignin metabolism by P. chrysosporium. Arch. Microbiol., 117: 177-85. 27. Ohmomo, S., M. Kainuma, S. Sirianuntapiboon, I. Aoshima and P. Atthasampunna, 1988. Adsorption of melanoidin to the mycelia of Aspergillus oryzae Y-2-32. Agric. Biol. Chem., 52: 381-386. 28. Miranda, M.P., G.G. Bento, N.S. Cristobal and C.H. Nieto, 1996. Color elimination from molasses wastewater by Aspergillus niger, Biores, Technol., 57: 229-235. 29. Gomaa, O., H. Abdel-karem, Z. Mattar and H. Hassanien, 2003. De-colorization of molasses waste water using A.niger. E.J. Biotech., 13: 1526.

1973

J. Appl. Sci. Res., 6(12): 1966-1974, 2010

30. Dahiya, J., D. Singh and P. Nigam, 2001. Decolourization of synthetic and spent wash meladonin using white rot fungus Phanerochaete chrysosporium JA4-40. Biores. Technol., 78(1): 9598. 31. Benito, G.G., P.M. Miranda, R.R. Ribn, C.M. Sanz and G. Cube, 2001. Development of biological treatment process with Tarmetes versicolar to decolorize waste water from alcoholic molasses. International water conference (IWC 2001) Proceedings. Porto (Potrugal), 26-29. 32. Gonzales, T., M.C. Terron, S. Yigue, E. Zapico, G.C. Galleti and A.E. Gonzales, 2000. Pyrolysis/ gas chromatography/mass spectrometry monitoring of fungal biotreated distillery wastewater using Tarmetes sp. 1-62(CET 20197). Rapid Commun. Mass Spectrum., 14(15): 1417-1424.

33. Ahalya, N., T.V. Ramachandra, R.D. Kanamadi, 2003. Biosorption of heavy metals. Res. J. Chem. Environ., 7(4): 71-78. 34. Vassanasak, L. and C. Pawinee, 2010. Decolorization of molasses melanoidins and palm oil mill effluent phenolic compounds by fermentative lactic acid bacteria J.of Envir. Sci.), 1209-1217. 35. Jiranuntipon, S., S. Chareonpornwattana, S Damronglerd, C. Albasi and Marie-line Delia, 2008. Decolorization of synthetic melanoidinscontaining wastewater by a bacterial consortium. J. Ind. Microniol. Biotechnol., 35: 1313-1321.

1974