A Title

A biological adsorption process for reduction of color, BOD, COD and TDS from distillery effluent.

Field of invention

This invention relates to a unique biological adsorption process for reduction of color, BOD, COD and TDS from distillery effluent.

Background of the need for invention

Pollution has been defined in various ways. It is considered as the release of unwanted substances to the environment by man in quantities that damage either the health or the resources itself. Water pollution involves the release of small amounts of substances directly through point sources or indirectly through non point sources. Industrial effluents from various industries like textile, dyestuffs, paper and pulp, distillery, olive oil mill and metal industries etc. are the major contributors to water pollution as they create more subtle effects on behavior, reproduction or even survival of biotic communities. The physical and chemical methods of industrial effluent treatment processes remove organic pollutants at low level; they are highly selective to the range of pollutants removed and prohibitively expensive. Control of pollution is one of the prime concerns of society today with economic constraints on pollution control processes, affordable and effective methods have become a necessity. Untreated or partially treated wastewaters and industrial effluent discharges into natural ecosystems pose a serious problem to the ecosystem and the life forms. Microbial treatment systems have advantage of being simple in design and low in cost (Baaat etal, 1996).

The alcohol distilleries in India generate around 45 billion liters of wastewater annually which is released in inland surface water or on land which is characterized by high BOD (45,000-60,000 mg/L), high COD (80,000-160,000 mg/L) and dark color.

Conventional waste water treatment processes are unable to remove the color from distillery spent wash. Due to dark brown color of this effluent, it has the

potential to block out light from contaminated waste water. Physical/Chemical treatments of distillery effluent lead to formation of large quantities of sludge, as well as simultaneous generation of other hazardous by products or pollutants and not cost effective. The biomethanation process is not efficient in degrading coloring compounds of distillery effluent. The recalcitrant nature of effluent is due to the presence of brown polymers.

In the absence of proper treatment methodologies to such difficult effluent, large tracts of land and water bodies have been severely affected to almost irreversible levels. With ever increasing demand on sugar, distillery, paper, textiles etc., quantity of such effluent is increasing at a very rapid pace. If not done so, then within a decade there will be no land left even to build houses. Such is the situation staring in our face.

Hence developing a cost effective and environmentally friendly efficient technology to decolorize distillery effluent is the need of the society and country. Conventional systems

Clarifiers, aerated lagoons, trickling filters, anaerobic lagooning, concentration, followed by incineration or concentration, followed by composting, biomethanation and other biological systems are the conventionally used systems for disposal of distillery effluent. All these treatments remove reasonably good amount of COD, BOD but not color toxicity, inorganic impurities and cost intensive. In composting, land requirement is high and chances of leaching out color are more, as well as there is formation of large quantities of sludge. Due to stringent environmental norms, such partially treated effluents of distillery industry are not allowed to mix in the natural stream. Non Conventional systems

Bioremediation of toxic industrial effluents by microorganisms serves as an effective method to substitute the conventional recovery and removal processes. Fungal biomasses have huge capability of treating effluents discharged from various industries.

Fungi, their biology, economic value and pathogenic capabilities are not new to human society. They have been used from fermentation of foods to production of pharmaceuticals. Fungi thrive well in inhospitable habitats with environmental extremes because of their enzyme system (Cooke, 1979). Fungi are involved in the

biodegradation of undesirable materials or compounds and convert them into harmless, tolerable or useful products. Many organisms are involved in the biodegradation of organic waste, which has resulted in the production of novel substances of biotechnological importance. Fungi are recognized for their superior aptitudes to produce a large variety of extra cellular proteins, organic acids and other metabolites, and for their capacities to adapt to severe environmental constraints (Lilly and Barnett, 1951; Cochrane, 1958). Fungal systems appear to be most appropriate in the treatment of colored and metallic effluents (Ezeronye and Okerentugba, 1999). Fungi not only produce various metabolites like citric acid, homogeneous proteins, heterogeneous proteins, peroxidases but have shown their effectiveness for removal, reduction and detoxification of industrial effluents ingredients. Bioremediation refers to the productive use of microorganisms to remove or detoxify pollutants, usually as contaminants of soil, water or sediments that otherwise threaten public health. Microorganisms have been used to remove organic matter and toxic chemicals from domestic and industrial waste discharged for many years (Gupta and Mukerji, 2001).

State of the art in the field

In the prior art, the reports of the research work on biological decolorization and reduction of COD, carried out by different workers using microbial cultures, for model melanoidin and melanoidin containing distillery waste water under laboratory conditions are described in brief herein below.. Commercialization of the biological decolorization method is difficult due to low biodegradability of wastewater colorants.

Ohmomo, S; Daengsubba, w; Yosikawa, H; YuIi, M; Nozaki, K; Nakajima, T and Nakaraura, I. (1988), Screening of anaerobic bacteria with the ability to decolorize molasses melanoidin. Journal of Agricultural biological chemistry, 52, 2429 -2435. In screening of the facultative anaerobic bacteria, a strain identified as Lactobacillus hilgardii gave 28% melanoidin decolorization activity. When the cells were immobilized in calcium alginate gel activity increased to 40%. Unlike organisms

previously tested, this bacterium rapidly decolorized the lower molecular weight fractions of melanoidin.

Kumar, V., Wati, L-, FitzGibbon, F., Nigam, P^ Banat, LM., Singh, D., Merchant, R., 1997b. Bioremediation and decolorization of anaerobically digested distillery spentwash. Biotechnology Letters. 19 (4), 311-314. These workers have isolated facultative anaerobic bacterial culture L-2, which was able to decolorize anaerobically treated effluent in absence of nitrogen source. The organism showed 31 % decolorization and 57 5 COD reduction for 12.5 % ( v/v), biodigested spent wash.

Sirianuntapiboon, S.; Somachai, P.; Sihanonth, P; Atthasampunna, P.

(1988), Microbial decolorization of molasses wastewater by Mycelia sterilia D-90. Journal of Agricultural biological chemistry,52, 393-398, A filamentous fungi Mycelia sterilia D-90, showed the highest decolorization yield (about 93%) when it was cultivated at 30 ⁰ C for 8 days in molasses pigment solution containing glucose 2.5% yeast extract 0.2%, KH ₄ PO ₄ 0.1% and MgSO ₄ . 7H ₂ O 0.05% and pH 6.0. In the fed- batch system this strain showed a constant decolorization yield of about 80% and caused a decrease in the biological oxygen demand value of about 70% during three times replacement (24 days).

Fahy, V., Fitzgibbon, F.J., McMuIIaaa, G.A.,Singh, D. and Merchant, R.,

(1997). Decolourization of molasses spentwash by Phanerachoete chrysosporium. Biotechnology Letters. 19(1), 97-99. Fahy et al, have isolated Phanerochoete chrysosporium strain capable of decolorizing molasses wastewater in absence of either glucose or peptone. This reported for the fist time that the fungus is capable of decolorizing molasses wastewater without supplementation of additional carbon source. 85 % decolorization in free cells reduced to 59 % with immobilized cells after 10 days of incubation.

Ohmomo, S^ Kainuma, M., Kamimura, K-, Sirianuntapiboon, S., Aoshima, I., Atthasampunna, P. 1988. Adsorption of melanoidin to the mycelia of Aspergillus oryzae Y-2- 32. Agric. Biol. Chem. 52, 381. Aspergillus oryzae Y-2-32 is a

thermophilic fungal strain. It shows the activity of decolorizing the distillery spent wash (43%) by adsorbtion of melanoidin to its mycelia in synthetic melanoidin medium. Autoclaved mycelium of Aspergillus oryzae adsorbed melanoidin, especially lower molecular weight Fractions and the degree of the adsorbtion were influenced by the kind of sugars utilized for growth. The melanoidin adsorbing activity of mycelia was observed to be repressed by a high concentration of salt. Furthermore, it decreased to half the initial level on washing with 0.1% sodium dodecyl solution.

Wafea M. Abd El-Rahim, Dr.- 17 JuI 2006, Assessment of textile dye remediation using biotic and abiotic agents, Journal of Basic Microbiology, Volume 46, Issue 4 , 318 — 328, (Agricultural Microbiology Department, National Research Centre, Dokki, Cairo, Egypt). Removal of dyes and their derivatives from aqueous solutions was investigated using sugarcane bagasse, sawdust, rice straw, charcoal and fungal biomass as dye removing agents. Seven fungal strains known to have high capacity in removing textile dyes were used. Results of this study indicated that Penicillium commune, P. freii, and P. allii removed 96, 64 and 65%, respectively, of direct violet dye after two hours of incubation. In addition, the use of rice straw was shown to be more efficient in dye removal, than was bagasse or sawdust. Rice støw was effective in removing 72% of direct violet dye within 24 hours. However, with reactive dyes, removal activity was reduced to 27%. Similar trends were recorded with the other tested biotic agents, fast removal of reactive dye was not found after 48 hours of contact time. Results of this study indicate that low-cost, renewable, bioadsorption agents are relatively effective in removing textile dyes from solution.

Deepika Swami, D. Buddhi, 2006; Removal of contaminants from industrial waste water through various non-conventional technologies: a review, International Journal of Environment and Pollution (IJEP), Vol. 27, No. 4. It is difficult to separate industrial growth from environmental pollution but it can be minimised through cost- effective approaches of pollution abatement. To reach the full objectives of zero pollution, adoption of alternative technologies which suit the situation of low capital availability, minimum man-power and limited energy consumption are necessary. Adsorption through agricultural products such as rice husk, sugarcane bagasse,

soybean hulls, saw dust, coconut shell, groundnut shell, apple-waste, fly-ash etc., has been demonstrated to be a useful alternative to the conventional treatment systems for the removal of toxic metals such as dyes/colour, chromium (Cr), mercury (Hg), copper (Cu), nickel (Ni), etc-, from aqueous solution. It could be considered as an eco-friendly device to the existing relatively more expensive treatment technologies. Various biological species such as algae, fungi and bacteria were found to be in extensive use for the removal of contaminants. In this review, an extensive list of sorbents literature has been compiled to provide a summary of available information on a wide range of potentially low-cost non-conventional sorbents and their effectiveness is only available to individual subscribers or to users at subscribing institutions.

G.K. Parshetti, S.I>, Kaime, $,S« Gomare and S,P, Govindwan December 2007, Bϊodegradation of Reactive blue-25 by Aspergillus oehraceus NClM-1146, Bioresource Technology, Volume 98, Issue 18, 3638-3642. The study dealt with the decolorization and degradation of textile dye Reactive blue-25 (0.1 g F ¹ ) by mycelium of Aspergillus oehraceus NCIM-1146. Spectrophotometric and visuaL examinations showed that the dechlorination was through fungal adsorption, followed by degradation. Shaking condition was found to be better for complete and fester adsorption (7 h) and decolorization (20 days) of dye Reactive blue-25 (100 mg F ¹ ) as compared to static condition. Presence of glucose in medium showed faster adsorption (4 h) and decolorization of dye from bound (7 days) mycelium. FTIR and GCMS analysis study revealed biodegradation of Reactive blue-25 into two metabolites phthalimide and di-isobutyl phthalate.

Chulhwan Park, Myunggu Lee, Byuaghwaa Lee, Seang-Wook Kim, Howard A. Chase, Jinwon Lee and Sangyong Kim; 15 August 2007, Biodegradation and biosorptiσn for decolorization of synthetic dyes byFunalia trogii Biochemical Engineering Journal, Volume 36, Issue 1, 59-65. The scientists investigated two major mechanisms for decolorization of dyes by cultivation of fungi on either a solid or in a liquid phase and to confirm the possibility of practical application via repeated-batch cultivation. The decolorization of six commercial dyes

with 10 fungal strains was studied. Enzyme activity, decolorization trends and decolorization mechanisms were monitored. Under the experimental conditions, extracellular laccase and manganese peroxidase (MnP), but not lignin peroxidase (LiP) _T were detected. The decolorization mechanisms by F. trogii ATCC 200800 involved a complex interaction of enzyme activity and biosorption. This study suggests that it is possible to decolorize a high concentration of commercial dyes, which would be a great advance in the treatment of dye containing wastewater. These methods may have a potential application for dye decolorization and for textile effluent treatment

Ozfer Yesilada, Dilek Asma and Seval Cing; 31 January 2003, Decolorization of textile dyes by fungal pellets, Process Biochemistry, Volume 38 _f Issue 6, 933-938. Decolorization of various dyes by pellets of white rot fungi was studied. All fungal pellets used could remove more than 75% of the color of these dyes in 24 h. Effect of various conditions such as initial pH, concentration of dye, amount of pellet, temperature and agitation on Astrazone blue dye decolorization activity of Ftmalia trogii was tested and the longevity of this decolorizatioii activity under optimum conditions was investigated in repeated-batch mode. An increase in the amount of pellet positively affected the longevity of the decolorization activity while a decrease in dye decolorization capability of pellets occurred with increasing dye concentration in repeated-batch mode. Spectrophotometrie and microscopic examinations of F. trogii pellets showed that the process involved decolorization through microbial metabolism but not biosorption. The effect Qf glucose concentration and cheese whey on longevity of decolorization activity was also tested. The percentage of decolorization at a dye concentration of 264 mg/1 remained high after 10 days operation especially in culture media with cheese whey. This study showed that white rot fungal pellets eould effectively be used as an alternative to the traditional physicochemical process.

Object of the invention

1. The principal object of the this invention is to provide a process for reducing color, BOD, COD and TDS of distillery effluent and make it suitable for reuse or fit for land disposal.

2. Another object of this invention is to provide a low cost, easy to use, eco friendly process for object as mentioned in 1 «

3. A further object is to provide a process for treating all kinds of colored effluents like Textile, Paper and pulp (black liquor), Tannery etc., and make the treated water suitable for reuse or for disposal.

Statement of the invention

The present invention provides a novel method for decoloration and reduction of BOD, COD and TDS of distillery effluent without diluting the effluent or without addition of any chemicals and is carried out at ambient temperatures. The treated water can be reused or used for inland disposal. Addition of chemicals is limited to pH correction only. As there is no need of dilution of effluent, load on fresh water requirement and former sizing of equipments, pumps, handling and operation is greatly limited. As chemical usage is very least, need for procurement, storage, handling of hazardous chemicals and special training to do so is eliminated. There is no additional sludge burden created further eliminating need for costly sludge disposal methodologies. The entire process is carried out at ambient temperature thus the system is energy efficient The biomass can be reused; hence there is less recurring eost.

Summary of the invention

Several methods of decolorizing spent wash have been reported up till now, including physical treatments using bone char or activated carbon, chemical

treatments using flocculaats and biological treatments using fungi and bacteria, Physical/Chemical treatments lead to formation of large quantities of sludge as well as simultaneous generation of other hazardous by products or pollutants and are highly costly. To develop cost effective and environmentally friendly efficient technology to decolorize distillery effluent is the need of the society and country.

The present invention relates to the use of a biological adsorption process for the treatment of distillery effluent by using the fungal biomass of Aspergillus oryzae, which comprises steps of preparation of fungal biomass and mixing the pre-grown biomass with, the pH corrected undiluted distillery effluent, allowing the contact of biomass with the effluent at ambient temperature for the contact time needed for maximum decolorization and reduction of BOD, COD and TDS of the effluent. The reduction of color and other parameters like BOD, COD and TDS of the effluent takes place by simple adsorption technique. It is seen that the mycelia retains the efficiency of adsorbing the coloring pigment in any form such as wet, autoclaved and dried biomass in the decolorization experiments. The biomass saturated with the adsorbed color pigments could be regenerated for decolorization of effluent again by washing it with dilute NaOH. After the complete exhaustion of the capacity of the biomass, the biomass in the form of sludge is used as manure after composting or disposed off as required. Thus the present invention provides a safe, economical, compact, easy to use and eco-friendly solution to the burning problem of treatment and disposal of colored industrial effluents like distillery, textile, paper and pulp (black liquor) and tannery effluents.

Detailed description of the invention

This invention provides a biological system which consists of utilization of adsorption property of fungal biomass of Aspergillus oryzae found commonly in soil or water and treatment of pH corrected undiluted effluent with the fungal biomass by allowing the contact of biomass with the effluent at ambient temperature for the contact time needed for maximum decolorization and reduction of BOD, COD and

TDS of the effluent. The treated effluent with reduced color, BOD, COD and TDS is recycled for the industrial need or disposed off as required.

Study on optimization of the growth medium parameters for maximum growth of Aspergillus oryzae, showed that the requirement of carbon source was the growth limiting parameter in the medium and glycerol was found to be the optimum carbon source at 5g% concentration. Peptone was found to be the best nitrogen source at 0.5g% concentration, pH 6, temperature ambient and rotational speed 150 rpm were found to be other important optimum parameters.

The Preparation of biomass of Aspergillus aryzae- The growth of the fungal biomass is obtained by inoculating the spores of Aspergillus aryzae in a sterile liquid medium containing glycerol 5%, peptone 0.5%, potassium dihydrogeπ phosphate 0.1 %, magnesium sulphate 0.05% and distilled water. The initial pH of the medium is adjusted to 6 and incubated at ambient temperature for 4-6 days on rotary shaker (150 rpm) for small scale growth and in fermentor for large scale growth. The mycelial growth is harvested after filtration.

The pH corrected effluent (100 ml) at pH 4.5 is inoculated with, the biomass (12.5 gm wet weight) and incubated for one hour on rotary shaker (150 rpm) at ambient temperature. After filtering mycelial biomass through four layers of cheese cloth, decolorization assay is carried out for filtrates to determine percent decolorizatioπ and analyzed for different pollution parameters.

Quantitative analysis of the pollution parameters of different distillery effluents after treatment with pre-grown wet biomass of Aspergillus oryzae fox one hour and the optimization of the process parameters for pre grown biomass based decolorization of distillery effluent by using Aspergillus oryzae, shows that the culture has strong adsorption property for the color pigments and it can adsorb the color of effluent without need of any dilution of the effluent as well as can reduce some important pollution parameters such as BOD, COD, sulphates, metals (kon, copper) and TDS efficiently.

The reduction of color and other parameters like BOD, COD and TDS of the effluent takes place by adsorption of color pigments and other organic and inorganic substances from the effluent, on the cell wall of the fungus Aspergillus oryzae.

The fungal biomass of Aspergillus oryzae is used till the saturation level of the fungal cells for adsorption and the saturated biomass is reused after conducting standard regeneration processes such as treatment with heat, alkali wash and acid wash.

After the complete exhaustion of the capacity of the biomass, the biomass in the form of sludge is used as manure after composting or disposed off as required.

In another embodiment of the invention, the fungal biomass of Aspergillus oryzae is used in combination with other biological (bacteria, fungus, algae) or non biological materials (physical or chemical agents used in the treatment of effluent).

In another embodiment of the invention, the fungal biomass is packed in a single or multiple contact containers placed in series, in which effluent enters serially in all containers.

In another embodiment of the invention, the fungal biomass is packed in any horizontal or vertical containers of any shape.

In another embodiment of the invention, the fungal biomass used is in wet or dried or live or dead form.

In another embodiment of the invention, the fungaL biomass is packed in any horizontal or vertical containers of any shape.

In another embodiment of the invention, the fugal biomass is obtained by growing the spores of the fungus on solid substrate such as bagass, press mud, wheat bran etc...for its maximum growth.

In another embodiment of the invention, the fungal biomass is obtained by growing the spores of the fungus in a liquid medium supplemented with any carbon source such as glucose, glycerol, raffinose etc

In another embodiment of the invention, the fungal biomass is obtained by growing the spores of the fungus in a liquid medium at pH ranging from 2 to 9 (optimum pH 6).

In. another embodiment of the invention, the fungal biomass is obtained by growing the spores of the fungus in a liquid medium and incubating it at any temperature ranging from 5 ⁰ C to 80 ⁰ C.

In another embodiment of the invention, the fungal biomass is obtained by growing the spores of the fungus in a liquid medium and incubating it for a period ranging from 2 days to 15 days.

In another embodiment of the invention, the fungal biomass is obtained by growing tiie spores of the fungus in a liquid medium and incubating it under stationary condition.

In another embodiment of the invention, the effluent is selected from any industry releasing colored effluents like Textile, Paper and pulp (black liquor), Tannery etc.

In another embodiment of the invention, the effluent is corrected for pH ranging from 2 to 9 (optimum pH 4.5).

In another embodiment of the invention, the effluent used for treatment is diluted to any dilution leveL

In another embodiment of the invention, the contact time for the contact of biomass with the effluent is from 5 minutes to 5 days.

Scope and/or ambit of the invention

In general the invented methodology is used wherever there is necessity of decolorization and reduction of pollution parameters like BOD, COD and TDS in the treatment of colored effluent from any industry like Textile, Paper and pulp (black liquor), Tannery etc. before disposal.

In particular the invented methodology is used for distillery effluent which shows around 80-99 % color reduction, around 70- 85% BOD reduction, around 85- 92% COD reduction and around 70- 80% TDS reduction in the distillery effluent after treatment.

Although the invention has been described with reference to specific embodiments, this description is not meant to be construed in a limiting sense. Various modifications of the disclosed embodiments, as well as alternate embodiments of the invention, will become apparent to persons skilled in the art upon reference to the description of the invention. It is therefore contemplated that such modifications can be made without departing from the spirit or scope of the present invention as defined.

Advantages of the invention

1. The present invention involves the treatment of effluent without need of dilution of the effluent.

2. The present invention involves the treatment of effluent without adding any chemicals; however addition of chemicals is limited to pH correction only.

3. The present invention involves the treatment of effluent with simple adsorption technique thus easy to operate.

4. The present invention involves the treatment of effluent in a compact format which saves huge amount of invaluable space required otherwise.

5. The present invention involves the treatment of effluent which requires least capital, operation and maintenance cost.

6. The present invention involves the treatment of effluent at ambient temperatures and least power consuming methodologies, thus saving huge energy cost which would be needed otherwise.

7. The present invention involves the treatment of effluent with, the fungal biomass of Aspergillus oryzae which is used till the saturation level of the fungal cells for adsorption and the saturated biomass is reused after conducting standard regeneration processes such as treatment with heat, alkali wash and acid wash.

8. The present invention involves the treatment of effluent without need of hazardous chemicals thus it is safe.

9. The present invention involves the treatment of effluent which provides highly decolorized and safe water to the industry by recycling the treated effluent.

10. The present invention involves the treatment of effluent which, helps in conservation of water in national interest.

11. The present invention involves the treatment of effluent which would give a highly economical and eco-ftiendly solution to the disposal of colored effluent from any industry in general and distillery effluent in particular.

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2O.Wafaa M. Abd El-Rahim, Dr.; 17 JuL 2006, Assessment of textile dye remediation using biotic and abiotic agents, Journal of Basic Microbiology, Volume 46, Issue 4 , 318 - 328.

21. Deepika Swami, D. Buddhi, 2006; Removal of contaminants from industrial waste water through various non-conventional technologies: a review, International Journal of Environment and Pollution (IJEP), Vol. 27, No.4.

22. GJC. Parshetti, S J). Kalme, S.S. Gomare and SJ*. Govindwar; December 2007, Biodegradation of Reactive blue-25 by Aspergillus ochraceus NCIM- 1146, Bioresource Technology, Volume 98, Issue 18, 3638-3642.

23. Chulhwan Park, Myunggu Lee, Byunghwan Lee, Seung-Wook Kim, Howard A. Chase, Jinwon Lee and Sangyong Kim; 15 August 2007, Biodegradation and bfosorption for decolorization of synthetic dyes by Funalia trogii Biochemical Engineering Journal, Volume 36, Issue 1, 59-65.

24. Qzfer Yesilada, Dilek Asma and Seval Cing; 31 January 2003, Decolorization of textile dyes by fungal pellets, Process Biochemistry, Volume 38, Issue 6, 933-938.