Title of Invention	AN IMPROVED PROCESS FOR THE BIODEGRADATION OF INDUSTRIAL WASTE USING A CONSORTIUM OF BACTERIA AND FUNGUS
Abstract	Present invention provides an improved process for the biodegradation of Industrial waste using a consortium of bacteria and fungus selected from curd bacteria and tea fungus . The process comprising the steps of preparing 1 : 1 industrial waste: water having concentration of aromatic compounds 1.4 g/L, treating the waste with a consortium of fungus and bacteria , in presence of assimible carbon source at a concentration 0.5 to 3.0%, having pH in the range of 4.0 to 5.8, at a temperature 28° - 37°C, for 24 h at non-sterile condition under agitation to get biodegraded industrial waste devoid of aromatic compounds and colour.

Title of Invention

AN IMPROVED PROCESS FOR THE BIODEGRADATION OF INDUSTRIAL WASTE USING A CONSORTIUM OF BACTERIA AND FUNGUS

Abstract

Present invention provides an improved process for the biodegradation of Industrial waste using a consortium of bacteria and fungus selected from curd bacteria and tea fungus . The process comprising the steps of preparing 1 : 1 industrial waste: water having concentration of aromatic compounds 1.4 g/L, treating the waste with a consortium of fungus and bacteria , in presence of assimible carbon source at a concentration 0.5 to 3.0%, having pH in the range of 4.0 to 5.8, at a temperature 28° - 37°C, for 24 h at non-sterile condition under agitation to get biodegraded industrial waste devoid of aromatic compounds and colour.

Full Text	This invention relates to an improved process for the removal of colour and degradation of industrial waste mainly containing aromatic compounds. More particularly, it relates to an improved process for the treatment of paper and pulp industry waste. The invention relates to an improved method of biodegradation and colour removal by using bacteria, fungi and yeast singly, or in consortium. When grown in waste supplemented with easily assimilable carbon source medium, the biomass strongly absorbs the colouring matter and the degraded organic pollutants including lignin, and lignin degradation compounds. Lignin is the characteristic-cementing constituent between the cell walls of woody tissues. The spaces between cellulose fibrils in woody vascular tissues are filled with lignin and hemicellulose, thus lignin is naturally available in abundance. One of the main constraints in this area is the colour of the effluent, which varies from light tan to deep brown or black. The source of colour in an integrated bleached kraft mill is mainly due to lignin derivatives produced from the extraction stage of the bleach plant. This effluent waste contains upto 3-6 % lignin. The industrial waste is highly coloured and typically accounts for 80% of the colour, 30% of the BOD (Biological oxygen demand) and 60% of the COD (Chemical Oxygen demand) of the mill pollution load. The proportion of colour and lignin are further concentrated by the practice of recycling of the effluents to conserve fresh water. In paper mill effluent treatment total dissolved solids are removed and used as energy source in fuel. Biomethenation has also been attempted. All these do not meet the standards of pollution control for colour, total dissolved solids, BOD and COD. Since the delignification of the starting material for paper and pulp manufacture is a continuous process, the industrial waste released into the environment cannot be completely avoided. In recent years regulatory agencies have become more stringent with respect to the discharge of coloured effluents to the surface waters. Hence some concrete efforts in the direction of colour removal from the spent wash are required. Lignin, its derivatives and phenolics are in general difficult to degrade by physical, chemical or biological methods. This is mainly due to the complex linkages within the molecules especially the biphenyl type carbon to carbon linkage present in lignin. Because, such compounds are not easily biodegradable, conventional biological treatment methods are only moderately effective in decreasing effluent COD. Consequently, paper industry, and other industries generating phenolic waste cannot satisfy the effluent discharge limits for COD and BOD that have been imposed by environmental pollution control board. In the prior art following processes are used for removal of lignin and colour from industrial wastes using bacterial and fungal cultures and their enzymes hitherto and described in brief herein below. A continuous biological process to decolourize black plant effluent. Chang, H.W., Compbeil, A. Biotechnol. Adv. 2(2) 301-308 (1985): The coloured waste stream from the extraction stage of a Kraft bleaching plant was treated with Phanerochaetae chrysosporium in a treatment scheme called the MyCoR process. Under optimal condition 2000 colour units /1 per day were removed alongwith concomitant removal of COD and BOD. Chlorolignins foraged were dechlorinated. Chemical characterization and biological decolourization by fungus of effluents from wheat straw alkaline pulping. Terron M.C., Rijksuniv genet: 56(49) 1569-1571 (1991). Tramates versicolour, a white rot fungus immobilized on different supports, along with granular activated carbon process achieved 80% colour removal from the effluent waste. Drawback: Required addition of activated carbon which adds to biomass, also increases the cost of the treatment. Photobleaching and biobleaching of kraft effluent. Duran Nelson Dezott; Mareia Rodiguez Jaime, J. Photochem Photobiol, A. 62(10) 269-279 (1991): Use of immobilized mycelial pellet of a fungus Chrysonilia sitophila in nylon provided effective means to decolourize lignin-containing Kraft wastewater. The immobilized microbial pellet, absorbed 25-30% colour of which 60-65% autooxidized in the presence of HjC^ in 6h. Kraft effluent is oxidatively decomposed by a semiconductor. Photocatalysed reaction in an aqueous solution containing oxygen and semiconductor powder. Under continuous illumination, the solution becomes colourless and the molecular weight distribution indicated almost total mineralization in 2h. Drawback: The process required IHkOz, special chemicals for immobilization, sterile conditions for immobilization. With all these additions, the color removal was only 25-30% Comparison and combination of ozone and fungal treatments of a kraft bleaching effluent. Roy Acrand L; Archibald F.S., Briere F. Tappi J. 74(9) 211-218 (19^1): In Kraft bleaching most of the colour and much of the toxicity is contained in the effluent from the alkaline extraction stages. Effluent was treated with ozone and the fungus Tramates (coricolus) varsicolour. Among the two treatttietits, the ftmgal treatment resulted in greater decolourization as and COD removal. Colour removal from kraft belach-plant effluents by Trichoderma sp. PMsad D.Y.,and Joyce T.W., Tappi, J.74 (1) 165-169 ) (1991): Trichoderma sp. is capable of degrading lignin and declolorizing the hardwood extraction-stage bleach-plant effluent. The optimum pH and carbon source for the fungus was determined. The results show that under optimal conditions, total Colour and COD (chemical oxygen demand) decrease by almost 85% and 25%, respectively, after cultivation for three days. Addition of nitrogen did not simulate the decolourization process, indicating that it is not a rate-limiting factor. Glucose was the most effect-carbohydrate utilized by the fungus, as a stimulated substantial colour reduction without any increase in COD. Drawback: The method required glucose, which is more costly as compared to commercial sucrose. Reported COD removal was only 25% Absorption effect in the dicolourization of a Kraft bleach effluent by Phanerochaete chrysosporium. Jaspers C.J. and Penninckx, M.Y., Biotechnology Letters 18(11) 1257- 1260 (1996). Biotechnol. Letters. 18 (11) 1257-1260(1996:. The fungal pellets inoculated in the effluent, without addition of nutrients, strongly absorb colour (1% glucose was added as a quickly metabolizing carbon source for growth of the fungus). Sixty- percent decolourization was achieved at 37 °C and 2.0 pH. Drawback: Required lots of acid for adjusting the pH to 2 before fungal treatment. .It appeared that reported color removal of 60% could be due to the acid. Degradation of lignin and decolourization of paper mill bleach plant effluent (BPE) by marine fungi. Raghukumar C.D., Chandramohan D., Michel F.C., and Reddy C.A. Biotechnology Letters 18(1) 105-106 (1996): Isolation of a lignin decolourizing bacterium. Moril Hiroshi, Nakamiya Kunichika and Kiroshita Shinichi. J. Fermentation and Bio-engineering 80(3) 296-299 (1995): A bacterial strain from soil namely Azatobacter spp. degraded and decolourized lignin very rapidly under optimum conditions. However the maximum color removal was observed in presence water, on of dead organic matter and are the principal life forms exposed to xenobiotics in the environment. Thus biotechnological applications use these life forms chiefly bacteria and fungi in hazardous waste treatment. A "Tea fungus" commonly designated as "Kombucha" in China, Japan, USA or "Bhakari" in India, is a symbiotic culture of atleast three microoganisms [Blanc J. Philppe. Biotechnology Letters 18(2): 139-142 (1996)]: the acetic acid bacterium Acetobacter xylimim and two yeasts Zygosaccharomyces roitxii and Candida sp. forming a floating cellulose mat in sugar tea. [Hesseltine C.W. A millenium of fungi, food and fermentation: In Mycologia 57(2): 149-197 (1965);. "A Tea fungus was obtained locally and maintained on sugar tea for seven days under non sterile conditions. "Lactic acid bacteria" in "curd culture" were maintained by 8hr subculture on milk under non-sterile conditions. Purity of these cultures under non-sterile conditions was maintained by virtue of the presence of antimicrobial compounds present in "Bhakari" and "Curd" cultures. In hitherto known processes main drawbacks are that 1. The microbial cultures are grown under sterile conditions containing basal salts and other nutrients, increasing the cost of the treatment. 2. In most of the cases the fungus or bacteria are immobilized on some support. 3. Sometimes treated with ozone or hydrogen peroxide, which is not cost effective. 4.1n majority of the process, % colour removal is not mentioned .In some processes colour removal was in the range of 80- 92% but details are not mentioned. 5. COD reduction is the range of 25-80%. 6. The colour removal was performed under aseptic conditions. 7. All these cultures use glucose as the additional carbon source. There is a continuous interest on the development of new improved biological processes for removal of colour and waste mainly containing aromatic compounds. The main objective of the present invention is to provide an improved process for the biological removal of colour and degradation of industrial waste mainly containing aromatic waste produced in the processing of paper, distillery and phenolic waste using these novel cultures. Another objective of the present invention is to use a mixture of bacteria and yeast commercially known as curd culture and Tea Fungus. The curd bacteria essentially contain Lactobacillus sp. Dominating in the sour culture. Tea fungus essentially is a mixture of a bacterium, Acetobacter Xylimum, and yeasts Candida sp and Zygosaccharomyces rouxii in a sugar tea. Yet another objective is to use these cultures without using sterile conditions either for maintenance or for treatment. Accordingly, the present invention provides an improved process for the biodegradation of Industrial waste using a consortium of bacteria and fungus selected from qard bacteria and tea fungus such as herein described, which comprises preparing 1 : 1 industrial waste: water having concentration of aromatic compounds 1.4 g/L, treating the said industrial waste with a consortium of fungus and bacteria as defined above , in presence of assimible carbon source such as herein described , at a concentration 0.5 to 3.0%, having pH in the range of 4.0 to 5.8, at a temperature 28° - 37°C, at least for 24 h at non-sterile condition under agitation, and getting biodegraded industrial waste devoid of aromatic compounds and colour. In one of the embodiments of the present invention the microbial cultures may be the consortium of "Tea fungus" and "curd culture." In another embodiment "Curd culture consists of Lactobacillus bulgaricus, Lactobacillus acidophilus and Streptococus lactis and the "Tea fungus" consortium consists of Acetobacter xylimum, Zygosacharomyces rouxii and Candida sp. In still another embodiment, the easily assimilating carbon source may be sucrose, glucose or glycerol, preferably, sucrose. In yet another embodiment, of the concentration of carbon source may be 0.5 to 3.0 % WAV, preferably 0.5%. In additional embodiment, the pH of the industrial waste containing medium may be 4.0 to 5.6, preferably, 5.3. In a further embodiment, the incubation temperature may be 28 to 37°C preferably 28°C. In still further embodiment, the growth of the cultures in industrial waste medium is under aerobic conditions. In a feature of the present invention, the fermentation was carried under non-sterile conditions. In another feature the effluent containing medium along with the respective microbial culture was agitated. In a feature of the present invention the cultures were incubated between 28-40°C for 24-60h. In another feature the industrial waste was obtained from paper mills using different pulping processes. In another feature of the present invention the treated effluent was found to remove almost all colour and aromatic pollutants and bring down BOD and COD values required to meet the environmental control standards (Figl). The invention is illustrated by the following examples, which should not be construed to limit the scope of present invention. EXAMPLE 1 This experiment was done to optimize the concentration of sucrose required for maximum color removal. The industrial waste was diluted with tap water in 1:1 proportion, sucrose was added in 0 to 2 % w/v concentration as an easily assimilating carbon source. The industrial waste medium was adjusted to pH 5.3. The flasks were inoculated with mixed culture. The minimum sucrose concentration required for colour removal was 0.5%. No color was removed in the absence of sucrose. EXAMPLE 2 This experiment was done to optimize the concentration of glycerol required for maximum color removal. The industrial waste was diluted with tap water in 1:1 proportion, and glycerol was added in 1, 5,10% v/v concentrations. The industrial waste medium was adjusted to pH 5.3, inoculated with mixed culture and incubated on a rotary shaker at 28-3 0°C for 48h. The colour removal was observed in the presence of 1% v/v glycerol and decreased with increasing concentrations of glycerol. Reduction in colour was 98%, reduction in COD was 95% and that in BOD is 98% respectively in presence of 1.0% glycerol.(Table 1). Table: 1 (Table Removed) EXAMPLE 3 This experiment was done to optimize the effect of pH on color removal from the industrial waste. The industrial waste was diluted with tap water in 1:1 proportion and 0.5% sucrose was added as an easily assimilating carbon source. The pH of the medium was adjusted in the range of 4.0, 5.0, 5.3, 5.5, 6.0 and 7.0. The flasks were inoculated with mixed culture and incubated on a rotary shaker at 28~30°C incubated for 48h. The maximum colour removal was observed at pH5.3 in case of mixed consortium of tea fungus and curd culture. The color removal was decreased on either side of this pH. The maximum colour removal was 98% EXAMPLE 4 Industrial waste was diluted with tap-water in 1:0, 1:1, 1:2, and 1:3 proportions for optimization of the color removal.; after addition of 0.5% sucrose, the medium was adjusted to pH 5.3, inoculated with mixed culture and incubated on a rotary shaker at 28-3 0°C for 48h. Maximum colour removal was observed in all flasks with 1: Iminimum dilution. The culture failed to remove colour in undiluted industrial waste. EXAMPLE 5 This experiment was done to optimize the effect of inoculum size on the color removal. Industrial waste was diluted with tap water in 1:1 proportion. After addition of 0.5% sucrose, the medium was adjusted to pH 5.3, inoculated with different amounts of inoculum, particularly, Tea fungus" was added by taking different fresh weights in the range of 1.0-10 g/lOOml medium with 100 mg "curd bacteria". The flasks were incubated on a rotary shaker at 28-30°C and incubated on a rotary shaker at 28~30°C for 48h. The culture failed to remove colour in absence of the inoculum. The colour removal increased linearly with increasing inoculum. Maximum colour removal was observed in flasks receiving 6.0g fresh weight of tea fungus. However, there was no further increase in colour removal was observed beyond 6.0g inoculum EXAMPLE 6 This experiment was done to optimize the effect of the ratio of volume of the medium to the flask capacity on color removal. The industrial waste was diluted with tap water in 1:1 proportion, after addition of 0.5% sucrose, the medium was adjusted to 5.3. The medium was distributed as follows: 50ml in 250ml, 50ml in 100ml, 100ml in 500ml and 150ml in 1000ml capacity conical flask. The ratio of the medium to flask was 1:5, 1:10, 1:5 and 1:6.6. The flasks were inoculated with equal inoculum of the mixed culture of Tea fungus and Curd bacteria and incubated on a rotary shaker for 48h. Maximum growth and colour removal was observed in 1:5 ratio. Reduction in COD was 94%, that in BOD and color is 98% (Table 2). Table 2. (Table Removed) The Fig 1, accompanying this specification, shows comparative data of reduction of the aromatic pollutants measured as percentage of the ultra violet absorbing material present in the black liquor medium (C) using "Tea fungus" (F) and "curd culture"(L) consortia singly oxjm mixture (F +L). The compaiitive tlata show that the mixture of "Tea fungus" and "Curd culture" removes the aromatic^ollutants to about 96% (Table2) complying the standards set by Statutory Water Selfertion Board. The main advantages of the present improved process are: 1. The process does not require sterile conditions for color removal. 2. Special chemicals are not required. 3. Commercial sucrose used as the additional carbon source is comparatively cheap and hence the process is cost effective. 4. The cultures used in this process are GRAS clear and nontoxic to living organisms. 5. The important natural resource such as water is conserved by the recycling of the treated effluent for diluting the effluent for the next batch. 6. Biomass generated as a bi product of the effluent treatment can be used as a fertilizer or a soil conditioner. 7 In the process of colour removal, some important chemicals such as organic acids, phenolic acids such as ferulic and vanillic acids and atechol can be obtained from black liquor under the controlled conditions of fermentation. We Claim: 1. An improved process for the biodegradation of Industrial waste using a consortium of bacteria and fungus selected from end bacteria and tea fungus such as herein described, which comprises preparing 1 : 1 industrial waste: water having concentration of aromatic compounds 1.4 g/L, treating with a consortium of fungus and bacteria as defined above , in presence of assimible carbon source , at a concentration 0.5 to 3.0%, having pH in the range of 4.0 to 5.8, at a temperature 28° - 37°C, at least for 24 h at non-sterile condition under agitation, and getting biodegraded industrial waste devoid of aromatic compounds and colour. 2. An improved process as claimed in claim 1 wherein the end bacteria used is selected from Lactobacillus bulgaricus, Lactobacillus acidophilus and Streptococus lactis and Tea fungus used is selected from the group comprising Acetobacter xylinum, Zygosacharomyces rouxii and Candida sp. and mixture thereof. 3. An improved process as claimed in claims 1 to 2 wherein the assimilating carbon source used is selected from sucrose, glucose or glycerol preferably sucrose. 4. An improved process for the biodegradation of Industrial waste using a consortium of bacteria and fungus substantially as herein described with reference to the examples.

Full Text

This invention relates to an improved process for the removal of colour and degradation of industrial waste mainly containing aromatic compounds. More particularly, it relates to an improved process for the treatment of paper and pulp industry waste. The invention relates to an improved method of biodegradation and colour removal by using bacteria, fungi and yeast singly, or in consortium. When grown in waste supplemented with easily assimilable carbon source medium, the biomass strongly absorbs the colouring matter and the degraded organic pollutants including lignin, and lignin degradation compounds.
Lignin is the characteristic-cementing constituent between the cell walls of woody tissues. The spaces between cellulose fibrils in woody vascular tissues are filled with lignin and hemicellulose, thus lignin is naturally available in abundance. One of the main constraints in this area is the colour of the effluent, which varies from light tan to deep brown or black. The source of colour in an integrated bleached kraft mill is mainly due to lignin derivatives produced from the extraction stage of the bleach plant. This effluent waste contains upto 3-6 % lignin. The industrial waste is highly coloured and typically accounts for 80% of the colour, 30% of the BOD (Biological oxygen demand) and 60% of the COD (Chemical Oxygen demand) of the mill pollution load. The proportion of colour and lignin are further concentrated by the practice of recycling of the effluents to conserve fresh water. In paper mill effluent treatment total dissolved solids are removed and used as energy source in fuel. Biomethenation has also been attempted. All these do not meet the standards of pollution control for colour, total dissolved solids, BOD and COD. Since the delignification of the starting material for paper and pulp manufacture is a continuous process, the industrial waste released into the environment cannot be completely avoided.

In recent years regulatory agencies have become more stringent with respect to the discharge of coloured effluents to the surface waters. Hence some concrete efforts in the direction of colour removal from the spent wash are required.
Lignin, its derivatives and phenolics are in general difficult to degrade by physical, chemical or biological methods. This is mainly due to the complex linkages within the molecules especially the biphenyl type carbon to carbon linkage present in lignin. Because, such compounds are not easily biodegradable, conventional biological treatment methods are only moderately effective in decreasing effluent COD. Consequently, paper industry, and other industries generating phenolic waste cannot satisfy the effluent discharge limits for COD and BOD that have been imposed by environmental pollution control board.
In the prior art following processes are used for removal of lignin and colour from industrial wastes using bacterial and fungal cultures and their enzymes hitherto and described in brief herein below.
A continuous biological process to decolourize black plant effluent. Chang, H.W., Compbeil, A. Biotechnol. Adv. 2(2) 301-308 (1985):
The coloured waste stream from the extraction stage of a Kraft bleaching plant was treated with Phanerochaetae chrysosporium in a treatment scheme called the MyCoR process. Under optimal condition 2000 colour units /1 per day were removed alongwith concomitant removal of COD and BOD. Chlorolignins foraged were dechlorinated. Chemical characterization and biological decolourization by fungus of effluents from wheat straw alkaline pulping. Terron M.C., Rijksuniv genet: 56(49) 1569-1571 (1991). Tramates versicolour, a white rot fungus immobilized on different supports, along with granular activated carbon process achieved 80% colour removal from the effluent waste.

Drawback: Required addition of activated carbon which adds to biomass, also increases the cost of the treatment.
Photobleaching and biobleaching of kraft effluent. Duran Nelson Dezott; Mareia Rodiguez Jaime, J. Photochem Photobiol, A. 62(10) 269-279 (1991): Use of immobilized mycelial pellet of a fungus Chrysonilia sitophila in nylon provided effective means to decolourize lignin-containing Kraft wastewater. The immobilized microbial pellet, absorbed 25-30% colour of which 60-65% autooxidized in the presence of HjC^ in 6h. Kraft effluent is oxidatively decomposed by a semiconductor. Photocatalysed reaction in an aqueous solution containing oxygen and semiconductor powder. Under continuous illumination, the solution becomes colourless and the molecular weight distribution indicated almost total mineralization in 2h. Drawback: The process required IHkOz, special chemicals for immobilization, sterile conditions for immobilization. With all these additions, the color removal was only 25-30%
Comparison and combination of ozone and fungal treatments of a kraft bleaching effluent. Roy Acrand L; Archibald F.S., Briere F. Tappi J. 74(9) 211-218 (19^1): In Kraft bleaching most of the colour and much of the toxicity is contained in the effluent from the alkaline extraction stages. Effluent was treated with ozone and the fungus Tramates (coricolus) varsicolour. Among the two treatttietits, the ftmgal treatment resulted in greater decolourization as and COD removal. Colour removal from kraft belach-plant effluents by Trichoderma sp. PMsad D.Y.,and Joyce T.W., Tappi, J.74 (1) 165-169 ) (1991):
Trichoderma sp. is capable of degrading lignin and declolorizing the hardwood extraction-stage bleach-plant effluent. The optimum pH and carbon source for the fungus was determined. The results show that under optimal conditions, total Colour

and COD (chemical oxygen demand) decrease by almost 85% and 25%, respectively,
after cultivation for three days. Addition of nitrogen did not simulate the
decolourization process, indicating that it is not a rate-limiting factor. Glucose was the
most effect-carbohydrate utilized by the fungus, as a stimulated substantial colour
reduction without any increase in COD.
Drawback: The method required glucose, which is more costly as compared to
commercial sucrose. Reported COD removal was only 25%
Absorption effect in the dicolourization of a Kraft bleach effluent by Phanerochaete
chrysosporium. Jaspers C.J. and Penninckx, M.Y., Biotechnology Letters 18(11) 1257-
1260 (1996). Biotechnol. Letters. 18 (11) 1257-1260(1996:.
The fungal pellets inoculated in the effluent, without addition of nutrients, strongly
absorb colour (1% glucose was added as a quickly metabolizing carbon source for
growth of the fungus). Sixty- percent decolourization was achieved at 37 °C and 2.0 pH.
Drawback: Required lots of acid for adjusting the pH to 2 before fungal treatment. .It
appeared that reported color removal of 60% could be due to the acid.
Degradation of lignin and decolourization of paper mill bleach plant effluent (BPE) by
marine fungi. Raghukumar C.D., Chandramohan D., Michel F.C., and Reddy C.A.
Biotechnology Letters 18(1) 105-106 (1996):
Isolation of a lignin decolourizing bacterium. Moril Hiroshi, Nakamiya Kunichika and
Kiroshita Shinichi. J. Fermentation and Bio-engineering 80(3) 296-299 (1995):
A bacterial strain from soil namely Azatobacter spp. degraded and decolourized lignin
very rapidly under optimum conditions. However the maximum color removal was
observed in presence water, on of dead organic matter and are the principal life forms
exposed to xenobiotics in the environment. Thus biotechnological applications use
these life forms chiefly bacteria and fungi in hazardous waste treatment.

A "Tea fungus" commonly designated as "Kombucha" in China, Japan, USA or "Bhakari" in India, is a symbiotic culture of atleast three microoganisms [Blanc J. Philppe. Biotechnology Letters 18(2): 139-142 (1996)]: the acetic acid bacterium Acetobacter xylimim and two yeasts Zygosaccharomyces roitxii and Candida sp. forming a floating cellulose mat in sugar tea. [Hesseltine C.W. A millenium of fungi, food and fermentation: In Mycologia 57(2): 149-197 (1965);.
"A Tea fungus was obtained locally and maintained on sugar tea for seven days under non sterile conditions. "Lactic acid bacteria" in "curd culture" were maintained by 8hr subculture on milk under non-sterile conditions. Purity of these cultures under non-sterile conditions was maintained by virtue of the presence of antimicrobial compounds present in "Bhakari" and "Curd" cultures.
In hitherto known processes main drawbacks are that
1. The microbial cultures are grown under sterile conditions containing basal salts and

other nutrients, increasing the cost of the treatment.
2. In most of the cases the fungus or bacteria are immobilized on some support.
3. Sometimes treated with ozone or hydrogen peroxide, which is not cost effective.
4.1n majority of the process, % colour removal is not mentioned .In some processes colour removal was in the range of 80- 92% but details are not mentioned.
5. COD reduction is the range of 25-80%.
6. The colour removal was performed under aseptic conditions.
7. All these cultures use glucose as the additional carbon source.
There is a continuous interest on the development of new improved biological processes for removal of colour and waste mainly containing aromatic compounds.

The main objective of the present invention is to provide an improved process for the biological removal of colour and degradation of industrial waste mainly containing aromatic waste produced in the processing of paper, distillery and phenolic waste using these novel cultures.
Another objective of the present invention is to use a mixture of bacteria and yeast commercially known as curd culture and Tea Fungus. The curd bacteria essentially contain Lactobacillus sp. Dominating in the sour culture. Tea fungus essentially is a mixture of a bacterium, Acetobacter Xylimum, and yeasts Candida sp and Zygosaccharomyces rouxii in a sugar tea.
Yet another objective is to use these cultures without using sterile conditions either for maintenance or for treatment.
Accordingly, the present invention provides an improved process for the biodegradation of
Industrial waste using a consortium of bacteria and fungus selected from qard bacteria and
tea fungus such as herein described, which comprises preparing 1 : 1 industrial waste: water having concentration of aromatic compounds 1.4 g/L, treating the said industrial waste with a consortium of fungus and bacteria as defined above , in presence of assimible carbon source such as herein described , at a concentration 0.5 to 3.0%, having pH in the range of 4.0 to 5.8, at a temperature 28° - 37°C, at least for 24 h at non-sterile condition under agitation, and getting biodegraded industrial waste devoid of aromatic compounds and colour.
In one of the embodiments of the present invention the microbial cultures may be the consortium of "Tea fungus" and "curd culture."
In another embodiment "Curd culture consists of Lactobacillus bulgaricus, Lactobacillus acidophilus and Streptococus lactis and the "Tea fungus" consortium consists of Acetobacter xylimum, Zygosacharomyces rouxii and Candida sp.
In still another embodiment, the easily assimilating carbon source may be sucrose, glucose or glycerol, preferably, sucrose.

In yet another embodiment, of the concentration of carbon source may be 0.5 to 3.0 %
WAV, preferably 0.5%.
In additional embodiment, the pH of the industrial waste containing medium may be 4.0
to 5.6, preferably, 5.3.
In a further embodiment, the incubation temperature may be 28 to 37°C preferably
28°C.
In still further embodiment, the growth of the cultures in industrial waste medium is
under aerobic conditions.
In a feature of the present invention, the fermentation was carried under non-sterile
conditions.
In another feature the effluent containing medium along with the respective microbial
culture was agitated.
In a feature of the present invention the cultures were incubated between 28-40°C for
24-60h.
In another feature the industrial waste was obtained from paper mills using different
pulping processes.
In another feature of the present invention the treated effluent was found to remove
almost all colour and aromatic pollutants and bring down BOD and COD values
required to meet the environmental control standards (Figl).
The invention is illustrated by the following examples, which should not be construed to
limit the scope of present invention.
EXAMPLE 1
This experiment was done to optimize the concentration of sucrose required for maximum color removal. The industrial waste was diluted with tap water in 1:1 proportion, sucrose was added in 0 to 2 % w/v concentration as an easily assimilating

carbon source. The industrial waste medium was adjusted to pH 5.3. The flasks were inoculated with mixed culture. The minimum sucrose concentration required for colour removal was 0.5%. No color was removed in the absence of sucrose.
EXAMPLE 2
This experiment was done to optimize the concentration of glycerol required for maximum color removal. The industrial waste was diluted with tap water in 1:1 proportion, and glycerol was added in 1, 5,10% v/v concentrations. The industrial waste medium was adjusted to pH 5.3, inoculated with mixed culture and incubated on a rotary shaker at 28-3 0°C for 48h. The colour removal was observed in the presence of 1% v/v glycerol and decreased with increasing concentrations of glycerol. Reduction in colour was 98%, reduction in COD was 95% and that in BOD is 98% respectively in presence of 1.0% glycerol.(Table 1).
Table: 1
(Table Removed)

EXAMPLE 3
This experiment was done to optimize the effect of pH on color removal from the industrial waste. The industrial waste was diluted with tap water in 1:1 proportion and 0.5% sucrose was added as an easily assimilating carbon source. The pH of the medium was adjusted in the range of 4.0, 5.0, 5.3, 5.5, 6.0 and 7.0. The flasks were inoculated

with mixed culture and incubated on a rotary shaker at 28~30°C incubated for 48h. The maximum colour removal was observed at pH5.3 in case of mixed consortium of tea fungus and curd culture. The color removal was decreased on either side of this pH. The maximum colour removal was 98%
EXAMPLE 4
Industrial waste was diluted with tap-water in 1:0, 1:1, 1:2, and 1:3 proportions for optimization of the color removal.; after addition of 0.5% sucrose, the medium was adjusted to pH 5.3, inoculated with mixed culture and incubated on a rotary shaker at 28-3 0°C for 48h. Maximum colour removal was observed in all flasks with 1: Iminimum dilution. The culture failed to remove colour in undiluted industrial waste.
EXAMPLE 5
This experiment was done to optimize the effect of inoculum size on the color removal. Industrial waste was diluted with tap water in 1:1 proportion. After addition of 0.5% sucrose, the medium was adjusted to pH 5.3, inoculated with different amounts of inoculum, particularly, Tea fungus" was added by taking different fresh weights in the range of 1.0-10 g/lOOml medium with 100 mg "curd bacteria". The flasks were incubated on a rotary shaker at 28-30°C and incubated on a rotary shaker at 28~30°C for 48h. The culture failed to remove colour in absence of the inoculum. The colour removal increased linearly with increasing inoculum. Maximum colour removal was observed in flasks receiving 6.0g fresh weight of tea fungus. However, there was no further increase in colour removal was observed beyond 6.0g inoculum
EXAMPLE 6
This experiment was done to optimize the effect of the ratio of volume of the medium to the flask capacity on color removal. The industrial waste was diluted with tap water in 1:1 proportion, after addition of 0.5% sucrose, the medium was adjusted to 5.3. The

medium was distributed as follows: 50ml in 250ml, 50ml in 100ml, 100ml in 500ml and 150ml in 1000ml capacity conical flask. The ratio of the medium to flask was 1:5, 1:10, 1:5 and 1:6.6. The flasks were inoculated with equal inoculum of the mixed culture of Tea fungus and Curd bacteria and incubated on a rotary shaker for 48h. Maximum growth and colour removal was observed in 1:5 ratio. Reduction in COD was 94%, that in BOD and color is 98% (Table 2).
Table 2.
(Table Removed)

The Fig 1, accompanying this specification, shows comparative data of reduction of the aromatic pollutants measured as percentage of the ultra violet absorbing material present in the black liquor medium (C) using "Tea fungus" (F) and "curd culture"(L) consortia singly oxjm mixture (F +L). The compaiitive tlata show that the mixture of "Tea fungus" and "Curd culture" removes the aromatic^ollutants to about 96% (Table2) complying the standards set by Statutory Water Selfertion Board. The main advantages of the present improved process are:
1. The process does not require sterile conditions for color removal.
2. Special chemicals are not required.
3. Commercial sucrose used as the additional carbon source is comparatively cheap and
hence the process is cost effective.
4. The cultures used in this process are GRAS clear and nontoxic to living organisms.

5. The important natural resource such as water is conserved by the recycling of the treated effluent for diluting the effluent for the next batch.
6. Biomass generated as a bi product of the effluent treatment can be used as a fertilizer or a soil conditioner.
7 In the process of colour removal, some important chemicals such as organic acids, phenolic acids such as ferulic and vanillic acids and atechol can be obtained from black liquor under the controlled conditions of fermentation.

We Claim:
1. An improved process for the biodegradation of Industrial waste using a consortium of
bacteria and fungus selected from end bacteria and tea fungus such as herein described, which comprises preparing 1 : 1 industrial waste: water having concentration of aromatic compounds 1.4 g/L, treating with a consortium of fungus and bacteria as defined above , in presence of assimible carbon source , at a concentration 0.5 to 3.0%, having pH in the range of 4.0 to 5.8, at a temperature 28° - 37°C, at least for 24 h at non-sterile condition under agitation, and getting biodegraded industrial waste devoid of aromatic compounds and colour.
2. An improved process as claimed in claim 1 wherein the end bacteria used is
selected from Lactobacillus bulgaricus, Lactobacillus acidophilus and
Streptococus lactis and Tea fungus used is selected from the group comprising
Acetobacter xylinum, Zygosacharomyces rouxii and Candida sp. and mixture
thereof.
3. An improved process as claimed in claims 1 to 2 wherein the assimilating carbon
source used is selected from sucrose, glucose or glycerol preferably sucrose.
4. An improved process for the biodegradation of Industrial waste using a
consortium of bacteria and fungus substantially as herein described with
reference to the examples.

Documents:

83-del-2001-abstract.pdf

83-del-2001-claims.pdf

83-del-2001-correspondence-others.pdf

83-del-2001-correspondence-po.pdf

83-del-2001-description (complete).pdf

83-del-2001-drawings.pdf

83-del-2001-form-1.pdf

83-del-2001-form-19.pdf

83-del-2001-form-2.pdf

83-del-2001-form-3.pdf

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Patent Number

217114

Indian Patent Application Number

83/DEL/2001

PG Journal Number

13/2008

Publication Date

31-Mar-2008

Grant Date

25-Mar-2008

Date of Filing

31-Jan-2001

Name of Patentee

COUNCIL OF SCIENTIFIC AND INDUSTRIAL RESEARCH

Applicant Address

RAFI MARG, NEW DELHI-110001, INDIA.

Inventors:

#	Inventor's Name	Inventor's Address
1	NIRMALA AVINASH SAHASRABUDHE	NATIONAL CHEMICAL LABORATORY, PUNE- 411008, MAHARASHTRA, INDIA.

PCT International Classification Number

C02F 3/34

PCT International Application Number

N/A

PCT International Filing date

PCT Conventions:

#	PCT Application Number	Date of Convention	Priority Country
1			NA