Title of Invention	A METHOD OF TREATING INDUSTRIAL WASTE WATER CONTAINING METAL TRACES AND METAL CONTAMINATED AQUEOUS SOLUTIONS
Abstract	A method of treating industrial waste water containing metal traces and metal contaminated aqueous solution,including metal polluted drinking water,the said method comprises treating the above defined metal contaminated waste water in a column containing immobilized metal wequetering substance,the said substances being prideced from stychnos potatorum (l) seeds which have ability to reduce metal load from PPM to PPB level form, metal containing water the siad method comprising passing the metal containing water through the said column containing immobilized metalo sequestering substances,so that the toxic metal substances are removed from the metal contaminated water.

Title of Invention

A METHOD OF TREATING INDUSTRIAL WASTE WATER CONTAINING METAL TRACES AND METAL CONTAMINATED AQUEOUS SOLUTIONS

Abstract

A method of treating industrial waste water containing metal traces and metal contaminated aqueous solution,including metal polluted drinking water,the said method comprises treating the above defined metal contaminated waste water in a column containing immobilized metal wequetering substance,the said substances being prideced from stychnos potatorum (l) seeds which have ability to reduce metal load from PPM to PPB level form, metal containing water the siad method comprising passing the metal containing water through the said column containing immobilized metalo sequestering substances,so that the toxic metal substances are removed from the metal contaminated water.

Full Text	This invention in general relates to tne neia particular this invention pertains to a method of preventing environmental degradation by metal pollution. This invention is the result of a research and development project of Girijan Co-operative Corporation Limited, an undertaking of State Government of Andhra Pradesh, India. Tribals and villagers uses these seeds for clarification of muddy water which was known from ancient times, hence these seeds are termed as cleaning nut seeds. This research work is aimed to develop value added biotechnology products from Strychnos potatorum (L.)seeds. Metal sequestering property of Strychnos potatorum (L.) seed constituents was identified and this invention led to the development of metal sequestering substances for metal pollution control. In broad embodiment this present invention is a method of removal of heavy metals from aqueous solution using metal sequestering substances from Strychnos potatorum (L.) seeds for pollution control. This invention further relates to identification, production and utility methods of metal sequestering substances from the seeds of Strychnos potatorum (L.) This invention is useful for heavy metal pollution control using biotechnological approach for environmental protection. Although many plant substances (such as peptides) are known but production on large scale for industrial usage, by using molecular biological techniques is a costly approach. The metal sequestering substances abundantly present in Strychnos potatorum (L) seeds offers potential industrial application for metal pollution control as they can be produced in large quantities at relatively low cost It is relevant to quote the work done by Prof ClementKFurlong, University of Washington, Seattle, Washington, USA, regarding utilising peptides from plant sources for removal of metal ions from aqueous solution, (U.S.Patent no.4,909,944,(1988), Removal of metal ions from aqueous solution. Jackson, P.J., Delhaize, E., Robinson, N.J., Unkefer, C.J., and Furlong, C.E.). The objective of the invention is to identify the metal sequestering substances from Strychnos potatorum (L,) seed constituents. It is also the object of the invention to develop viable methods for production and utilization of metal sequestering substances from Strychnos potatorum (L.). seeds for pollution control. Thus according to this invention the peptides present in Strychnos Potatorum(L.) seeds are mainly responsible for metal sequestering activity. Glutamic Acid, Aspartic Acid, Arginine, Leucine and Glycine amino acids comprises about 56% of these peptides. The flocculating, fibrous, carbohydrates present in seed provide structural stability to these metal sequestering peptides by preventing denaturation at various aqueous reaction conditions. For production and utility of metal sequestering substances, technically feasible and economically viable methods were developed. It is therefore a primary object of the invention to invent a method/technique to reduce industrial metal pollution and to protect the environment It is another object of the invention to invent a method by virtue of which industrial waste water containing toxic metal substances are converted into nontoxic metal free water by using biodegradable plant derived substances. It is another object of the invention to develop process methods for metal sequestering peptides containing substances to control environmental pollution. It is another object of the invention to device a method to reduce environmental pollution by using plant derived metal sequestering substances. Further object of the invention will be apparent from the ensuing description. Accordingly the invention envisages a method of treating industrial waste water containing metal traces and metal contaminated aqueous solution, the said method comprises treating the above defined metal polluted waste water in a column containing immobilized metal sequestering substances, the said substances obtained from biological plant sources which have ability to reduce metal load from PPM to PPB level from metal polluted industrial waste, the said metal comprising passing the waste water through the said column containing immobilized metal sequestering substances. Further the invention envisages a method of treating industrial waste water containing metal traces as claimed in claim 1 wherein the said metal sequestering peptides containing substances are obtained from Strychnos potatorum (L.) seeds by process of centrifugation or frothing methods. Metal sequestering peptides containing substances were immobilized on polyester resia Now the invention will be described in detail with respect to various examples so as to illustrate and explain the salient features of the inventioa As shown in Figure(l) the fourteen peptides identified in the seeds of Strychnos potatorum (L.) are mainly responsible for metal sequestering activity in aqueous solution. Based on electrophoresis of seed constituents and purified metal sequestering substances (as described in this invention) on 10-18% gradient SDS PAGE gel (Figure 1), the molecular weight (Daltons) of these peptides (shown in Table 1): (1) 61,700 (2) 56,500 (3) 53,200 (4) 51,500 (5) 44,600 (6) 39,900 (7) 37,200 (8) 31,700 (9) 30,200 (10) 20,000 (11) 18,700 (12) 14,800 (13) 13,600 (14) 7,080. When these peptides are digested or separated or removed and or reduced from seed constituents by various methods using proteases digestion (such as protinase k, or pepsin) or alkali treatment (such as 1.0 M NaOH) or extraction with denaturing agents ( 3uch as 3.0M urea) reduces 70 - 80 % metal sequestering activity. Hie Strychnos potatorum (L.) seeds contains carbohydrates as major ;onstituents. These carbohydrates are divided into two parts as flocculating and non flocculating. The flocculating carbohydrates are fibrous in nature. Tlie fourteen metal sequestering peptides are entrapped in these fibrous structures of carbohydrates. There is no chemical bond between these :arbohydrates and peptides. If not disturbed by vigorous homogenization hese carbohydrates travel along with these metal sequestering fourteen peptides for flocculating in an aqueous medium at neutral P** range. These netal sequestering fourteen peptides entrapped in fibrous carbohydrates ;omprises about 4-5% of total seed constituents. Table 2 shows the amino acid composition(%) of metal sequestering peptides. Amino acid analysis of metal sequestering peptides containing substances revealed that Glutamic Acid, Aspartic Acid, Argnine, Leucine and Glycine comprises about 56% of these fourteen peptides. The flocculating carbohydrates are not important directly in relation to metal sequestering activity but indirectly these carbohydrates provide structural base to the metal sequestering peptides therefore preventing denaturation of metal sequestering peptides at various reaction conditions in a solution. Strychnos potatorum(L.) seeds are one of the hardest known seeds in plant kingdom and this toughness of seed structure is due to these fibrous carbohydrates and peptides internal assembly. Now this invention will describe a method of producing metal sequestering peptides containing substances from Strychons potatorumfL.) seeds by centrifugation method. Micronization of seed constituents below SO-SO microns size was found suitable for starting the process for production of said substances (metal sequestering substances). Micronized seed powder was homogenized with water at a ratio of 1:75 to 1:100 (Wt/V.). Seed substances have gummy nature and requires minimum 20 times of water (seed material: water = 1:20 Wt./V.) for mixing to start any process. By adding chloroform 3-5%(V./V.) to seed powder containing water after homogenization, the said substances were precipitated by centrifuging at 5000 to 6000 RPM (for 30-40 minutes) and obtained precipitate was dried either by using solvent (such as acetone) or in a drier (at 80°-85° C). By this method the product yield comprising 20-22 % weight of total seed constituents was obtained. Now this invention will describe the method of producing immobilized metal sequestering substances. The said substances were immobilized on polyester (Bisphenol) resins after thorough mixing of said substances with the resin(l:4 or 1:5 = resin; metal sequestering substances, V./Wt.). Metal catalysts were avoided in the process of resin immobilization. Detailed investigation on immobilized said substances on column mode revealed that these substances have repeated use and gave consistant performance of metal sequestering substances. The said substances containing immobilized resins below 200 microns are mostly suitable for batch method. The said substances containing of coarse resins (above 200 microns size) are suitable for column mode. For obtaining granulation of coarse resins addition of celite (3-4 %, wt/wt), to said substances before immobilization was found useful. The said substances after immobilization when tested through column mode(using coarse resins of above 200 microns size) sequestered metals from aqueous solution (example: the said substances containing resin sequestered 0.3 mg. Zinc per ml. of resin in a column when solution was passed with a metal load of 10 ug/ml at a flow rate of 1.8 ml/min.). The finer size immobilized resins containing said substances (below 50 micron size) when tested through batch method sequestered 0.7 mg. of Zinc per a gram of resin used in a solution at neutral pH In the following description another method of production of the said substances will be described in detail. Using frothing property of proteins, the said substances were obtained by collecting the froth at the surface layer of the homogenate alter vigorous stirring at high speed (8000 to 10000 RPM for 40-60 minutes) with homogenizers the said substances containing precipitate was washed with water at acidic PH(l-2) to remove bound metals followed by neutralization with repeated washings with water or with addition of (small amount ) alkali (such as 0.1 M NaOH). By this method the product yield comprising 18-20% weight of seed constituents was obtained. These said substances obtained from frothing method were immobilized on polyester resins (as described earlier) and the resulting products sequestered metals in similar manner as the products obtained from centrifugation method. In the following description another method namely batch method of using metal sequestering substances for removal of metal traces from aqueous solution will be ^described in detail. Batch method: Addition of metal sequestering substances in metal traces containing solution by mixing ( gently and thoroughly without vigorous stirring) at a suitable PH for maximum sequestration of that particular metal present in solution and later followed by removal of metal bound said substance by way of centrifugation or precipitation. Most of the bound metal from the said substances were eluted at 0.8 to 1.0 PH using mineral acids including sulfuric, nitric, and hydrochloric. The metal sequestering property of said substances (metal sequestering substances) from Strychnos potatorum(L.) seeds will be described with the following experiment using batch method. A gram of metal sequestering substances ( produced by centrifugation method as described earlier without immobilization) was mixed gently and thoroughly without vigorous stirring in a reaction volume of 50 ml at neutral P1 in a metal containing solution. The supernatant was removed by centrifuging at 5,000 to 6,000 RPM for 30 minutes. Total metal amount sequestered by said substances was assayed by Spectrophotometry or ICP method. The metal amount absorbed by said substances was measured by assaying the metal content in supernatant and as well as bound metal in said substances after eluting with mineral acids including sulfuric, nitric and hydrochloric (metal elution from said substances was carried out with acid at 0.8 P* after washing the said substances with enough water to remove the excess unbound metal content). As shown in Table(3), the said substances ( metal sequestering substances from Strychnos potatorum(L.) seeds) sequestered metals in aqueous medium at neutral P1. The experimental illustration of the invention (as shown in Table.3) in relation to metal sequestering property of metal sequestering substance should not be constructed to limit to the scope of invention, as metal sequestering substances from Strychnos potatorum(L.) seed constituents have ability to sequester many metals in aqueous medium at various conditions. Now this invention will describe the important parameters for metal sequestering and bound metal elution. This said substances effective use is dependant on suitable P* for metal sequestration. Each metal has its own binding property based on the PH of the reaction medium. The said substances sequestering ability is maximum at neutral r for most of the metals tested The said substances (metal sequestering substances) can not bind and or sequester many metals at acidic PK( 2-3 ) except few such as iron and some heavy metals. Most of the bound metals in the said substances (after reacting with a particular metal in a solution) can be eluted with mineral acids including sulfuric, nitric and hydrochloric at 0.8 to 1.0 r . The immobilized metal sequestering substance are suitable for repeated usage where as non immobilized metal sequestering substances can be used for 3-4 times. The metal sequestering capacity of non immobilized metal sequestering substances is more than the immobilized metal sequestering substances. One of the salient features of the invention is the identification of metal binding or sequestering substances present in Strychnos potatorum(L.) seeds constituents. The fourteen peptides identified in seed constituents are mainly responsible for metal sequestering and or binding and or removal of metals in an aqueous medium. The molecular weight of these peptides. (1) 61,700 (2) 56,500 (3) 53,200 (4) 51,500 (5) 44,600 (6) 39,900 (7) 37,200 (8) 31,700 (9) 30,200 (10) 20,000 (11) 18,700 (12) 14,800 (13) 13,600 (14) 7,080. The Strychnos potatorum(L.) seed contains carbohydrates as major constituents. These carbohydrates are divided into two parts as flocculating and non flocculating. The flocculating carbohydrates comprises about 15 to 17 % of total seed constituents. These flocculating carbohydrates are fibrous in nature. The fourteen metal sequestering peptides are entrapped in these fibrous structures of carbohydrates. There is no chemical bond between these carbohydrates and peptides. If not disturbed by vigorous homogenization these carbohydrates travel along with these metal sequestering fourteen peptides for flocculation in an aqueous medium at neutral P^ range. These metal sequestering fourteen peptides entrapped in fibrous carbohydrates comprises about 4 to 5 % of total seed constituents. Amino acid analysis of metal sequestering peptides containing substances revealed that Glutamic Acid, Aspartic Acid, Arginine, Leucine and Glycine comprises about 56 % of these fourteen peptides. The flocculating carbohydrates are not important directly in relation to metal sequestering activity but indirectly these carbohydrates are important as they provide structural base to the metal sequestering peptides therefore preventing denaturation of metal sequestering peptides at various reaction conditions in a solutioa Another salient feature of this invention is regarding the production and utility methods of metal sequestering substances form Strvchnos potatorum(L.) seeds. For production of metal sequestering substances from seed constituents centrifugation or frothing methods were developed. Immobilization of metal sequestering substances was achieved using polyester resins. Most of the metal sequestering substances are useful to remove metal traces using a batch method or column method. The products produced by centrifugation or frothing are immobilized said substances (metal sequestering substance) are useful through batch method to remove or sequester various metals from PPM to PPB level in an aqueous medium. The metal bound said substances from batch method are removed through centrifugation or precipitation. The immobilized said substances (above 200 micron size) are useful on column mode and suitable for repeated usage on many cycles to remove or sequester various metals from PPM to PPB level. Another salient feature of the invention is the process of metal sequestration and elution by using suitable P^. The said substances effective use depends on suitable PH for metal sequestration in an aqueous medium. Each metal has its own binding property based on the P of the reaction medium. The said substance sequestering ability is maximum at neutral PH range for the most of the metals tested The said substances (metal sequestering substances) can not bind and or sequester many metals at acidic PH ( 2 to 3) except few such as iron and few heavy metaJs. Most of the bound metals in said substances after reacting with the particular metal in a solution can be eluted with mineral acids including sulfuric, nitric and hydrochloric at 0.8 to 1.0 PH The immobilized metal sequestering substances are suitable for repeated usage where as non immobilized metal sequestering substances can be used for 3-4 times. The metal sequestering capacity of non immobilized metal sequestering substances is more than the immobilized metal sequestering substances. The metal sequestering substances have extensive industrial applications. They can be effectively used for pollution control to protect environment from heavy metal contamination. The wide application of the said substance is the subject matter of this invention. Accordingly, the invention pertains to a method of treating industrial waste water containing metaJ traces, the said method comprises treating the above defined waste (containing toxic heavy metal) water in a column containing immobilized metal sequestering substances, the said substances being obtained from plant sources which have ability to reduce metal load from PPM to PPB level from metal polluted industrial waste water, the said method comprising passing the metal traces containing waste water through the said column containing immobilized metal sequestering substances, so that the toxic metal substances are removed from the industrial waste water. Advantages: The metal sequestering substances from the seeds of Strychnos potatorum(L.) are capable of metal load removal from PPM to PPB level and these substances are non toxic biodegradable plant materials for easy disposal which can be produced at relatively cheaper cost compared to other plant derived metal sequestering substances. This economically viable and technically feasible metal pollution control method using metal sequestering substances from Strychnos potatorum(L.) seeds is useful for environmental protection and helps tribals to get more income as they collect these seeds from the forest. The molecular details of metal sequestering peptides as herein before described and as shown in Figure 1 (electrophoresis of metal sequestering peptides on SDS PAGE, 10-18% gradient gel), Table 1 (molecular weight of fourteen metal sequestering peptides) and Table 2 (amino acid composition (%) of metal sequestering peptides ) are enclosed with this complete specification. It is to be noted that an embodiment of the invention is described in detail in the complete specification so as to illustrate salient features of the invention, further it is envisaged that within the scope of the invention various modifications of the invention are permissible. It is evident that description will in no way delimit the scope of the invention. The scope of the invention is described in the ensuing pages. WE CLAIM: (1) A method of treating industrial waste water containing metal traces and metal contaminated aqueous solution, including metal polluted drinking water, the said method comprises treating the above defined metal contaminated waste water in a column containing immobilized metal sequestering substance, the said substances being produced from Stn/chnos potatorum (L.) seeds which have ability to reduce metal load from PPM to PPB level from metal containing water, the said method comprising passing the metal containing water through the said column containing immobilized metal sequestering substances, so that the toxic metal substances are removed from the metaJ contaminated water. (2) A method of removal of metal ions from aqueous solution as claimed in claim 1 wherein the said metal sequestering peptides containing substances are produced from Strychnos potatorum (L.) seeds by process of centrifugation method or frothing method. Immobilization of metal,sequestering peptides containing substances on polyester resin was found useful to reduce the metal content from PPM to PPB level. (3) The method as claimed in claim 1 that the method of the sequestering the metal load from metal containing aqueous solution uses a batch method, by addition of metal sequestering substances in metal traces containing aqueous medium by mixing at a suitable PH for maximum sequestration of that particular metal present in solution and later followed by removal of metal bound said substances by way of centrifugation or precipitation. (4) The method as claimed in claim 1 that the method of the sequestering the metal load from metal containing aqueous solution uses a column method (5) The method as claimed in claim 3 and 4 where in the PPM to PPB metal content is obtained by regulating the process conditions such as the suitable P1* conditions of that particular metal binding and e tut ion. (6) A method of removal of metal ions from aqueous solution as described in this specification and as explained with the help of examples.

Full Text

This invention in general relates to tne neia particular this invention pertains to a method of preventing environmental degradation by metal pollution.
This invention is the result of a research and development project of Girijan Co-operative Corporation Limited, an undertaking of State Government of Andhra Pradesh, India. Tribals and villagers uses these seeds for clarification of muddy water which was known from ancient times, hence these seeds are termed as cleaning nut seeds. This research work is aimed to develop value added biotechnology products from Strychnos potatorum (L.)seeds. Metal sequestering property of Strychnos potatorum (L.) seed constituents was identified and this invention led to the development of metal sequestering substances for metal pollution control. In broad embodiment this present invention is a method of removal of heavy metals from aqueous solution using metal sequestering substances from Strychnos potatorum (L.) seeds for pollution control.
This invention further relates to identification, production and utility methods of metal sequestering substances from the seeds of Strychnos potatorum (L.) This invention is useful for heavy metal pollution control using biotechnological approach for environmental protection. Although many plant substances (such as peptides) are known but production on large scale for industrial usage, by using molecular biological techniques is a costly approach. The metal sequestering substances abundantly present in Strychnos potatorum (L) seeds offers potential industrial application for metal pollution control as they can be produced in large quantities at relatively low cost It is relevant to quote the work done by Prof ClementKFurlong, University of Washington, Seattle, Washington, USA, regarding utilising peptides from plant sources for removal of metal ions from aqueous solution, (U.S.Patent no.4,909,944,(1988), Removal of metal ions from aqueous solution. Jackson, P.J., Delhaize, E., Robinson, N.J., Unkefer, C.J., and Furlong, C.E.).
The objective of the invention is to identify the metal sequestering substances from Strychnos potatorum (L,) seed constituents. It is also the object of the invention to develop viable methods for production and utilization of metal sequestering substances from Strychnos potatorum (L.). seeds for pollution control. Thus according to this invention the peptides present in Strychnos Potatorum(L.) seeds are mainly responsible for metal sequestering activity. Glutamic Acid, Aspartic Acid, Arginine, Leucine and Glycine amino acids comprises about 56% of these peptides. The flocculating, fibrous, carbohydrates present in seed provide structural stability to these metal sequestering peptides by preventing denaturation at various aqueous reaction conditions. For production and utility of metal sequestering substances, technically feasible and economically viable methods were developed.

It is therefore a primary object of the invention to invent a method/technique to reduce industrial metal pollution and to protect the environment
It is another object of the invention to invent a method by virtue of which industrial waste water containing toxic metal substances are converted into nontoxic metal free water by using biodegradable plant derived substances.
It is another object of the invention to develop process methods for metal sequestering peptides containing substances to control environmental pollution.
It is another object of the invention to device a method to reduce environmental pollution by using plant derived metal sequestering substances.
Further object of the invention will be apparent from the ensuing description.
Accordingly the invention envisages a method of treating industrial waste water containing metal traces and metal contaminated aqueous solution, the said method comprises treating the above defined metal polluted waste water in a column containing immobilized metal sequestering substances, the said substances obtained from biological plant sources which have ability to reduce metal load from PPM to PPB level from metal polluted industrial waste, the said metal comprising passing the waste water through the said column containing immobilized metal sequestering substances.
Further the invention envisages a method of treating industrial waste water containing metal traces as claimed in claim 1 wherein the said metal sequestering peptides containing substances are obtained from Strychnos potatorum (L.) seeds by process of centrifugation or frothing methods. Metal sequestering peptides containing substances were immobilized on polyester resia
Now the invention will be described in detail with respect to various examples so as to illustrate and explain the salient features of the inventioa
As shown in Figure(l) the fourteen peptides identified in the seeds of Strychnos potatorum (L.) are mainly responsible for metal sequestering activity in aqueous solution. Based on electrophoresis of seed constituents and purified metal sequestering substances (as described in this invention) on 10-18% gradient SDS PAGE gel (Figure 1), the molecular weight (Daltons) of these peptides (shown in Table 1): (1) 61,700 (2) 56,500 (3) 53,200 (4) 51,500 (5) 44,600 (6) 39,900 (7) 37,200 (8) 31,700 (9) 30,200 (10) 20,000 (11) 18,700 (12) 14,800 (13) 13,600 (14) 7,080. When these peptides are digested or separated or removed and or reduced from seed constituents by various methods using proteases digestion (such as protinase k, or pepsin) or

alkali treatment (such as 1.0 M NaOH) or extraction with denaturing agents ( 3uch as 3.0M urea) reduces 70 - 80 % metal sequestering activity.
Hie Strychnos potatorum (L.) seeds contains carbohydrates as major ;onstituents. These carbohydrates are divided into two parts as flocculating and non flocculating. The flocculating carbohydrates are fibrous in nature.
Tlie fourteen metal sequestering peptides are entrapped in these fibrous structures of carbohydrates. There is no chemical bond between these :arbohydrates and peptides. If not disturbed by vigorous homogenization hese carbohydrates travel along with these metal sequestering fourteen peptides for flocculating in an aqueous medium at neutral P** range. These netal sequestering fourteen peptides entrapped in fibrous carbohydrates ;omprises about 4-5% of total seed constituents.
Table 2 shows the amino acid composition(%) of metal sequestering peptides. Amino acid analysis of metal sequestering peptides containing substances revealed that Glutamic Acid, Aspartic Acid, Argnine, Leucine and Glycine comprises about 56% of these fourteen peptides. The flocculating carbohydrates are not important directly in relation to metal sequestering activity but indirectly these carbohydrates provide structural base to the metal sequestering peptides therefore preventing denaturation of metal sequestering peptides at various reaction conditions in a solution. Strychnos potatorum(L.) seeds are one of the hardest known seeds in plant kingdom and this toughness of seed structure is due to these fibrous carbohydrates and peptides internal assembly.
Now this invention will describe a method of producing metal sequestering peptides containing substances from Strychons potatorumfL.) seeds by centrifugation method. Micronization of seed constituents below SO-SO microns size was found suitable for starting the process for production of said substances (metal sequestering substances). Micronized seed powder was homogenized with water at a ratio of 1:75 to 1:100 (Wt/V.). Seed substances have gummy nature and requires minimum 20 times of water (seed material: water = 1:20 Wt./V.) for mixing to start any process. By adding chloroform 3-5%(V./V.) to seed powder containing water after homogenization, the said substances were precipitated by centrifuging at 5000 to 6000 RPM (for 30-40 minutes) and obtained precipitate was dried either by using solvent (such as acetone) or in a drier (at 80°-85° C). By this method the product yield comprising 20-22 % weight of total seed constituents was obtained.
Now this invention will describe the method of producing immobilized metal sequestering substances. The said substances were immobilized on polyester (Bisphenol) resins after thorough mixing of said substances with the resin(l:4 or 1:5 = resin; metal sequestering substances, V./Wt.). Metal catalysts were avoided in the process of resin immobilization. Detailed investigation on immobilized said substances on column mode revealed that these substances have repeated use and gave consistant performance of metal sequestering substances.

The said substances containing immobilized resins below 200 microns are mostly suitable for batch method. The said substances containing of coarse resins (above 200 microns size) are suitable for column mode. For obtaining granulation of coarse resins addition of celite (3-4 %, wt/wt), to said substances before immobilization was found useful. The said substances after immobilization when tested through column mode(using coarse resins of above 200 microns size) sequestered metals from aqueous solution (example: the said substances containing resin sequestered 0.3 mg. Zinc per ml. of resin in a column when solution was passed with a metal load of 10 ug/ml at a flow rate of 1.8 ml/min.). The finer size immobilized resins containing said substances (below 50 micron size) when tested through batch method sequestered 0.7 mg. of Zinc per a gram of resin used in a solution at neutral
pH
In the following description another method of production of the said
substances will be described in detail. Using frothing property of proteins, the said substances were obtained by collecting the froth at the surface layer of the homogenate alter vigorous stirring at high speed (8000 to 10000 RPM for 40-60 minutes) with homogenizers the said substances containing precipitate was washed with water at acidic PH(l-2) to remove bound metals followed by neutralization with repeated washings with water or with addition of (small amount ) alkali (such as 0.1 M NaOH). By this method the product yield comprising 18-20% weight of seed constituents was obtained. These said substances obtained from frothing method were immobilized on polyester resins (as described earlier) and the resulting products sequestered metals in similar manner as the products obtained from centrifugation method.
In the following description another method namely batch method of using metal sequestering substances for removal of metal traces from aqueous solution will be ^described in detail. Batch method: Addition of metal sequestering substances in metal traces containing solution by mixing ( gently and thoroughly without vigorous stirring) at a suitable PH for maximum sequestration of that particular metal present in solution and later followed by removal of metal bound said substance by way of centrifugation or precipitation. Most of the bound metal from the said substances were eluted at 0.8 to 1.0 PH using mineral acids including sulfuric, nitric, and hydrochloric.
The metal sequestering property of said substances (metal sequestering substances) from Strychnos potatorum(L.) seeds will be described with the following experiment using batch method. A gram of metal sequestering substances ( produced by centrifugation method as described earlier without immobilization) was mixed gently and thoroughly without vigorous stirring in a reaction volume of 50 ml at neutral P*1 in a metal containing solution. The supernatant was removed by centrifuging at 5,000 to 6,000 RPM for 30 minutes. Total metal amount sequestered by said substances was assayed by Spectrophotometry or ICP method. The metal amount absorbed by said substances was measured by assaying the metal content in supernatant and as

well as bound metal in said substances after eluting with mineral acids including sulfuric, nitric and hydrochloric (metal elution from said substances was carried out with acid at 0.8 P** after washing the said substances with enough water to remove the excess unbound metal content).
As shown in Table(3), the said substances ( metal sequestering substances from Strychnos potatorum(L.) seeds) sequestered metals in aqueous medium at neutral P*1. The experimental illustration of the invention (as shown in Table.3) in relation to metal sequestering property of metal sequestering substance should not be constructed to limit to the scope of invention, as metal sequestering substances from Strychnos potatorum(L.) seed constituents have ability to sequester many metals in aqueous medium at various conditions.
Now this invention will describe the important parameters for metal sequestering and bound metal elution. This said substances effective use is dependant on suitable P** for metal sequestration. Each metal has its own binding property based on the PH of the reaction medium. The said substances sequestering ability is maximum at neutral r for most of the metals tested The said substances (metal sequestering substances) can not bind and or sequester many metals at acidic PK( 2-3 ) except few such as iron and some heavy metals. Most of the bound metals in the said substances (after reacting with a particular metal in a solution) can be eluted with mineral acids including sulfuric, nitric and hydrochloric at 0.8 to 1.0 r . The immobilized metal sequestering substance are suitable for repeated usage where as non immobilized metal sequestering substances can be used for 3-4 times. The metal sequestering capacity of non immobilized metal sequestering substances is more than the immobilized metal sequestering substances.
One of the salient features of the invention is the identification of metal binding or sequestering substances present in Strychnos potatorum(L.) seeds constituents. The fourteen peptides identified in seed constituents are mainly responsible for metal sequestering and or binding and or removal of metals in an aqueous medium. The molecular weight of these peptides. (1) 61,700 (2) 56,500 (3) 53,200 (4) 51,500 (5) 44,600 (6) 39,900 (7) 37,200 (8) 31,700 (9) 30,200 (10) 20,000 (11) 18,700 (12) 14,800 (13) 13,600 (14) 7,080. The Strychnos potatorum(L.) seed contains carbohydrates as major constituents. These carbohydrates are divided into two parts as flocculating and non flocculating. The flocculating carbohydrates comprises about 15 to 17 % of total seed constituents. These flocculating carbohydrates are fibrous in nature. The fourteen metal sequestering peptides are entrapped in these fibrous structures of carbohydrates. There is no chemical bond between these carbohydrates and peptides. If not disturbed by vigorous homogenization these carbohydrates travel along with these metal sequestering fourteen peptides for flocculation in an aqueous medium at neutral P^ range.
These metal sequestering fourteen peptides entrapped in fibrous carbohydrates comprises about 4 to 5 % of total seed constituents. Amino acid analysis of metal sequestering peptides containing substances revealed that

Glutamic Acid, Aspartic Acid, Arginine, Leucine and Glycine comprises about 56 % of these fourteen peptides. The flocculating carbohydrates are not important directly in relation to metal sequestering activity but indirectly these carbohydrates are important as they provide structural base to the metal sequestering peptides therefore preventing denaturation of metal sequestering peptides at various reaction conditions in a solutioa
Another salient feature of this invention is regarding the production and utility methods of metal sequestering substances form Strvchnos potatorum(L.) seeds. For production of metal sequestering substances from seed constituents centrifugation or frothing methods were developed. Immobilization of metal sequestering substances was achieved using polyester resins. Most of the metal sequestering substances are useful to remove metal traces using a batch method or column method. The products produced by centrifugation or frothing are immobilized said substances (metal sequestering substance) are useful through batch method to remove or sequester various metals from PPM to PPB level in an aqueous medium. The metal bound said substances from batch method are removed through centrifugation or precipitation. The immobilized said substances (above 200 micron size) are useful on column mode and suitable for repeated usage on many cycles to remove or sequester various metals from PPM to PPB level.
Another salient feature of the invention is the process of metal sequestration and elution by using suitable P^. The said substances effective use depends on suitable PH for metal sequestration in an aqueous medium. Each metal has its own binding property based on the P of the reaction medium. The said substance sequestering ability is maximum at neutral PH range for the most of the metals tested The said substances (metal sequestering substances) can not bind and or sequester many metals at acidic PH ( 2 to 3) except few such as iron and few heavy metaJs. Most of the bound metals in said substances after reacting with the particular metal in a solution can be eluted with mineral acids including sulfuric, nitric and hydrochloric at 0.8 to 1.0 PH
The immobilized metal sequestering substances are suitable for repeated usage where as non immobilized metal sequestering substances can be used for 3-4 times. The metal sequestering capacity of non immobilized metal sequestering substances is more than the immobilized metal sequestering substances.
The metal sequestering substances have extensive industrial applications. They can be effectively used for pollution control to protect environment from heavy metal contamination. The wide application of the said substance is the subject matter of this invention. Accordingly, the invention pertains to a method of treating industrial waste water containing metaJ traces, the said method comprises treating the above defined waste (containing toxic heavy metal) water in a column containing immobilized metal sequestering substances, the said substances being obtained from plant

sources which have ability to reduce metal load from PPM to PPB level from metal polluted industrial waste water, the said method comprising passing the metal traces containing waste water through the said column containing immobilized metal sequestering substances, so that the toxic metal substances are removed from the industrial waste water.
Advantages: The metal sequestering substances from the seeds of Strychnos potatorum(L.) are capable of metal load removal from PPM to PPB level and these substances are non toxic biodegradable plant materials for easy disposal which can be produced at relatively cheaper cost compared to other plant derived metal sequestering substances. This economically viable and technically feasible metal pollution control method using metal sequestering substances from Strychnos potatorum(L.) seeds is useful for environmental protection and helps tribals to get more income as they collect these seeds from the forest.
The molecular details of metal sequestering peptides as herein before described and as shown in Figure 1 (electrophoresis of metal sequestering peptides on SDS PAGE, 10-18% gradient gel), Table 1 (molecular weight of fourteen metal sequestering peptides) and Table 2 (amino acid composition (%) of metal sequestering peptides ) are enclosed with this complete specification.
It is to be noted that an embodiment of the invention is described in detail in the complete specification so as to illustrate salient features of the invention, further it is envisaged that within the scope of the invention various modifications of the invention are permissible. It is evident that description will in no way delimit the scope of the invention. The scope of the invention is described in the ensuing pages.

WE CLAIM:
(1) A method of treating industrial waste water containing metal traces
and metal contaminated aqueous solution, including metal polluted
drinking water, the said method comprises treating the above defined
metal contaminated waste water in a column containing immobilized
metal sequestering substance, the said substances being produced
from Stn/chnos potatorum (L.) seeds which have ability to reduce
metal load from PPM to PPB level from metal containing water, the
said method comprising passing the metal containing water through
the said column containing immobilized metal sequestering
substances, so that the toxic metal substances are removed from the
metaJ contaminated water.
(2) A method of removal of metal ions from aqueous solution as
claimed in claim 1 wherein the said metal sequestering peptides
containing substances are produced from Strychnos potatorum (L.)
seeds by process of centrifugation method or frothing method.
Immobilization of metal,sequestering peptides containing substances
on polyester resin was found useful to reduce the metal content from
PPM to PPB level.
(3) The method as claimed in claim 1 that the method of the sequestering
the metal load from metal containing aqueous solution uses a batch
method, by addition of metal sequestering substances in metal traces
containing aqueous medium by mixing at a suitable PH for maximum
sequestration of that particular metal present in solution and later
followed by removal of metal bound said substances by way of
centrifugation or precipitation.
(4) The method as claimed in claim 1 that the method of the sequestering
the metal load from metal containing aqueous solution uses a
column method
(5) The method as claimed in claim 3 and 4 where in the PPM to PPB
metal content is obtained by regulating the process conditions such
as the suitable P1* conditions of that particular metal binding and
e tut ion.
(6) A method of removal of metal ions from aqueous solution as
described in this specification and as explained with the help of
examples.

Documents:

141-mas-1999- abstract.pdf

141-mas-1999- descripition complete duplicate.pdf

141-mas-1999- descripition complete original.pdf

141-mas-1999- drawings.pdf

141-mas-1999-claims duplicate.pdf

141-mas-1999-claims original.pdf

141-mas-1999-correspondence other.pdf

141-mas-1999-correspondence po.pdf

141-mas-1999-form 1.pdf

« Previous Patent

Next Patent »

Patent Number

206769

Indian Patent Application Number

141/MAS/1999

PG Journal Number

26/2007

Publication Date

29-Jun-2007

Grant Date

11-May-2007

Date of Filing

04-Feb-1999

Name of Patentee

GIRIJAN CO-OPERATIVE CORPORATION LIMITED

Applicant Address

VISAKHAPATNAM-530 017(A.P)

Inventors:

#	Inventor's Name	Inventor's Address
1	YANDAPALLI DURGA PRASAD	21-339-5, FIRST ROAD, BHASKARAPURAM, MACHILIPATABAM, KRISHNA DIST (A.P)

PCT International Classification Number

B01J20/24

PCT International Application Number

N/A

PCT International Filing date

PCT Conventions:

#	PCT Application Number	Date of Convention	Priority Country
1			NA