FORM 2
THE PATENTS ACT, 1970
(39 of 1970)
and
THE PATENTS RULES, 2003
COMPLETE SPECIFICATION
[See section 10 and rule 13]
l.Title: BIOCLEANING PROCESS FOR WATER OR SOIL
CONTAMINATED BY HEXAMETHYLENETETRAMINE
2.AppIicant:
Name : KANORIA CHEMICALS & INDUSTRIES LIMITED
Nationality : Indian
Address : 3407, G.I.D.C, Industrial Estate,
Ankleshwar-393 002,
Gujarat, India
The following specification particularly describes the invention and the manner
in which is to be performed.

FIELD OF THE INVENTION
The present invention relates to a biocleaning process for water contaminated by Hexamethylenetetramine ((CH2)6N4), (Hexamine).
More particularly, the invention relates to a novel bacterial KAN-X-012 capable of remarkably decomposing a Hexamethylenetetramine ((CH2)6N4), (Hexamine) of high concentration recalcitrant toxic chemicals difficult to be degraded that are isolated from waste soil and water, the recalcitrant toxic chemicals having a chemical oxygen demand (COD) of 10,000 to 20,000 ppm , Ammonical nitrogen 1300 to 1500 ppm, and comprising chemicals, such as Hexamethylenetetramine ((CH2)6N4), (Hexamine), at concentration 3000 to 5000 ppm and the like.
BACKGROUND OF THE INVENTION
Many producers and processing plants which use large amounts of water render this water unsuitable for reuse and undesirable for release into the biosphere due to the pollution problems which result when it is discharged untreated. Biological systems, such as aerated lagoons and activated sludge systems are the generally accepted methods of processing these wastewaters prior to reuse or discharge to receiving bodies of water.
While biological processes occurring during such a biological treatment provide the ability, with most wastewaters, to produce effluent which has both low biological oxygen demand (BOD), low chemical oxygen demand (COD), and low total suspended solids (TSS), unfortunately, conventionally employed biological treatment systems depend on a temperature range favorable to biological activity.

Often, lagoon efficiency suffers due to concentration build up during winter months when microorganisms are less active. In some cases, because of reduced lagoon efficiency the rate of discharge of effluents into the lagoons must be decreased during.winter months.
With increased concern as to minimization of the problems arising from pollution, biological processes are being employed in industry in an increasing amount, and a large amount of activity in research and development is occurring presently to develop new microbial strains capable of use in wastewater treatment industrially, municipally and domestically. Even with this increased activity in investigating and developing strains of microorganisms to solve particular waste removal problems, treatment at low temperatures still remains a problem.
Research has been conducted in biocleaning methods in which contaminants are efficiently decomposed and detoxified by microorganisms. Since these methods utilize the decomposition mechanism of microorganisms, they do not require a large amount of energy as compared with the above-mentioned physical methods. They are also able to completely decompose and detoxify contaminants without causing secondary contamination. Moreover, the cleaning can be performed even at low concentrations of contaminants, thus enabling decontamination to be performed over a wide area at the original location and creating significant expectations of low costs.
Examples of methods used to purify contaminated soil by microorganisms include a solid phase treatment in which microorganisms are mixed into excavated soil with nutrient sources such as phosphorous and nitrogen to promote, decomposition of contaminants, a slurry treatment in which microorganisms are mixed into excavated soil with water and nutrient sources to treat the soil in the liquid state and promote decomposition of contaminants, and an original location treatment in

which air, nutrient sources and so forth are injected into contaminated soil without excavating to promote decomposition of contaminants by microorganisms present in the soil.
Among the above-mentioned biotreatment techniques, since soil excavation is required and the application range is limited in the case of the solid phase treatment and slurry treatment method, treatment and equipment costs are relatively high.
On the other hand, the original location treatment method involves relatively low costs and allows treatment over wide area. However, the cleaning rate is slow since the absolute number of soil microorganisms is low. In the case of compounds that are difficult to decompose such - - as Hexamine in particular, there is a possibility that microorganisms being able to decompose contaminants in the soil may not be present in the soil, thus making cleaning impossible. In this case, acquiring microorganisms that are able to decompose Hexamine and inoculating them into the soil enables the cleaning rate to be improved and soil to be purified even though microorganisms being able to decompose the contaminants are not present in the soil
Environmental contaminations, which are caused by recalcitrant toxic substances, often lead to (i) degradation of food stuffs for humans related to the food chain in the biosphere, (ii) prevalence of infection diseases due to deteriorated immunity, and (iii) spread of chronic diseases and energy exhaustion. Once the ecosphere is radically destroyed, it may take several hundred millions of years for the environment of the ecosphere to recover. Degradation of food stuffs for human related to the food chain in the biosphere, prevalence of infectious diseases due to deteriorated immunity, spread of chronic diseases and energy exhaustion. Once the

ecosphere is radically destroyed, it may take several hundred millions of years for recovery of the environment of ecosphere.
There has been an attempt to remedy recalcitrant toxic substances contained in the industriaLwastewater or noxious waste materials dumped. into the Sea normally, using physical and chemical methods such as incineration, landfill, chemicals, electrolysis, membrane separation or the like. However, these conventional methods also involve economical and environmental problems. The most efficient and safe method that meets requirements in every aspect of environmentology, sanitation, ecology and economy is the biological treatment method. Many studies have been made on the biological methods, for example, using the natural ecosystem such as silt at an estuary. However, these methods could not be a solution of treating a great mass of industrial waste materials.
SUMMARY OF THE INVENTION
Accordingly, an object of this invention is to provide a novel microorganism KAN-X-012 which is effective in degrading Hexamethylenetetramine.
A further object of this invention is to provide a treatment for industrial, municipal and domestic wastewaters utilizing a microorganism KAN-X-012, alone or in combination with other microorganisms, to degrade and remove Hexamethylenetetramine.
Another object of this invention is to provide a biological process for removing hexamine from wastewater using a microorganism KAN-X-012 in conjunction with a specific combination of other microorganisms.

An even further object of this invention is to provide a biological treatment process effective even at low temperatures for treating industrial, municipal and domestic wastewaters containing Hexamine and rendering such wastewaters suitable, for discharge into the biosphere, thereby minimizing problems of pollution.
An additional object of this invention is to provide a process of screening of novel microorganism KAN-X-012.
An additional object of this invention provides a process for treating wastewater containing Hexamine, which comprises treating wastewater containing Hexamine with a novel microorganism KAN-X-012.
It is, therefore, an object of the present invention to provide bioremediation to remedy biologically recalcitrant toxic chemicals contaminating industrial wastewater, waste materials and soils, etc., using a trace of useful specific bacterial that exists in the ecological system.
BRIEF DESCRIPTION OF THE DRAWINGS
Fig. 1 graph 1 shows C.O.D reduction with respect to time/dates.
Fig. 2 is a graph indicating the reduction % of Ammonical nitrogen with respect to feed.
Fig. 3 is a graph indicating cell viability in the aerobic tank over a period of 2.5 months, the cell were viable throughout with MLSS values at an average of 350

mg/Itr.Only initially culture addition was done in the tank, later on no addition of nutrients or culture was done.
Fig. 4 is a graph indicating depicts the pH range, of aerobic tank which was. maintained toward 7.4 to 8.0 throughout the trial indicating healthy aerobic degradation of Hexamine effluent.
DETAILED DESCRIPTION OF THE INVENTION
Hexamethylenetetramine ((CH2)6N4), (Hexamine) has multiple uses. For example it is useful in curing phenol formaldehyde and resorcinol formaldehyde resins, as a rubber to textile adhesive, as a protein modifier, as an ingredient of high explosive cyclonite, as fuel tablets, as corrosion inhibitor, as a shrink-proofing agent in textiles. In addition hexamine also has application in organic synthesis and pharmaceutical compositions.
In the synthesis of Hexamine, water effluent is generated, which contains free ammonia, traces of formaldehyde and some % of hexamine itself.
Effective treatment of hexamine effluent has been problem for hexamine producers. Currently the known method of treating hexamine effluent is mainly of chemical origin (FENTON, Cation raisin adsoption, reverse osmosis etc). These chemical methods for hexamine effluent treatment is costly and unsafe to handle. Biological method if adopted for degradation of hexamine effluent would be comparatively cheaper and safer.
Successful Biodegradation of Hexamine effluent is not been reported till date. Also micro-organism capable for degrading hexamine effluent is not being

isolated nor reported in technical research papers till date. Few Scientists have tried to isolate micro-organism capable to degrade Hexamine effluent but were not successful to degrade it efficiently.
Hexamine effluent is known to be resistant to biodegradation. Also the chemical constitution of hexamine is addition of formaldehyde and ammonia which are also toxic for micro -organisms.
In this study an attempt was made to isolate micro organisms which would grow in hexamine effluent and successively degrade it lowering down the C.O.D and Ammonical nitrogen values in the effluent.
The novel microorganism Pseudomonas sp. KAN-X-012 is isolated from a soil at a large chemical manufacturing plant.
The microorganism of the present invention is belonging to the genus of Pseudomonas. This microorganism is a new strain isolated from nature such as rivers and soil, and their isolation method and taxonomical characteristics are specifically described. This strain KAN-X-012 were deposited to MTCC and identified as Pseudomonas sp by 16 S rRNAgene sequencing. The microorganism of the present invention is designated as Pseudomonas KAN-X-012, respectively by MTCC.
The microorganisms of the present invention can be cultured in the presence of routinely used carbon sources and nitrogen sources in a medium containing inorganic salt, vitamins and other trace elements as necessary. Any carbon source can be used provided it is a carbon source that is preferentially assimilated by the microorganisms of the present invention. Although varying according to the type of carbon source, the concentration of carbon source in the medium is preferably,

for example,. 0. \ to 0.5 g/L. Examples of nitrogen sources that can be used include organic nitrogen sources such as yeast extract, peptone and meat extract, while examples of inorganic nitrogen sources include ammonium salts and nitrates.
In another embodiment of this invention, this invention provides a novel strain of the species Pseudomonas KAN-X-012 having the characteristics described AS KAN-X-012 isolated from soil a novel Gram-negative, rod-shaped.
This microorganism KAN-X-012 has been found to be capable of degrading Hexamine and at a rate faster and has the characteristics described below.
The present invention is directed to a novel bacteria KAN-X-012 strain and a method for remedying biologically recalcitrant toxic chemicals Hexamethylenetetramine ((CH2)6N4), (Hexamine) compounds contained in industrial wastewater, waste materials, soils, or the like using the bacterial KAN-X-012 strain. More particularly, the invention is directed to a novel bacterial KAN-X-012 strain capable of remarkably degrading Hexamine compounds of high concentration and alkaline recalcitrant toxic chemicals that are isolated from soil and wastewater having COD of 10,000 to 20,000 ppm, such as contained in the industrial wastewater nitrogen compounds'are amines such as. Hexamine, and the like.
The inventor isolated a novel bacterial KAN-X-012 strain having a characteristic of rapidly degrading recalcitrant waste materials Hexamine compounds, from the soils and wastewater collected from the industrial complex and the petroleum chemical industrial complex. The bacterial KAN-X-012 strain is pseudomonas spices. It was found that the bacterial KAN-X-012 degrades Hexamine recalcitrant waste materials having the COD of 10,000 to 20,000 ppm and Hexamine content 3000 to 5,000 ppm contained in the industrial wastewaters in an aerobic manner.

Accordingly, the present invention is to provide such a novel bacterial KAN-X-012 strain capable of degrading recalcitrant toxic chemicals and specifically hexamine, and a method using the abacterial KAN-X-012 strain in remedying., biologically recalcitrant toxic chemicals contaminating industrial wastewater, waste materials and soils.
Now, a description will be made to isolation, identification and activity of the novel bacterial KAN-X-012 strain.
1. Isolation of Novel Bacterial KAN-X-012 strain
(1) Isolation of Strains from Soil samples collected from various regions The soil and wastewater from the industrial complex were dissolved in sterile water and plated on nutrient agar plates. The organisms grown on nutrient agar plates were isolated purified and screened on plates containing 600, 1800, 3000, and 5000 ppm Hexamine content. Microbial colony growing on these selective plates were isolated and used in this study.
Procedures
The aerobic tank was commissioned using screened micro-organisms dissolved in water, small amounts of nutrients(urea 1 ppm) were added in the tank initially, and hexamine effluent was feeded slowely,initially 3000 ppm of Hexamine containing effluent (Diluted 40:60 with process water) was feeded and then the concentration was exceeded slowly up till 5000 ppm.
The pH of the diluted effluent was maintained 8.0 to 8.5 with addition of Sulfuric acid

500 ml of diluted effluent was feeded daily into the aerobic tank, and overflow of the tank was taken up into another aerobic tank of 05 Itrs capacity.
Daily samples were .drawn, from the tank and analyzed for parameters, such ,as C.O.D, Ammonical nitrogen, MLSS, pH, also samples were plated on nutrient agar plates and selective plates containing 5000 ppm % v/v hexamine.
2. Optimum Conditions for Growth of Bacteria KAN-X-012
The bacterial KAN-X-012 had the best growth in the nutrient agar plates as well as selective media plates containing Hexamine effluent (Hexamine cone up till 5000 ppm) at pH 7 to 9 and 25 to 35° C. The bacterial KAN-X-012 was also well grown in the successive transfer culture under the same conditions,
3. Identification of Bacterial KAN-X-012
Bacterial KAN-X-012 is a gram negative rod shaped pseudomonas sp. Bacterial KAN-X-012 having sequence listing as follows [SEQ ID NO:l]:
The isolated bacterial strain was deposited to MTCC Chandigarh for identification. Based on sequencing of 16 S rRNA gene strain it was been identified as Pseudomonas sp. With 99.156 pair wise similarity with Pseudomonas Taiwanesis strain BCRC 17751 (T).

Hexamine
(concentration) Reduction in COD
Ppm
2400 44.49
3000 41.30
3600 47.25
5000 56.9

As shown in Table Results indicate the reduction in C.O.D is 40 to 57 % with increase in Hexamine effluent concentration up till 5000 ppm the C.O.D reduction increases.
(2) Decomposition of Ammonical nitrogen in hexamine Compound by bacteria KAN-X-012
Batches of hexamine effluent with an increasing concentration of 3000 ppm, 3600 ppm, and 5000 ppm of Hexamine were subjected to degradation by bacteria KAN-X-012. After inoculation of the bacterial KAN-X-012, the degradation of Ammonical Nitrogen was measured.
Results indicate the Ammonical nitrogen reduction is 40 to 57 %, with increase in feed concentration reduction in Ammonical nitrogen was found increasing.

Hexamine
(concentration)
Ppm Reduction in Ammonical Nitrogen %
2400 41.80
3000 42.77
3600 59.83
4200 56.45
Treatment of Recalcitrant Industrial Wastewater with bacterial KAN-X-012
1. The isolated micro-organism KAN-X-012 was able to successfully degrade hexamine effluent till 5000 ppm of Hexamine concentration.
2. Bacterial cultures, Pseudomonas sp was. prominent to degrade hexamine effluent this Pseudomonas sp cultures are gram negative long rods. The

identification was done by MTCC Chandigarh India based on sequencing of 16 S rRNA gene.
3. Treatment to run under aerobic condition with temperature maintained 28 to 36._deg C.
4. Hexamine effluent to be diluted in the ratio 70:30 with process water as to maintain Hexamine concentration around 5000 ppm, pH to be adjusted 8.0 to 8.5 and feeded in the aerobic tank.
5. Hydraulic retention time of 9 to 10 days should be given for effective degradation up till 60 %.
EXPERIMENTAL EXAMPLE 1
Use of Bacterial KAN-X-012 in Treatment of a hexamine contained in Industrial Wastewater
The aerobic tank was commissioned using screened micro-organisms dissolved in water, small amounts of nutrients(urea 1 ppm) were added in the tank initially, and hexamine effluent was feeded slowely,initially at 3000 ppm concentration of Hexamine v/v(Diluted 40:60 with process water) effluent was feeded and then the concentration was exceeded slowly up till 5000 ppm. The pH of the diluted effluent was maintained 8.0 to 8.5 with addition of Sulfuric acid, 500 ml of diluted effluent was feeded daily into the aerobic tank, and overflow of the tank was taken up into another aerobic tank of 05 Itrs capacity. Daily samples were drawn from the tank and analyzed for parameters such as C.O.D, Ammonical nitrogen, MLSS, pH, also samples were plated on nutrient agar plates and selective plates containing 5000 ppm v/v hexamine.
For a reference, the analytical methods used in the experimental examples of the present invention were as follows:

1) COD Analysis
The chemical oxygen demand (COD) was measured by the COD reflux method as
per standard method APHA 1998.
2). Ammonical nitrogen values in the feed and samples drawn from overflow of
aerobic tank was analyzed by phenate method using standard methods APHA
1998
3) The pH. suspended solid (SS), Sludge volume index (SVI), volatile suspended solids (VSS), dissolved oxygen (DO) were analyzed using standard methods(APHA1998).
4) CFU Examination
The counts of indigenous bacteria and inocula of the individual treated samples were analyzed by the plate count method according to the standard method for bacterial examination, to measure the growth of the bacteria at the beginning of the test and with an elapse of time by a decrease in the noxious substances.

We claim;
1. A novel microorganism Pseudomonas species said microorganism being
capable upon culturing in wastewater containing hexamine.
2. A novel microorganism belonging to the genus Pseudomonas and having an ability to decompose hexamine, which are able to decompose 57 % or more of 5000 ppm of hexamine in 22 days.
3. A novel microorganism according to claim 1 or 2 that are Pseudomonas KAN-X-012

4. A novel microorganism according to claim 1 or 2 that are Pseudomonas KAN-X-012 shown in the sequence listing under SEQ ID NO: 1.

KAN-X-012(1426 bp)

5. A novel microorganism belonging to the species Pseudomonas KAN-X-012 having the ability to decompose hexamine.
6. An isolated bacterial KAN-X-012 which grows under culture in Hexamine nutrient medium at pH 7 to 9, at a temperature of 25 to 36° C, wherein the

bacterial KAN-X-012 grows in a successive transfer culture under the same conditions.
7. A method for removing biologically recalcitrant, chemicals from wastewater
containing said biologically recalcitrant chemicals, comprising contacting the
wastewater with an isolated bacterial KAN-X-012, wherein the wastewater
contains hexamine.
8. Microorganism according to claim 1 to 6 wherein said hexamine is
Hexamethylenetetramine.
9. A process for cleaning of water or soil containing hexamine comprising the
steps of strains of the microorganisms according to any of claims 1 through 6 is
added to water or soil containing hexamine.
10. A process according to claim 9 wherein microorganism activator is added to
the water or soil with said added microorganisms.
DATE and SIGNATURE
Date: 4th April 2013
Signature:
Name: Bhavik B. Patel
(Applicants Agent)