FORM 2
THE PATENT ACT 1970
&
THE PATENTS RULES, 2003
COMPLETE SPECIFICATION
(See section 10 and rule 13)
1. TITLE OF THE INVENTION PROCESS FOR BIOLOGICAL TREATMENT OF WASTEWATER
2. APPLICANT
(a) NAME: PRAJ INDUSTRIES LIMITED
(b) NATIONALITY: Indian Company
(c) ADDRESS: PRAJ House, Bavdhan, Pune-411021, INDIA
3. PREAMBLE TO THE DESCRIPTION
The following specification particularly describes the invention and the manner in which it is to be performed.

4. DESCRIPTION
FIELD OF THE INVENTION
The invention relates to a method for the treatment and removal of organic pollutants from an effluent wastewater specifically containing contaminants like xylene, toluene, epichlorohydrin and similar compounds. More particularly it relates to removing said organic contaminants using a set of microorganisms to make said wastewater reusable or ecologically safe.
■■*.-BACKGROUND
Chemical industry is one of the largest of all industries, which contributes substantially to the economic development of several, communities worldwide. Worldwide chemical factories produce millions of tons of effluent wastewater from its plants that needed to be treated and purified before release to natural streams, recycled or reused.
In recent times due to increased need of preservation of water and its use for more important agricultural applications than chemical production, there is significant pressure on chemical factories to reduce water consumption and recycle the process wastewater generated in plants more effectively. To this end, several technologies have been presented on the treatment, recovery and recycling of process wastewater. However, in many cases

these technologies have achieved limited success due to composition of wastewater coming out of chemical factories. In many cases, the effluent wastewater generated at a particular chemical plant has specific composition of contaminants; such wastewater can only be treated when customised biological treatment is developed for it. Due to complexity of organic contaminants and its tolerance by microorganisms each case of special effluent requires creating of new consortium of useful microorganisms for biological treatment of said effluent.
At epoxy resin production plants wastewater effluents contain chlorohydrins, glycerin, polyglycerides, inorganic, salts of alkali and alkaline earth metals and other organic pollutes. These effluents have high CODs. Before this stream can be discharged into environment or sea water, the organic contents must be substantially reduced in order to meet regulatory standards as disposal of such wastewater without prior treatment is known to adversely affect the environment. Disposal of such waste streams containing significant amounts of metal salts like sodium chloride and calcium chloride, significant amounts of water soluble organic materials such as glycerin, toluene, epichlorohydrin, xylene and polyglycerides is both difficult and expensive. These elements cause corrosion or fouling problems for disposal by incineration. TABLE 1 list a typical composition of wastewater effluent from an epoxy resin production plant.

TABLE 1: TYPICAL COMPOSITION OF SAID WASTEWATER FEED STREAM

Sr. No. PARAMETER UNIT VALUE
1 pH - 6 to 8
2 COD PPM 7500 - 9000
3 Xylene % 0.001 -0.005
4 Glycerol % 0.01-0.13
5 Toluene % 0.01 -0.05
5 Epichrolohydrin % 0.01 - 0.05
6 NaCI % 1.6-3.0
Biological treatment is preferred due to consistent COD removal efficiency and good performance of microbes without disturbing ecosystem. It is also environmentally benign and sustainable. But due to presence of alkali and alkaline earth metals and their salts as well as organic pollutes, water becomes toxic to both aerobic and anaerobic bacteria. Conventional biological treatment systems are known to be insufficient in removal of COD from saline wastewaters because of the adverse effects of salt on microbial flora. Microorganisms requiring or tolerating salt for growth are designated halophilic. The intracellular salt concentration of halophilic microorganisms is low and they maintain an osmotic balance of their cytoplasm with the external medium by accumulating high concentration of various organic osmotic solutes. Therefore, utilization of salt tolerant

microorganisms in biological wastewater treatment systems could be a solution for COD removal from saline wastewater.
The invention presented herein discloses a method for treatment of an effluent with high levels of salts and COD with organic pollutants having several advantages over known methods. Besides it provides recycled water at the end of treatment that is directly usable in plant processes or released to natural streams without any adverse effect on the environment.
BRIEF DESCRIPTION OF THE DRAWINGS
Particular examples of a method in accordance with this invention will now be described with reference to accompanying drawings, in which:
FIGURE 1 is a schematic diagram of the mass flow in the disclosed process. An effluent wastewater stream is subjected to a dilution step and nutrient additives are added. Then, said consortium of halophilic microorganisms is added and biological treatment allowed at desired conditions so that COD of the stream reduced up to 90% that of initial amount. Next, said treated stream is filtered through a membrane filter to separate said microorganisms forming a clean water stream, while removed microorganisms are recycled for effective substance of said microbial consortium.

DETAILED DESCRIPTION OF THE INVENTION
In one embodiment of the invention a wastewater effluent generated during manufacture of epoxy resins contained about 1.6 - 3.0 % sodium chloride, up to 0.13 % glycerol, about 0.01 - 0.05 % toluene, about 0.001 - 0.005 % xylene, and about 0.01 - 0.05 % epichlorohydrin. Said effluent having pH between about 6.5 to about 8 and COD between about 7000 to about 9000 PPM. Next, to said effluent a seed culture prepared from a consortium of halophilic microorganisms is added and biological treatment is allowed for up to 7 days at desired temperature between 25 - 45 °C with aeration as required to achieve a COD reduction of 90 % in said period.
In another embodiment, said seed culture is prepared of a consortium of halophilic microorganisms isolated from natural salt water locations. Each isolate of said consortium having certain ability to bio-degrade or bio-convert organic pollutants in the wastewater effluents. To support the robust growth of said consortium, said seed culture is supplemented with nutrients like dried brewer's yeast, calcium chloride, sodium nitrate, magnesium sulphate and potassium dihydrogen phosphate. Said seed culture is allowed to grow at 33 °C for 2 days before use for said biological treatment. After several passages in said effluent containing seed medium, said consortium is well tolerant and biotransforms said organic pollutants effective to no harmful products.

In yet another embodiment,.after reaching the optimum level of microbial growth in wastewater in 5-7 days, COD of the wastewater stream is measured and, if required said biological treatment is continued till COD is reduced to between about 70 % to about 90 %. Then treated effluent is subjected to membrane filtration to remove microbial consortium to form a clean stream. This clean stream is permeated to natural water or reused and the microorganisms are reused for further treatment of wastewater.
Embodiments provided above provide wider utility of the invention without any limitations as to the variations that may be appreciated by a person skilled in the art. A non-limiting summary of various embodiments is given in the examples and tables, which demonstrate the advantageous and novel aspects of the process disclosed herein.
EXAMPLE 1: ANALYSIS OF WASTEWATER
Composition of wastewater generated at an epoxide manufacturing plant was analyzed using standard HPLC methods. Said wastewater effluent was found to contain about 3.0 % sodium chloride, about 0.13 % glycerol, about 0.05 % toluene, about 0.005 % xylene, about 0.05 % epichlorohydrin and pH about 8. Said wastewater further had COD of about 7000 PPM as measured by a close-reflux colorimetric method.

EXAMPLE 2: SAMPLING AND ISOLATION OF MICROORGANISMS
For isolation of halophilic microorganisms eight samples were collected randomly from separate locations on marine salt pans of coastal area around Mumbai. These samples were diluted to 10, 102 and 103 times with sterile distilled water and showed ability to grow and survive in high salt concentration of up to 10 % by weight. These enriched samples were further diluted up to 1014 and subjected to spread plate technique. About 12 isolates were found growing efficiently in 10X diluted above wastewater sample at 33 °C. These isolates were maintained on nutrient agar plates containing 3 % NaCI as the master isolates. These isolates were mainly members of halophilic bacterial and archaeal genus not specifically characterized. For biological treatment of said wastewater as disclosed herein a consortium containing said master isolates was prepared before any large scale operation.
EXAMPLE 3: WASTEWATER TREATMENT
A microbial consortium was prepared by mixing 12 master isolates mentioned above in a nutrient broth containing 3 % NaCI. The nutrient broth was incubated at 37 °C at 150 RPM for about 24 hours to get optical density [OD600] about 2.0 of growing cells. Next, about 1 L of wastewater was diluted to about 10 times with process condensate. Additives like dried brewer's yeast (0.05 %), calcium chloride (0.03 %), sodium nitrate (0.02

%), magnesium sulfate (0.01 %), and potassium dihydrogen phosphate (0.005 %) which support growth of microorganisms were added to the this medium. About 100 ml_ of master inoculum containing said microbial consortium was inoculated in said 1 L of wastewater and growth was allowed at temperature of 35 °C, aeration of 0.4 WM and agitation at 300 RPM for about 7 days to grow the cells to reach OD of about 1.7. This batch formed a seed culture for the next large scale treatment of wastewater. Next, about 100 L of effluent wastewater was diluted to about 10X with process condensate. Above mentioned additives were added to this diluted wastewater. Then said seed culture was added to it and biological treatment was allowed over next 7 days. COD of effluent under treatment was periodically measured and at the end of 7-day period it was reduced from about 7000 PPM to about 1480 PPM. This treatment achieved COD reduction of about 80 %. This treated effluent was then passed through a filter of 1 micron to remove suspended solids and then through a 0.2 micron filter to remove halophiles for reuse in next round of biological treatment of said wastewater effluent. Clean water separated at the end was either recycled for industrial uses or discharged to nature water stream.

5. CLAIMS
WE CLAIM:
1. A method for treating a wastewater using halophilic microorganisms comprising:
. (a) providing a wastewater effluent stream comprising NaCI and organic pollutants;
(b) collecting said effluent stream in a bioreactor and diluting it forming a first stream with optimum amount of said organic pollutants;
(c) adding one or more chemicals supporting growth of said microorganisms forming a second stream;
(d) contacting said second stream with said microorganisms for desired temperature, aeration, agitation and time to form a third stream;
(e) removing said consortium of halophilic microorganisms from said third stream by membrane filtration forming a fourth stream;
(f) subjecting said forth stream to quality check; and
(g) releasing said forth stream to a natural water stream.

2. The method of claim 1, wherein said wastewater effluent stream is diluted with water up to 10 times.
3. The method of claim 1, wherein said halophilic microorganisms comprise one or more of bacteria and archaea that are tolerant to salt concentrations of up to 10% by weight forming the consortium.
4. The method of claim 1, wherein said halophilic microorganisms require one or more of dry brewer's yeast, calcium chloride, sodium nitrate, magnesium sulphate and potassium hydrogen phosphate in nutrient medium for optimum growth.
5. The method of claim 1, wherein said organic pollutants comprise toluene, xylene or epichlorohydrin.
6. The method of claim 1, wherein chemical oxygen demand of said wastewater effluent stream is reduced up to about 90%.
7. The method of claim 1, wherein a microaerophilic condition is maintained during treatment of wastewater by supply of air at a rate between 0.01-0.4 WM.
8. The method of claim 1, wherein agitation is maintained during treatment of wastewater at a rate between 10 - 300 RPM.

9. The method of claim 1, wherein temperature is maintained during treatment of wastewater at between 25 - 45 °C.
10. The method of claim 1, wherein said microorganisms are tolerant to any wastewater stream comprising organic pollutants toluene, xylene and epichlorohydrin.
11. The method of claim 1, wherein said microorganisms are reused for treating said second stream after recovery using membrane filtration from said third stream