Title of Invention	A PROCESS FOR THE PREPARATION OF A NOVEL IMMOBILIZED MICROBIAL MEMBRANE
Abstract	An immobilized microbial consortium is formulated which comprises of a synergistic mixture of the isolated bacterial namely, Aeromonas hydrophila, Pseudomonas aeruginosa, Yersinia enterocolitica, Serratia liquefaciens, Pseudomonas fluorescens, Enterobacter cloaca, Klebsielfa oxytoca, Citrobacter amalonat"cus and Enterobacter sakazaki. The formulated microbial consortium is immobilized on charged nylon membrane. The said immobilized microbial consortium is attached to dissolved oxygen probe for the preparation of electrode assembly. The prepared electrode assembly is used for rapid and reliable BOD estimation. The prepared electrode assembly is used for the monitoring of BOD load of synthetic sample i.e., Glucose-Glutamic acid (GGA) used as a reference standard in BOD analysis and industrial effluents; covering a range from low to rvgh biodegradable organic matter.

Title of Invention

A PROCESS FOR THE PREPARATION OF A NOVEL IMMOBILIZED MICROBIAL MEMBRANE

Abstract

An immobilized microbial consortium is formulated which comprises of a synergistic mixture of the isolated bacterial namely, Aeromonas hydrophila, Pseudomonas aeruginosa, Yersinia enterocolitica, Serratia liquefaciens, Pseudomonas fluorescens, Enterobacter cloaca, Klebsielfa oxytoca, Citrobacter amalonat"cus and Enterobacter sakazaki. The formulated microbial consortium is immobilized on charged nylon membrane. The said immobilized microbial consortium is attached to dissolved oxygen probe for the preparation of electrode assembly. The prepared electrode assembly is used for rapid and reliable BOD estimation. The prepared electrode assembly is used for the monitoring of BOD load of synthetic sample i.e., Glucose-Glutamic acid (GGA) used as a reference standard in BOD analysis and industrial effluents; covering a range from low to rvgh biodegradable organic matter.

Full Text	FIELD OF THE INVENTION The present invention relates to a process for the preparation of a novel immobilized microbial membrane. More particularly, the present invention relates to an immobilized microbial consortium and a process for the preparation of the said immobilized microbial consortium useful for rapid and reliable BOD estimation. Description of the prior art Rapid analytical devices have attracted tremendous interest and attention in science and technology for their wide range of possible application as an alternative to conventional analytical techniques. Analytical devices are sensitive to biological parameters and consist of a biological sensing element such as microbes, enzymes, etc., in close contact with a physico-chemical transducer such as an electrode, which converts biological signal to a quantitative response. These devices have several unique features such as compact size, simple to use, one step reagent-less analysis, low cost and quick real time results. Rapid analytical devices, termed as biosensors, have the potential for a major impact in the human health care, environmental monitoring, food analysis and industrial process control. Among these, microbial biosensors (the devices using microbes as biological component), have great potentiai in environmental monitoring. Recent trends in biotechnology suggest that mon.torirg and control of pollutant by means of microbial biosensors may be of crucia: importance. Such microbial sensors, constructed by entrapping the required micro-organisms in sultable-pelvmeric matrices.and Attached to a transducer Function on the basis of assimilatory capacity of the micro-organisms. In addition, microbial biosensors are more stable and inexpensive for the determination of compounds of interest as compared to enzyme-based biosensors; where enzymes employed in enzyme-based biosensors require costly extraction and purification prior to use as biocatalysts. Further, micro-organisms employed in microbial biosensors show a high degree of stability as compared to enzymes. The vast majority of micro-organisms are relatively easy to maintain in pure cultures, grow and harvest at low cost. Moreover, the use of microbes in biosensor field have opened up new possibilities and advantages such as ease of handling, preparation and low cost of the device. Such devices will help in monitoring the compounds of environmental interest such as Biochemical Oxygen Demand (BOD), heavy metals, pesticides, phenols, etc. Among the environmental parameters, the potential demand for rapid BOD monitoring device is higher, since, BOD is a parameter which is measured most frequently by many industries for measuring the level of pollution of waste-waters. BOD provides information about the amount of biodegradable substances in waste-waters. Conventional BOD test takes 3-5 days and as a consequence, is unsuitable for use in direct process control. A more rapid estimation of BOD is possible by developing a BOD biosensor. Such BOD biosensors are able to reduce the time of BOD test upto a great extent. A number of microbial BOD sensors have been developed nationally and internationally (Rajasekar et al, 1992 and Karube, 1977). A number of pure cultures, eg., Trichosporon cutaneum, Hansenula anamola, Bacillus cereus, Bacillus subtilis, Klebsiella oxytoca, Pseudomonas sp., etc., individually, have been used by many workers for the construction of BOD biosensor (Preinenger et al, 1994; Hyun et ai, 1993, Li and Chu 1991; Riedel et al, 1989 and Sun and Kiu, 1992). Karube et al, (1992) developed a BOD biosensor by utilizing thermophilic bacteria isolated from Japanese hot spring. On the other hand, most of the workers have immobilized activated sludge (Vanrolleghem et al 1990; Kong et al 1993; Vanrolleghem et al, 1984), or a mixture of two or three bacterial species (Iki, 1992 and Galindo et al 1992) on various membranes for the construction of BOD biosensor. The most commonly used membranes were polyvinyl alcohol, porous hydrophilic membranes, etc. Riedel et al, (1988), have used polyvinyl alcohol for the immobilization of Bacillus subtilis or Trichosporon cutaneum which are used for the development of BOD biosensor. Vinegar (1993) immobilized Klebsiella oxytoca on porous hydrophilic membranes such as nitrocellulose, acetyl cellulose, polyvinylidene flouride or polyether sulfone, 50-2000 micrometer thick. Cellulose acetate membrane was used for the immobilization of Lipomya-s kononankoae and Asperillus niger (Hartmeier et al, 1993). The drawback of such developed BOD biosensors which are constructed by using either single, pure culture or activated sludge is that they do not give reproducible results, as single microbe is not able to assimilate/ degrade all the organic compounds and therefore may not respond for the total organic matter present in the test sample (eg., carbohydrates, proteins, fats, grease, etc.) Moreover, in the activated sludge either non-specific predominating microorganisms are present thereof or microorganisms with antagonistic effects are present which may produce erratic results. On the other hand, randomly selected mixtures of two or three micro-organisms also do not give reproducible, comparable BOD results. The reproducibility of the BOD biosensor can be obtained by formulating a defined microbial composition. To avoid the discrepancies in BOD results as well as to get instant BOD values using rapid analytical devices, in the present invention, a defined microbial composition is formulated by conducting a systematic study, i.e., pretesting of selected micro-organisms for use as a seeding material in BOD analysis of a wide variety of industrial effluents. The formulated microbial consortium is capable of assimilating most of the organic matter present in different industrial effluents. The formulated microbial consortium has been immobilized on suitable membrane i.e., charged nylon membrane useful for BOD estimation. Suitability of the charged nylon membrane lies in the specific binding between the negatively charged bacterial cell and positively charged nylon membrane. So, the advantages of the used membrane over other membranes are the dual binding i.e., adsorption as well as entrapment, thus resulting in a more stable immobilized membrane. Such specific microbial consortium based BOD analytical devices, may find great application in on-line monitoring of the degree of pollutional strength, in a wide variety of industrial waste-waters within a very short time (from 3-5 days to within an hour), which is very essential from pollution point of view. For solving the aforementioned problems, the applicants have realized that there exists a need to provide a process for the preparation of a defined synergistic microbial consortium immobilized on a suitable support i.e., charged nylon membrane, useful for BOD estimation. The said microbial consortium is capable of assimilating most of the organic matter present in different industrial effluents Objects of the invention The main object of the present invention is to provide a microbial consortium and a process for the preparation of the microbial consortium immobilized on a suitable support useful for BOD estimation. The formulated microbial consortium comprises of cultures of the following bacteria viz., Aeromonas hydrophila, Pseudomonas aeruginosa, Yersinia enterocolitica, Serratia liquefaciens, Pseudcomonas fluorescens, Enterobacter-cloaca, Klebsiella oxytoca, Citrobacter amalonaticus and Enterobacter sakazaki, The individual bacteria of microbial consortium are pre-tested by using them as a seeding material in BOD analysis of a wide variety of industrial effluents. The micro-organisms have been selected for the formulation of microbial consortium on the basis of pre-testing. The formulated microbial consortium is obtained by inoculating a suspension of these bacteria individually. Incubating at 37°C, mixing all bacterial cultures in equal proportions based on optical density and centrifuging. The resultant pellet is immobilized on suitable support, i.e., charged nylon membrane by entrapment and adsorption on the charged surface of the membrane. The said, charged immobilized microbial membrane has high viability, long stability and greater shelf-life as compared to the microbial consortium immobilized on conventional supports such as polyvinyl alcohol + nylon cloth. Accordingly, another object of the present invention, is to provide a process for the production of immobilized formulated microbial consortium useful for monitoring the BOD load of a wide range of industrial effluents with low, moderate and high BOD load. Summary of the invention The present invention provides an immobilized microbial consortium and a process for the preparation of the said immobilized microbial consortium, useful for rapid and reliable BOD estimation of a wide range of industrial effluents with low, moderate and high BOD load. Detailed description of the invention The microbial consortium provided according to the present invention contains bacteria consisting of: (Table Removed) which facilitate the process of testing, giving BOD results of a wide variety of industrial effluents, performed at any place. Above microorganisms are deposited at Centre for Biochemical Technology Culture Collection (CBTCC) designated as stated above and will be made available to public on request as per normal official procedures. The main characteristic features of all the bacterial cultures used for the invention which are similar to ATCC cultures are given below : Characteristic features of Aeromonas hydrophila (CBTCC/MICRO/10) Gram negative rods Motile by a single polar flagellum Metabolism of glucose is both respiratory and fermentative Oxidase positive Catalase positive Ferments salicin, sucrose and mannitol Produces hydrogen sulphide from cysteine Characteristic features of Pseudomonas aeruginosa (CBTCC/MICRO/3) Gram negative, aerobic rod shaped bacteria Have polar flagella Metabolism is respiratory, never fermentative Oxidase positive Catalase positive Denitrification positive Characteristic features of Yersinia enterocolitica (CBTCC/MICRO/4) Gram negative rods Facultative anaerobic, having both respiratory and fermentative type of metabolism Oxidase negative Motile Produces acid from sucrose, cellobiose, sorbose and sorbitol Characteristic features of Serratia liquefaciens (CBTCC/MICRO/7) Gram negative, facultative anaerobic rods Motile and have peritrichous flagella Produces acid from L-arabinose, D-xylose and D-sorbitol Tween 80 hydrolysis positive Lysine carboxylase and ornithine carboxylase positive Characteristic features of Pseudomonasfluorescens(CBTCC/MICRQ/11) Gram negative, aerobic rod shaped bacteria Have polar flagella Metabolism is respiratory, never fermentative Catalase positive Produces pyoverdin Gelatin liquefaction positive Characteristic features of Enterobacter cloaca (CBTCC/MICRO/1) Gram negative straight rods Motile by peritrichous flagella Facultative anaerobe Ferments glucose with production of acid and gas KCN and gelatinase positive Nitrate reductase positive Characteristic features of Klebsiella oxytoca (CBTCC/MICRO/5) Gram negative, facultative anaerobic rods Non-motile Oxidase negative Positive for Voges Proskauer test Utilizes citrate, m-hydroxybenzoate and degrades pectin Ferments L-arabinose, myoinositol, lactose, sucrose and raffinose Characteristic features of C/trobacter ama/onat/cus (CBJCC/MICRO/2) Gram negative, facultative anaerobic rods Facultative anaerobic Motile Indole production positive Utilizes malonate Esculin hydrolysis positive Characteristic features of Enterobacter sakazaki(CBTCC/MICRO/6) Gram-negative, facultative anaerobic rods Motile by peritrichous flagella Produces a non-diffusible yellow pigment at 25°C Utilizes citrate Gelatinase and p-xylosidase positive Produces acid from sucrose, raffinose and a-methylglucoside. The microbial consortium may contain the bacteria, in a preferred embodiment of the invention, in uniform amounts. The microbial consortium of the present invention is useful for BOD estimation. The bacterial cultures of the above microbial consortium are isolated from sewage. Sewage samples are collected from Okhia Coronation Plant near Okhia, New Delhi. Sewage is homogenized for 2 minutes and suspended in gram-negative culture broth. Incubation is carried out for 24 hours. Cultures are plated on Mac Conkey's agar. Colonies are mixed on a vortex mixer and all the cultures are isolated in pure form after several sub-cultures. The immobilization technique of formulated microbial consortium of the present invention is carried out by inoculating the individual strains of the above mentioned bacteria separately in nutrient broth containing (per litre), 5.0 g peptic digest of animal tissue, 5.0 g of sodium chloride, 1.5 g of beef extract, 1.5 g yeast extract and 0.2 ml tween-80. All the cultures are incubated preferably at 37°C for approximately 16-24 hours in an incubator shaker. For gentle shaking, the incubator shaker is maintained at an appropriate rpm, preferably at 75 rpm. After sufficient growth is obtained, the bacterial cells from these individual cultures are taken in equal proportions based on optical density and then mixed for formulating microbial consortium. The resultant bacterial suspension is centrifuged at an appropriate rpm, preferably at 10,000 rpm for a period of 20 minutes. The resultant pellet is washed by dissolving in minimum quantity of phosphate buffer, 0.05 M, pH 6.8 and recentrifuged at an appropriate rpm, preferably at 10,000 rpm for a period of approximately 20 minutes. During centrifugation, the temperature is maintained preferably at 4°C. The pellet thus obtained is immobilized on various membranes/supports such as charged nylon membrane and polyvinyl alcohol + nylon cloth. For the immobilization of formulated microbial consortium on charged nylon membrane, the pellet of formulated microbial consortium is dissolved in 2ml of phosphate buffer, 0.05M, pH 6.8 and filtered under vacuum. A number of immobilized microbial membranes are prepared under varying conditions of cell density and phase of cell growth. The immobilized microbial membranes thus obtained are left at room temperature for 4-6 hours to dry and stored at an appropriate temperature, preferably at 4°C. For immobilization of microbial consortium on polyvinyl alcohol (high molecular weight, i.e., 70,000 to 1,00,000 hot water soluble) + nylon cloth, a strip of nylon net (approx. 4x4 inch2) is tightly bound to a glass plate with the help of an adhesive. The pellet of formulated microbial consortium is dissolved in 2.0 ml phosphate buffer, 0.05M, pH 6.8 and mixed with 2% polyvinyl alcohol (PVA). The mixture of PVA and culture is poured onto a tightly bound nylon net. The mixture is spread with the help of glass rod thoroughly. A PVA + nylon cloth membrane without microorganisms is also prepared simultaneously, for control. The prepared membranes are left at room temperature for 4-6 hours to dry and then stored at an appropriate temperature, preferably at 4°C. The immobilized microbiai membranes thus obtained, are characterized with respect to cell density and phases of cell growth. For this, the individual microorganisms are grown for different time periods and a range of cell concentration is used for the immobilization on charged nylon membrane. The viability and stability of the immobilized microbial consortium is checked by storing at different pH and different temperatures. For checking the viability of immobilized microbial membranes, the membrane is placed on an agar plate in an inverted position and incubated at 37°C overnight. The colonies were observed for growth on agar plates. For the stability study, the prepared immobilized microbial membranes are stored at different temperatures i.e., 4°C, 15°C, 25°C & 37°C and different pH ranging from 6.4-7.2. The response of immobilized microbial membranes is observed at regular time intervals. To enhance the sensitivity of the response, an amperometric system is designed using dissolved oxygen (DO) probe and a highly sensitive multimeter. An external source of - 0.65 volts is applied to the system to get the actual reduction of oxygen at cathode. A suitable polarization voltage i.e., -0.65 volts between the anode and cathode selectively reduces oxygen at the cathode (Karube and Chang, 1991). For the preparation of electrode assembly, the immobilized microbial membranes are sandwiched between an oxygen permeated teflon membrane and a porous membrane, i.e., cellulose acetate membrane. The immobilized microbial membrane is fixed directly onto the platinum cathode of an commercially available O2 probe. The response characteristics of prepared immobilized microbial membranes is observed with synthetic sample i.e., glucose-glutamic acid (GGA), a reference standard used in BOD analysis. For this, the electrode assembly is dipped into a stirred P04-3 buffer solution. After a stable current was obtained, known strength of GGA was injected into the reaction assembly. Consumption of oxygen by the microbial cells immobilized on membrane caused a decrease in dissolved oxygen around the membrane. As a result, the values of dissolved oxygen decreased markedly with time until a steady state is reached. The steady state indicated that the consumption of oxygen by the immobilized microbial cells and the diffusion of oxygen from the solution to the membrane are in equilibrium. This value is recorded. Consumption of oxygen by the immobilized microorganisms is observed with multimeter in terms of current (nA). The change in current is linearly related to GGA standard over the range of 30 to 300 mg/l. Accordingly, the present invention provides a process for the preparation of a novel immobilized microbial membrane comprising a synergistic mixture of the isolated bacterial strains of the kind such as herein described present in equal proportions, the said process comprises: a) isolating a range of bacterial strains from sewage collected from sewage treatment plants; b) culturing the said strains on conventional nutrient media to get pure cultures; c) testing the said individual pure bacterial cultures for use as seeding material in BOD analysis using glucose-glutamic acid (GGA) as a reference standard by recording BOD values exhibited by individual strains; d) comparing the BOD values using the said bacterial strains with the BOD values using sewage as a seeding material; e) selecting the bacterial strains giving BOD values equal to or more than the BOD values using sewage from step (d); f) formulating the microbial consortium of selected bacterial strains obtained from step (e); g) comparing the BOD values using the formulated microbial consortium of step [f] as seeding material with the BOD values obtained using sewage as a seeding material; h) growing the individual bacterial strains comprising the formulated microbial consortium obtained in step [f] by inoculating bacterial strains individually, incubating the said bacterial strains, growing the said incubated strains and mixing them in equal proportions; i) centrifuging the resultant suspension to obtain pellets, washing the collected pellet by dissolving in PO4-3 buffer, 0.025 - 0.075 M, pH 6.4 -7.2 and recentrifuging the pellet; j) dissolving the pellet obtained from step (i) in 2.0 - 4.0 ml PO4- 3 buffer, 0.025-0.075 M, pH 6.4 - 7.2, to obtain cell slurry; k) immobilizing the cell slurry obtained from step [j] on commercially available charged nylon membrane under vacuum; I) drying the immobilized microbial membrane obtained from step (k); m)storing the dried immobilized microbial membrane obtained from step (I) preferably at 1 - 4°C in said PO4-3 buffer. The invention also provides a novel immobilized microbial membrane for estimating the BOD of industrial wastewaters, the process steps comprising: a) attaching the immobilized microbial membrane as claimed in claim 1 with a commercially available dissolved oxygen probe to obtain an electrode assembly; b) applying an external polarization voltage of -0.65 V to the electrode assembly obtained from step (a); c) stabilizing the electrode assembly obtained from step (b) in said PO4-3 buffer for 30-45 minutes; d) adding the industrial wastewater in the stabilized assembly of step [c]; e) observing the BOD values of the added wastewater in terms of change in current. f) In an embodiment of the present invention, the formulated microbial consortium is obtained by inoculating a suspension of the bacteria selected from a group consisting of Aeromonas hydrophila, Pseudomonas aeruginosa, Yersinia enterocolitica, Serratia liquefadens, Pseudomonas fluorescens, Enterobacter cloaca, Klebsiella oxytoca, Citrobacter amalonaticus and Enterobacter sakazakL In another embodiment of the present invention, the individual strains of the above mentioned bacteria are inoculated separately in a nutrient broth. In a further embodiment of the present invention, the incubation of bacterial strains is carried out by gentle agitation at approximately 75-100 rpm. In one of the embodiment of the present invention, the growth of incubated bacterial strains is carried out at a temperature ranging between 30-37°C for a period of 16-24 hours. In an embodiment of the present invention, the said individual strains are mixed in equal proportions. In a further embodiment of the present invention, the resultant microbial consortium is centrifuged at appropriate rpm preferably at 8,000-12,000 rpm for a period of approximately 20-30 minutes at a temperature ranging from 1-4°C In another embodiment of the present invention, the resultant pellet is washed by dissolving in an appropriate quantity of PO-T3 buffer, 0.025-0.075 M, pH 6.4-7.2 and recentrifuged at an approximate rpm in the range 8,000-12,000 rpm at a temperature preferably at 4 °C. In an embodiment of the present invention, the resultant cell pellet obtained is immobilized by dissolving in 1.0-2.0 ml of phosphate buffer ranging between 0.025-0.075 M, pH 6.4-7.2 to obtain cell slurry. In one of the embodiment of the present invention, the resulting cell slurry is filtered on charged nylon membrane under vacuum. In an embodiment of the present invention, the immobilized microbial membrane is dried at appropriate temperature, ranging between 25-35 *C, for a period ranging between 4-6 hours. In a further embodiment of the present invention, the dried immobilized membrane is stored in phosphate buffer, 0.05M, pH 6.8 at appropriate temperature ranging between 1-4°C In one of the embodiment of the present invention, the prepared immobilized microbial membrane is placed on nutrient agar plate and incubated at temperature ranging between 30°C-37°C for a period of 16-24 hours to observe the bacterial growth for viability of immobilized microorganisms. The invention further provides a method for the estimation of BOD which comprises of an immobilized microbial membrane. In one of the embodiment of the present invention, the dried immobilized microbial membrane is attached to dissolved oxygen probe with 0 ring for the preparation of electrode assembly. In an embodiment of the present invention, the stability of the immobilized microbial membrane stored at different temperatures ranging from 4°C-37°C was observed using electrode assembly. The response was observed in terms of change in current. In another embodiment of the present invention, the stable and viable immobilized microbial membrane was used for rapid and reliable BOD analysis using GGA as a reference standard in the concentration range of 30-300 mg/l. In a further embodiment of the present invention, the immobilized microbial membrane was used for rapid and reliable BOD analysis of industrial effluents ranging from low, moderate to high biodegradable organic matter. The invention, further described with references to the examples given below and shall not be construed, to limit the scope of the invention. Example I Two loops from agar plates of Aeromonas hydrophila, Pseudomonas aeruginosa, Yersinia enterocolitica, Serratia liquefadens, Pseudomonas fluorescens, Enterobacter cloaca, Klebsiella oxytoca, Citrobacter amalonaticus, and Enterobacter sakazaki were inoculated separately in 500 ml of nutrient broth. All the cultures were incubated at 37°C for 16-24 hours in an incubator shaker at 75 rpm. After incubation, optical density was measured at 650 nm. Optical density of all the bacteria was maintained to 0.5 either by diluting or concentrating the bacterial suspension. All the individual bacterial suspensions were mixed thoroughly and centrifuged at 10,000 rpm for 30 minutes at 4°C. The pellet was washed by dissolving it in small volume of phosphate buffer, 0.05 M, pH 6.8 and recentrifuged at 10,000 rpm for 30 minutes at 4°C. The pellet of microbial consortium prepared as described above was dissolved in 2.0 ml phosphate buffer, 0.05 M, pH 6.8 to obtain cell slurry. The cell slurry was mixed with 10.0 ml of 2% polyvinyl alcohol (mw. 70,000 to 1,00,000) in luke warm distilled water. A strip of nylon net (4x4") was tightly bound to a glass plate. The prepared solution of polyvinyl alcohol with cell slurry was spread onto the tightly bound nylon net. The immobilized microbial membrane was left for drying for 4-6 hours. The dried immobilized microbial membrane was stored in 0.05 M phosphate buffer, pH6.8 at 4°C. The prepared immobilized microbial membrane was not stable due to the low retaining capacity of the membrane. Example II Two loops from agar plates of Aeromonas hydrophila, Pseudomonas aeruginosa, Yersinia enterccolitica, Serratia liquefaciens, Pseudomonas fluorescens, Enterobacter cloaca, Klebsiella oxytoca, Citrobacter amalonaticus, and Enterobacter sakazaki were inoculated separately in 500 ml of nutrient broth. All the cultures were incubated at 37°C for 16-24 hours in an incubator shaker at 75 rpm. After incubation, optical density was measured at 650 nm. Optical density of all the bacteria was maintained to 0.5 either by diluting or concentrating the bacterial suspension. All the individual bacterial suspensions were mixed thoroughly and centrifuged at 10,000 rpm for 30 minutes at 4°C. The pellet was washed by dissolving it in small volume of phosphate buffer, 0.05 M, pH 6.8 and recentrifuged at 10,000 rpm for 30 minutes at 4°C. The pellet of microbial consortium prepared as described above was dissolved in 2.0 ml phosphate buffer, 0.05 M, pH 6.8 to obtain cell slurry. The cell slurry was filtered under vacuum on charged nylon membrane. The immobilized microbial membrane was left for drying for 4-6 hours. The dried immobilized microbial membrane was stored in 0.05 M phosphate buffer, pH6.8 at 4°C. The microbial consortium immobilized on charged nylon membrane was found to be stable , so this membrane was selected, for further study. Example III The selected immobilized microbial membrane was further characterized with respect to different phases of eel! growth as presented in Tablel(a-d)..For this, two loops from agar plates of Aeromonas hydrophila, Pseudomonas aeruginosa, Yersinia enterocolitica, Serratia liquefaciens, Pseudomonas fluorescens, Enterobacter cloaca, Klebsiella oxytoca, Citrobacter amalonaticus, and Enterobacter sakazaki were inoculated separately in 500 ml of nutrient broth. All the cultures were incubated at 37°C for different timings ranging between 4-16 hours in an incubator shaker at 75 rpm. After incubation, optical density was measured at 650 nm. Optical density of all the bacteria grown at different phases was maintained to 0.5 either by diluting or concentrating the bacterial suspension separately. All the bacterial suspensions were mixed thoroughly and centrifuged at 10,000 rpm for 30 minutes at 4°C. The pellets of bacterial cultures grown at different phases were washed by dissolving them in small volume of phosphate buffer, 0.05 M, pH 6.8 and recentrifuged at 10,000 rpm for 30 minutes at 4°C. The pellets of microbial consortium prepared at different phases of growth as described above were redissolved separately in 2.0 ml of phosphate buffer, 6.05 M, pH 6.8 to obtain cell slurry. The prepared-cell slurry of different growth phases were filtered on charged nylon membrane separately under vacuum. The immobilized microbial membranes of different phases of cell growth were dried for 4-6 hours. The dried immobilized microbial membranes were stored in 0.05 M phosphate buffer, pH 6.8 at 4°C . The said immobilized microbial membranes were used for the response study using GGA as a reference standard. The immobilized microbial membrane prepared using 8 hours grown microbial cells was giving better response in comparison to other immobilized microbial membranes and selected for further use. TABLE la : Characterization of immobilized microbial membrane with respect to different phases of cell growth (Table Removed) TABLE Ib : Characterization of immobilized microbial membrane with respect to different phases of cell growth (Table Removed) TABLE lc : Characterization of immobilized microbial membrane with respect to different phases of cell growth (Table Removed) TABLE Id : Characterization of immobilized microbial membrane with respect to different phases of cell growth (Table Removed) Example IV Table 2(a-c) represents the characterization of the selected immobilized microbial membrane with respect to cell density. For this, two loops from agar plates of Aeromonas hydrophila, Pseudomonas aeruginosa, Yersinia enterocolitica, Serratia Hqusfaciens, Pseudomonas fluorescens, Enterobacter cloaca, Klebsiella oxytoca, Citrobacter amalonaticus, and Enterobacter sakazaki were inoculated separately in 500 ml of nutrient broih. All the cultures were incubated at 37°C for 8 hours in an incubator shaker at 75 rpm. After incubation, optical density was measured at 650 nm. Optical density of all the bacteria was maintained to 0.5 either by diluting or concentrating the bacterial suspension separately. All the bacterial suspensions were mixed thoroughly and centrifuged at 10,000 rpm for 30 minutes at 4°C. The pellet of mixed bacterial cultures was washed by dissolving them in small volume of phosphate buffer, 0.05 M, pH 6.8 and recentrifuged at 10,000 rpm for 30 minutes at 4°C. The pellet of microbial consortium prepared as described above was redissolved separately in 2.0 ml of phosphate buffer, 0.05 M, pH 6-8 to obtain cell slurry. Five different aliquots ranging from 100µl to 1000µl of the prepared cell slurry were filtered on charged nylon membrane separately under vacuum. The immobilized microbial membranes having different cell density were dried for 4-6 hours. All the dried immobilized microbial membranes were stored in 0.05 M phosphate buffer, pH 6.8 at 4°C. The said immobilized microbial membranes were used for the response study using GGA as a reference-standard. The immobilized microbial membrane of 100µl cell density of 8 hours grown cells was giving best response and selected for further study. TABLE 2a Characterization of selected immobilized microbial membrane with 100µl cell slurry using a range of GGA concentrations (Table Removed) TABLE 2b haracterization of selected immobilized microbial membrane with 500µl cell slurry using a range of GGA concentrations (Table Removed) TABLE 2c Characterization of selected immobilized microbial membrane with 1000µl cell slurry using a range of GGA concentrations (Table Removed) Example V The Visibility study of the selected immobilized microb:al rnambrares of 3- hours grown microbial cells having cell slurry of 100µl scored at different temperatures ranging from 4oC-37°C, pH 6.8, were carried cut by observing the bacterial growth when the immobilized microbial membrane was placed on tre nutrient agar plate and incubated at 37°C for the desired time period. Table 3 represents the viability of immobilized microbia membranes stored at different temperatures. TABLE 3 Viability study of immobilized microbial membrane stored at different temperatures (Table Removed) ++++ excellent growth +++ very good growth ++ good growth + fair growth poor growth On storage, it was observed that the immobilized microbial membrane stored at a temperature of 4°C was viable for the longest time period. Example VI The viability study of the selected immobilized microbial membranes having cell slum/ of 100µl of 8 hours grown microbial cells, stored at different pH ranging from 6.4-7.2 and temperature 4°C was carried out by observing the bacterial growth when the immobilized microbial membrane was placed on the nutrient agar plate and incubated at 37°C for the desired time period. Table 4 represents the viability of microbial consortium immobilized on charged nylon membrane stored at different pH. TABLE 4 Viability study of immobilized microbia! membrane (Table Removed) On storage, it was observed that the immobilized microbial membrane stored in buffer of pH 6.8 was viable for the longest time interval. Example VII The electrode assembly was prepared by attaching the selected immobilized microbial membrane to dissolved oxygen probe. An external source of -0.65 V is applied to the system to get the actual reduction of oxygen at cathode. This prepared electrode assembly was used for checking the stability of immobilized microbial membrane. Example VIII Table 5 represents the stability study of the selecled microbial membrane immobilized on charged nylon membrane by storing at different temperatures for 180 days. For this, the immobilized microbial membrane of 8 hours grown microbial cells having 100µl cell slurry, stored at a temperature ranging from 4°C-37°C, pH 6.8 attached with dissolved oxygen probe for the response study using the prepared electrode assembly. Table 5 Stabilitv study of immobilized microbial membrane stored at different temperatures (Table Removed) ++++ Eexcellent growth +++ Very good growth ++ Good growth + Fair growth Poor growth On storage, it was observed that: the immobilized microbial membrane gave best response when stored at 4°C. Example IX The stability studies of the selected immobilized microbiai membrane of 8 hours grown microbial cells having 100µl cell slurry were carried out by storing in different pH ranging from 6.4-7.2. Table 6 represents the change in oxygen concentration in terms of current by immobilized microbia! membranes when stored at different pH values. Table 6 Stability study of immobilized microbial membrane (Table Removed) On storage, it was observed that the immobilized microbial membrane stored in pH 6.8 gave best response. Example X The prepared electrode assembly was used to observe the change in oxygen concentration in terms of current using GGA , as a reference standard in BOD analysis. Table 7 represents change in current of GGA concentration ranging between 30-300 mg/l at regular time intervals Table 7 depicts the change in oxygen concentration in terms of current with increasing GGA concentration. It is observed that higher is the GGA concentration, more is the change in current. This is indicative of the fact that at higher GGA concentration, there is more organic matter, thereby utilizing more oxygen for its oxidation. The utilization of oxygen leads to a decrease in oxygen concentration around the electrode assembly, until a steady state is reached. The steady state shows that the diffusion of oxygen from outside and its utilization are in equilibrium. Table 7 Change in current with GGA concentrations ranging between 30-300 mg/l at regular time intervals (Table Removed) CHANGE IN CURRENT (AI) OF INDUSTRIAL SAMPLE WITH HIGH Example XI BIODEGRADABLE ORGANIC LOADThe prepared immobilized microbial membrane of 8 hours grown microbial cells having 100µl cell slurry stored in 0.05 M phosphate buffer, pH 6.8 at a temperature of 4°C attached to the electrode assembly was used to observe the change in oxygen concentration in terms of current of various industrial effluents covering a range from 0.5-20.0% of low, moderate and high biodegradable effluents. Table 8 represents the change in oxygen concentration in terms of current for rapid and reliable BOD estimation by immobilized microbial membrane of various industrial effluents. The results indicate that the change in current is linearly proportional to the amount of biodegradabkle organic matter present in the sample. TABLE 8a CHANGE IN CURRENT (AI) OF INDUSTRIAL SAMPLE WITH HIGH BIODEGRADABLE ORGANIC LOAD (Table Removed) TABLE 8b CHANGE IN CURRENT (AI) OF INDUSTRIAL SAMPLE WITH MODERATE BIODEGRADABLE ORGANIC LOAD (Table Removed) TABLE 8c CHANGE IN CURRENT (AI) OF INDUSTRIAL SAMPLE WITH LOW BIODEGRADABLE ORGANIC LOAD (Table Removed) Advantages 1. The prepared microbial consortium, acting in a synergistic way is capable of biodegrading almost all kinds of organic matter present in a wide range of industrial effluents, thereby giving rapid and reproducible BOD values. 2. The prepared immobilized charged nylon membrane is more stable as compared to the existing immobilized microbial membranes. 3. The support used for the immobilization is charged nylon membrane which being positively charged binds specifically to the negatively charged bacterial cell by adsorption as well as entrapment. 4. The support used for immobilization is non-toxic to the micro-organisms. 5. The support i.e., charged nylon membrane used for the immobilization of microorganisms is novel for rapid and reliable BOD estimation. We Claim : 1. A process for the preparation of a novel immobilized microbial membrane comprising a synergistic mixture of the isolated bacterial strains of the kind such as herein described present in equal proportions, the said process comprises: a) isolating a range of bacterial strains from sewage collected from sewage treatment plants; b) culturing the said strains on conventional nutrient media to get pure cultures; c) testing the said individual pure bacterial cultures for use as seeding material in BOD analysis using glucose-glutamic acid (GGA) as a reference standard by recording BOD values exhibited by individual strains; d) comparing the BOD values using the said bacterial strains with the BOD values using sewage as a seeding material; e) selecting the bacterial strains giving BOD values equal to or more than the BOD values using sewage from step (d); f) formulating the microbial consortium of selected bacterial strains obtained from step (e); g) comparing the BOD values using the formulated microbial consortium of step [f] as seeding material with the BOD values obtained using sewage as a seeding material; h) growing the individual bacterial strains comprising the formulated microbial consortium obtained in step [f] by inoculating bacterial strains individually, incubating the said bacterial strains, growing the said incubated strains and mixing them in equal proportions; i) centrifuging the resultant suspension to obtain pellets, washing the collected pellet by dissolving in PO4-3 buffer, 0.025 - 0.075 M, pH 6.4 - 7.2 and recentrifuging the pellet; j) dissolving the pellet obtained from step (i) in 2.0 - 4.0 ml PO4-3 buffer, 0.025-0.075 M, pH 6.4 - 7.2, to obtain cell slurry; k) immobilizing the cell slurry obtained from step [j] on commercially available charged nylon membrane under vacuum; I) drying the immobilized microbial membrane obtained from step (k); m) storing the dried immobilized microbial membrane obtained from step (I) preferably at 1 - 4°C in said PO4-3 buffer. 2. A process as claimed in claim 1, wherein the formulated microbial consortium is obtained by inoculating a suspension of the bacteria selected from a group consisting of Aeromonas hydrophila, Pseudomonas aeruginosa, Yersinia enterocolitica, Serratia liquefaciens, Pseudomonas fluorescens, Enterobacter cloaca, Klebsiella oxytoca, Citrobacter amalonaticus and Enterobacter sakazaki. 3. A process as claimed in claim 1, wherein the individual strains of the above mentioned bacteria are inoculated separately in a nutrient broth. 4. A process as claimed in claim 1, wherein the incubation of bacterial strains is carried out by gentle agitation at approximately 75-100 rpm. 5. A process as claimed in claim 1, wherein the incubation of bacterial strains is carried out at a temperature ranging between 30°C - 37°C for a period of 16- 18 hours. 5. A process as claimed in claim 1, wherein the microbial consortium is centrifuged at appropriate rpm preferably at 8,000 -12,000 rpm for a period of approximately 20 - 30 minutes at a temperature of 1 - 4°C. 6. A process as claimed in claim 1, wherein the immobilized microbial membrane is dried for 4 - 6 hours at a temperature ranging between 25°C - 35°C. 7. A process for the preparation of a novel immobilized microbial membrane substantially as herein described with reference to the foregoing examples.

Full Text

FIELD OF THE INVENTION
The present invention relates to a process for the preparation of a novel immobilized microbial membrane. More particularly, the present invention relates to an immobilized microbial consortium and a process for the preparation of the said immobilized microbial consortium useful for rapid and reliable BOD estimation.
Description of the prior art
Rapid analytical devices have attracted tremendous interest and attention in science and technology for their wide range of possible application as an alternative to conventional analytical techniques. Analytical devices are sensitive to biological parameters and consist of a biological sensing element such as microbes, enzymes, etc., in close contact with a physico-chemical transducer such as an electrode, which converts biological signal to a quantitative response. These devices have several unique features such as compact size, simple to use, one step reagent-less analysis, low cost and quick real time results.
Rapid analytical devices, termed as biosensors, have the potential for a major impact in the human health care, environmental monitoring, food analysis and industrial process control. Among these, microbial biosensors (the devices using microbes as biological component), have great potentiai in environmental monitoring. Recent trends in biotechnology suggest that mon.torirg and control of pollutant by means of microbial biosensors may be of crucia: importance. Such microbial sensors, constructed by entrapping the required micro-organisms in sultable-pelvmeric matrices.and Attached to a transducer Function on the basis of assimilatory capacity of the micro-organisms. In addition, microbial biosensors are more stable and inexpensive for the determination of compounds of interest as compared to enzyme-based biosensors; where enzymes employed in enzyme-based biosensors require costly extraction and purification prior to use as biocatalysts. Further, micro-organisms employed in microbial biosensors show a high degree of stability as compared to enzymes.
The vast majority of micro-organisms are relatively easy to maintain in pure cultures, grow and harvest at low cost. Moreover, the use of microbes in biosensor field have opened up new possibilities and advantages such as ease of handling, preparation and low cost of the device. Such devices will help in monitoring the compounds of environmental interest such as Biochemical Oxygen Demand (BOD), heavy metals, pesticides, phenols, etc.
Among the environmental parameters, the potential demand for rapid BOD monitoring device is higher, since, BOD is a parameter which is measured most frequently by many industries for measuring the level of pollution of waste-waters. BOD provides information about the amount of biodegradable substances in waste-waters.
Conventional BOD test takes 3-5 days and as a consequence, is unsuitable for use in direct process control. A more rapid estimation of BOD is possible by developing a BOD biosensor. Such BOD biosensors are able to reduce the time of BOD test upto a great extent.
A number of microbial BOD sensors have been developed nationally and internationally (Rajasekar et al, 1992 and Karube, 1977). A number of pure cultures, eg., Trichosporon cutaneum, Hansenula anamola, Bacillus cereus, Bacillus subtilis, Klebsiella oxytoca, Pseudomonas sp., etc., individually, have been used by many workers for the construction of BOD biosensor (Preinenger et al, 1994; Hyun et ai, 1993, Li and Chu 1991; Riedel et al, 1989 and Sun and Kiu, 1992). Karube et al, (1992) developed a BOD biosensor by utilizing thermophilic bacteria isolated from Japanese hot spring. On the other hand, most of the workers have immobilized activated sludge (Vanrolleghem et al 1990; Kong et al 1993; Vanrolleghem et al, 1984), or a mixture of two or three bacterial species (Iki, 1992 and Galindo et al 1992) on various membranes for the construction of BOD biosensor. The most commonly used membranes were polyvinyl alcohol, porous hydrophilic membranes, etc. Riedel et al, (1988), have used polyvinyl alcohol for the immobilization of Bacillus subtilis or Trichosporon cutaneum which are used for the development of BOD biosensor. Vinegar (1993) immobilized Klebsiella oxytoca on porous hydrophilic membranes such as nitrocellulose, acetyl cellulose, polyvinylidene flouride or polyether sulfone, 50-2000 micrometer thick. Cellulose acetate membrane was used for the
immobilization of Lipomya-s kononankoae and Asperillus niger (Hartmeier et al, 1993).
The drawback of such developed BOD biosensors which are constructed by using either single, pure culture or activated sludge is that they do not give reproducible results, as single microbe is not able to assimilate/ degrade all the organic compounds and therefore may not respond for the total organic matter present in the test sample (eg., carbohydrates, proteins, fats, grease, etc.) Moreover, in the activated sludge either non-specific predominating microorganisms are present thereof or microorganisms with antagonistic effects are present which may produce erratic results. On the other hand, randomly selected mixtures of two or three micro-organisms also do not give reproducible, comparable BOD results. The reproducibility of the BOD biosensor can be obtained by formulating a defined microbial composition.
To avoid the discrepancies in BOD results as well as to get instant BOD values using rapid analytical devices, in the present invention, a defined microbial composition is formulated by conducting a systematic study, i.e., pretesting of selected micro-organisms for use as a seeding material in BOD analysis of a wide variety of industrial effluents. The formulated microbial consortium is capable of assimilating most of the organic matter present in different industrial effluents. The formulated microbial consortium has been immobilized on suitable membrane i.e., charged nylon membrane useful for BOD estimation. Suitability of the charged nylon membrane lies in the specific binding between the
negatively charged bacterial cell and positively charged nylon membrane. So, the advantages of the used membrane over other membranes are the dual binding i.e., adsorption as well as entrapment, thus resulting in a more stable immobilized membrane. Such specific microbial consortium based BOD analytical devices, may find great application in on-line monitoring of the degree of pollutional strength, in a wide variety of industrial waste-waters within a very short time (from 3-5 days to within an hour), which is very essential from pollution point of view.
For solving the aforementioned problems, the applicants have realized that there exists a need to provide a process for the preparation of a defined synergistic microbial consortium immobilized on a suitable support i.e., charged nylon membrane, useful for BOD estimation. The said microbial consortium is capable of assimilating most of the organic matter present in different industrial effluents
Objects of the invention
The main object of the present invention is to provide a microbial consortium and a process for the preparation of the microbial consortium immobilized on a suitable support useful for BOD estimation.
The formulated microbial consortium comprises of cultures of the following bacteria viz., Aeromonas hydrophila, Pseudomonas aeruginosa, Yersinia
enterocolitica, Serratia liquefaciens, Pseudcomonas fluorescens, Enterobacter-cloaca, Klebsiella oxytoca, Citrobacter amalonaticus and Enterobacter sakazaki, The individual bacteria of microbial consortium are pre-tested by using them as a seeding material in BOD analysis of a wide variety of industrial effluents. The micro-organisms have been selected for the formulation of microbial consortium on the basis of pre-testing. The formulated microbial consortium is obtained by inoculating a suspension of these bacteria individually. Incubating at 37°C, mixing all bacterial cultures in equal proportions based on optical density and centrifuging. The resultant pellet is immobilized on suitable support, i.e., charged nylon membrane by entrapment and adsorption on the charged surface of the membrane. The said, charged immobilized microbial membrane has high viability, long stability and greater shelf-life as compared to the microbial consortium immobilized on conventional supports such as polyvinyl alcohol + nylon cloth.
Accordingly, another object of the present invention, is to provide a process for the production of immobilized formulated microbial consortium useful for monitoring the BOD load of a wide range of industrial effluents with low, moderate and high BOD load.
Summary of the invention
The present invention provides an immobilized microbial consortium and a process for the preparation of the said immobilized microbial consortium, useful
for rapid and reliable BOD estimation of a wide range of industrial effluents with low, moderate and high BOD load.
Detailed description of the invention
The microbial consortium provided according to the present invention contains bacteria consisting of:
(Table Removed)
which facilitate the process of testing, giving BOD results of a wide variety of industrial effluents, performed at any place. Above microorganisms are

deposited at Centre for Biochemical Technology Culture Collection (CBTCC) designated as stated above and will be made available to public on request as per normal official procedures.
The main characteristic features of all the bacterial cultures used for the invention which are similar to ATCC cultures are given below :
Characteristic features of Aeromonas hydrophila (CBTCC/MICRO/10)
Gram negative rods
Motile by a single polar flagellum
Metabolism of glucose is both respiratory and fermentative
Oxidase positive
Catalase positive
Ferments salicin, sucrose and mannitol
Produces hydrogen sulphide from cysteine
Characteristic features of Pseudomonas aeruginosa (CBTCC/MICRO/3)
Gram negative, aerobic rod shaped bacteria
Have polar flagella
Metabolism is respiratory, never fermentative
Oxidase positive
Catalase positive
Denitrification positive
Characteristic features of Yersinia enterocolitica (CBTCC/MICRO/4)
Gram negative rods
Facultative anaerobic, having both respiratory and fermentative type of
metabolism
Oxidase negative
Motile
Produces acid from sucrose, cellobiose, sorbose and sorbitol
Characteristic features of Serratia liquefaciens (CBTCC/MICRO/7) Gram negative, facultative anaerobic rods Motile and have peritrichous flagella Produces acid from L-arabinose, D-xylose and D-sorbitol Tween 80 hydrolysis positive Lysine carboxylase and ornithine carboxylase positive
Characteristic features of Pseudomonasfluorescens(CBTCC/MICRQ/11) Gram negative, aerobic rod shaped bacteria Have polar flagella
Metabolism is respiratory, never fermentative Catalase positive Produces pyoverdin Gelatin liquefaction positive
Characteristic features of Enterobacter cloaca (CBTCC/MICRO/1) Gram negative straight rods Motile by peritrichous flagella Facultative anaerobe
Ferments glucose with production of acid and gas KCN and gelatinase positive Nitrate reductase positive
Characteristic features of Klebsiella oxytoca (CBTCC/MICRO/5) Gram negative, facultative anaerobic rods Non-motile Oxidase negative
Positive for Voges Proskauer test
Utilizes citrate, m-hydroxybenzoate and degrades pectin
Ferments L-arabinose, myoinositol, lactose, sucrose and raffinose
Characteristic features of C/trobacter ama/onat/cus (CBJCC/MICRO/2) Gram negative, facultative anaerobic rods Facultative anaerobic Motile
Indole production positive Utilizes malonate Esculin hydrolysis positive
Characteristic features of Enterobacter sakazaki(CBTCC/MICRO/6) Gram-negative, facultative anaerobic rods Motile by peritrichous flagella Produces a non-diffusible yellow pigment at 25°C Utilizes citrate
Gelatinase and p-xylosidase positive Produces acid from sucrose, raffinose and a-methylglucoside.
The microbial consortium may contain the bacteria, in a preferred embodiment of the invention, in uniform amounts.
The microbial consortium of the present invention is useful for BOD estimation.
The bacterial cultures of the above microbial consortium are isolated from sewage. Sewage samples are collected from Okhia Coronation Plant near Okhia,
New Delhi. Sewage is homogenized for 2 minutes and suspended in gram-negative culture broth. Incubation is carried out for 24 hours. Cultures are plated on Mac Conkey's agar. Colonies are mixed on a vortex mixer and all the cultures are isolated in pure form after several sub-cultures.
The immobilization technique of formulated microbial consortium of the present invention is carried out by inoculating the individual strains of the above mentioned bacteria separately in nutrient broth containing (per litre), 5.0 g peptic digest of animal tissue, 5.0 g of sodium chloride, 1.5 g of beef extract, 1.5 g yeast extract and 0.2 ml tween-80. All the cultures are incubated preferably at 37°C for approximately 16-24 hours in an incubator shaker. For gentle shaking, the incubator shaker is maintained at an appropriate rpm, preferably at 75 rpm. After sufficient growth is obtained, the bacterial cells from these individual cultures are taken in equal proportions based on optical density and then mixed for formulating microbial consortium. The resultant bacterial suspension is centrifuged at an appropriate rpm, preferably at 10,000 rpm for a period of 20 minutes. The resultant pellet is washed by dissolving in minimum quantity of phosphate buffer, 0.05 M, pH 6.8 and recentrifuged at an appropriate rpm, preferably at 10,000 rpm for a period of approximately 20 minutes. During centrifugation, the temperature is maintained preferably at 4°C. The pellet thus obtained is immobilized on various membranes/supports such as charged nylon membrane and polyvinyl alcohol + nylon cloth.
For the immobilization of formulated microbial consortium on charged nylon membrane, the pellet of formulated microbial consortium is dissolved in 2ml of phosphate buffer, 0.05M, pH 6.8 and filtered under vacuum. A number of immobilized microbial membranes are prepared under varying conditions of cell density and phase of cell growth. The immobilized microbial membranes thus obtained are left at room temperature for 4-6 hours to dry and stored at an appropriate temperature, preferably at 4°C.
For immobilization of microbial consortium on polyvinyl alcohol (high molecular weight, i.e., 70,000 to 1,00,000 hot water soluble) + nylon cloth, a strip of nylon net (approx. 4x4 inch2) is tightly bound to a glass plate with the help of an adhesive. The pellet of formulated microbial consortium is dissolved in 2.0 ml phosphate buffer, 0.05M, pH 6.8 and mixed with 2% polyvinyl alcohol (PVA). The mixture of PVA and culture is poured onto a tightly bound nylon net. The mixture is spread with the help of glass rod thoroughly. A PVA + nylon cloth membrane without microorganisms is also prepared simultaneously, for control. The prepared membranes are left at room temperature for 4-6 hours to dry and then stored at an appropriate temperature, preferably at 4°C.
The immobilized microbiai membranes thus obtained, are characterized with respect to cell density and phases of cell growth. For this, the individual microorganisms are grown for different time periods and a range of cell concentration is used for the immobilization on charged nylon membrane. The viability and stability of the immobilized microbial consortium is checked by
storing at different pH and different temperatures. For checking the viability of immobilized microbial membranes, the membrane is placed on an agar plate in an inverted position and incubated at 37°C overnight. The colonies were observed for growth on agar plates. For the stability study, the prepared immobilized microbial membranes are stored at different temperatures i.e., 4°C, 15°C, 25°C & 37°C and different pH ranging from 6.4-7.2. The response of immobilized microbial membranes is observed at regular time intervals.
To enhance the sensitivity of the response, an amperometric system is designed using dissolved oxygen (DO) probe and a highly sensitive multimeter. An external source of - 0.65 volts is applied to the system to get the actual reduction of oxygen at cathode. A suitable polarization voltage i.e., -0.65 volts between the anode and cathode selectively reduces oxygen at the cathode (Karube and Chang, 1991).
For the preparation of electrode assembly, the immobilized microbial membranes are sandwiched between an oxygen permeated teflon membrane and a porous membrane, i.e., cellulose acetate membrane. The immobilized microbial membrane is fixed directly onto the platinum cathode of an commercially available O2 probe.
The response characteristics of prepared immobilized microbial membranes is observed with synthetic sample i.e., glucose-glutamic acid (GGA), a reference standard used in BOD analysis. For this, the electrode assembly is dipped into a stirred P04-3 buffer solution. After a stable current was obtained,
known strength of GGA was injected into the reaction assembly. Consumption of oxygen by the microbial cells immobilized on membrane caused a decrease in dissolved oxygen around the membrane. As a result, the values of dissolved oxygen decreased markedly with time until a steady state is reached. The steady state indicated that the consumption of oxygen by the immobilized microbial cells and the diffusion of oxygen from the solution to the membrane are in equilibrium. This value is recorded. Consumption of oxygen by the immobilized microorganisms is observed with multimeter in terms of current (nA). The change in current is linearly related to GGA standard over the range of 30 to 300 mg/l.
Accordingly, the present invention provides a process for the preparation of a novel immobilized microbial membrane comprising a synergistic mixture of the isolated bacterial strains of the kind such as herein described present in equal proportions, the said process comprises:
a) isolating a range of bacterial strains from sewage collected
from sewage treatment plants;
b) culturing the said strains on conventional nutrient media to
get pure cultures;
c) testing the said individual pure bacterial cultures for use as
seeding material in BOD analysis using glucose-glutamic
acid (GGA) as a reference standard by recording BOD
values exhibited by individual strains;
d) comparing the BOD values using the said bacterial strains
with the BOD values using sewage as a seeding material;
e) selecting the bacterial strains giving BOD values equal to or
more than the BOD values using sewage from step (d);
f) formulating the microbial consortium of selected bacterial
strains obtained from step (e);
g) comparing the BOD values using the formulated microbial
consortium of step [f] as seeding material with the BOD
values obtained using sewage as a seeding material;
h) growing the individual bacterial strains comprising the formulated microbial consortium obtained in step [f] by inoculating bacterial strains individually, incubating the said bacterial strains, growing the said incubated strains and mixing them in equal proportions;
i) centrifuging the resultant suspension to obtain pellets,
washing the collected pellet by dissolving in PO4-3 buffer,
0.025 - 0.075 M, pH 6.4 -7.2 and recentrifuging the pellet; j) dissolving the pellet obtained from step (i) in 2.0 - 4.0 ml PO4-
3 buffer, 0.025-0.075 M, pH 6.4 - 7.2, to obtain cell slurry; k) immobilizing the cell slurry obtained from step [j] on
commercially available charged nylon membrane under
vacuum; I) drying the immobilized microbial membrane obtained from
step (k); m)storing the dried immobilized microbial membrane obtained
from step (I) preferably at 1 - 4°C in said PO4-3 buffer.
The invention also provides a novel immobilized microbial membrane for estimating the BOD of industrial wastewaters, the process steps comprising:
a) attaching the immobilized microbial membrane as claimed in
claim 1 with a commercially available dissolved oxygen
probe to obtain an electrode assembly;
b) applying an external polarization voltage of -0.65 V to the
electrode assembly obtained from step (a);
c) stabilizing the electrode assembly obtained from step (b) in
said PO4-3 buffer for 30-45 minutes;
d) adding the industrial wastewater in the stabilized assembly of
step [c];
e) observing the BOD values of the added wastewater in terms
of change in current.
f) In an embodiment of the present invention, the formulated microbial consortium is obtained by inoculating a suspension of the bacteria selected from a group consisting of Aeromonas hydrophila, Pseudomonas aeruginosa, Yersinia enterocolitica, Serratia liquefadens, Pseudomonas fluorescens, Enterobacter cloaca, Klebsiella oxytoca, Citrobacter amalonaticus and Enterobacter sakazakL
In another embodiment of the present invention, the individual strains of the above mentioned bacteria are inoculated separately in a nutrient broth.
In a further embodiment of the present invention, the incubation of bacterial strains is carried out by gentle agitation at approximately 75-100 rpm.
In one of the embodiment of the present invention, the growth of incubated bacterial strains is carried out at a temperature ranging between 30-37°C for a period of 16-24 hours.
In an embodiment of the present invention, the said individual strains are mixed in equal proportions.
In a further embodiment of the present invention, the resultant microbial consortium is centrifuged at appropriate rpm preferably at 8,000-12,000 rpm for a period of approximately 20-30 minutes at a temperature ranging from 1-4°C
In another embodiment of the present invention, the resultant pellet is washed by dissolving in an appropriate quantity of PO-T3 buffer, 0.025-0.075 M, pH 6.4-7.2 and recentrifuged at an approximate rpm in the range 8,000-12,000 rpm at a temperature preferably at 4 °C.
In an embodiment of the present invention, the resultant cell pellet obtained is immobilized by dissolving in 1.0-2.0 ml of phosphate buffer ranging between 0.025-0.075 M, pH 6.4-7.2 to obtain cell slurry.
In one of the embodiment of the present invention, the resulting cell slurry is filtered on charged nylon membrane under vacuum.
In an embodiment of the present invention, the immobilized microbial membrane is dried at appropriate temperature, ranging between 25-35 *C, for a period ranging between 4-6 hours.
In a further embodiment of the present invention, the dried immobilized membrane is stored in phosphate buffer, 0.05M, pH 6.8 at appropriate temperature ranging between 1-4°C

In one of the embodiment of the present invention, the prepared immobilized microbial membrane is placed on nutrient agar plate and incubated at temperature ranging between 30°C-37°C for a period of 16-24 hours to observe the bacterial growth for viability of immobilized microorganisms.
The invention further provides a method for the estimation of BOD which comprises of an immobilized microbial membrane.
In one of the embodiment of the present invention, the dried immobilized microbial membrane is attached to dissolved oxygen probe with 0 ring for the preparation of electrode assembly.
In an embodiment of the present invention, the stability of the immobilized microbial membrane stored at different temperatures ranging from 4°C-37°C was observed using electrode assembly. The response was observed in terms of change in current.
In another embodiment of the present invention, the stable and viable immobilized microbial membrane was used for rapid and reliable BOD analysis using GGA as a reference standard in the concentration range of 30-300 mg/l.
In a further embodiment of the present invention, the immobilized microbial membrane was used for rapid and reliable BOD analysis of industrial effluents ranging from low, moderate to high biodegradable organic matter.
The invention, further described with references to the examples given below and shall not be construed, to limit the scope of the invention.
Example I
Two loops from agar plates of Aeromonas hydrophila, Pseudomonas aeruginosa, Yersinia enterocolitica, Serratia liquefadens, Pseudomonas fluorescens, Enterobacter cloaca, Klebsiella oxytoca, Citrobacter amalonaticus, and Enterobacter sakazaki were inoculated separately in 500 ml of nutrient broth. All the cultures were incubated at 37°C for 16-24 hours in an incubator shaker at 75 rpm. After incubation, optical density was measured at 650 nm. Optical density of all the bacteria was maintained to 0.5 either by diluting or concentrating the bacterial suspension. All the individual bacterial suspensions were mixed thoroughly and centrifuged at 10,000 rpm for 30 minutes at 4°C. The pellet was washed by dissolving it in small volume of phosphate buffer, 0.05 M, pH 6.8 and recentrifuged at 10,000 rpm for 30 minutes at 4°C.
The pellet of microbial consortium prepared as described above was dissolved in 2.0 ml phosphate buffer, 0.05 M, pH 6.8 to obtain cell slurry. The cell slurry was mixed with 10.0 ml of 2% polyvinyl alcohol (mw. 70,000 to 1,00,000) in luke warm distilled water. A strip of nylon net (4x4") was tightly bound to a glass plate. The prepared solution of polyvinyl alcohol with cell slurry was spread onto the tightly bound nylon net. The immobilized microbial membrane was left for drying for 4-6 hours. The dried immobilized microbial membrane was stored in 0.05 M phosphate buffer, pH6.8 at 4°C. The prepared immobilized microbial membrane was not stable due to the low retaining capacity of the membrane.
Example II
Two loops from agar plates of Aeromonas hydrophila, Pseudomonas aeruginosa, Yersinia enterccolitica, Serratia liquefaciens, Pseudomonas fluorescens, Enterobacter cloaca, Klebsiella oxytoca, Citrobacter amalonaticus, and Enterobacter sakazaki were inoculated separately in 500 ml of nutrient broth. All the cultures were incubated at 37°C for 16-24 hours in an incubator shaker at 75 rpm. After incubation, optical density was measured at 650 nm. Optical density of all the bacteria was maintained to 0.5 either by diluting or concentrating the bacterial suspension. All the individual bacterial suspensions were mixed thoroughly and centrifuged at 10,000 rpm for 30 minutes at 4°C. The pellet was washed by dissolving it in small volume of phosphate buffer, 0.05 M, pH 6.8 and recentrifuged at 10,000 rpm for 30 minutes at 4°C.
The pellet of microbial consortium prepared as described above was dissolved in 2.0 ml phosphate buffer, 0.05 M, pH 6.8 to obtain cell slurry. The cell slurry was filtered under vacuum on charged nylon membrane. The immobilized microbial membrane was left for drying for 4-6 hours. The dried immobilized microbial membrane was stored in 0.05 M phosphate buffer, pH6.8 at 4°C. The microbial consortium immobilized on charged nylon membrane was found to be stable , so this membrane was selected, for further study.
Example III
The selected immobilized microbial membrane was further characterized with respect to different phases of eel! growth as presented in Tablel(a-d)..For this, two loops from agar plates of Aeromonas hydrophila, Pseudomonas aeruginosa, Yersinia enterocolitica, Serratia liquefaciens, Pseudomonas fluorescens, Enterobacter cloaca, Klebsiella oxytoca, Citrobacter amalonaticus, and Enterobacter sakazaki were inoculated separately in 500 ml of nutrient broth. All the cultures were incubated at 37°C for different timings ranging between 4-16 hours in an incubator shaker at 75 rpm. After incubation, optical density was measured at 650 nm. Optical density of all the bacteria grown at different phases was maintained to 0.5 either by diluting or concentrating the bacterial suspension separately. All the bacterial suspensions were mixed thoroughly and centrifuged at 10,000 rpm for 30 minutes at 4°C. The pellets of bacterial cultures grown at different phases were washed by dissolving them in small volume of phosphate buffer, 0.05 M, pH 6.8 and recentrifuged at 10,000 rpm for 30 minutes at 4°C.
The pellets of microbial consortium prepared at different phases of growth as described above were redissolved separately in 2.0 ml of phosphate buffer, 6.05 M, pH 6.8 to obtain cell slurry. The prepared-cell slurry of different growth phases were filtered on charged nylon membrane separately under vacuum. The immobilized microbial membranes of different phases of cell growth were dried for 4-6 hours. The dried immobilized microbial membranes were stored in 0.05 M phosphate buffer, pH 6.8 at 4°C . The said immobilized microbial membranes were used for the response study using GGA as a reference standard. The immobilized microbial membrane
prepared using 8 hours grown microbial cells was giving better response in comparison to other immobilized microbial membranes and selected for further use.
TABLE la : Characterization of immobilized microbial membrane with respect to different phases of cell growth

(Table Removed)
TABLE Ib : Characterization of immobilized microbial membrane with respect to different phases of cell growth

(Table Removed)
TABLE lc : Characterization of immobilized microbial membrane with respect to different phases of cell growth
(Table Removed)
TABLE Id : Characterization of immobilized microbial membrane with respect to different phases of cell growth

(Table Removed)
Example IV
Table 2(a-c) represents the characterization of the selected immobilized microbial membrane with respect to cell density. For this, two loops from agar plates of Aeromonas hydrophila, Pseudomonas aeruginosa, Yersinia enterocolitica, Serratia Hqusfaciens, Pseudomonas fluorescens, Enterobacter cloaca, Klebsiella oxytoca, Citrobacter amalonaticus, and Enterobacter sakazaki were inoculated separately in 500 ml of nutrient broih. All the cultures were incubated at 37°C for 8 hours in an incubator shaker at 75 rpm. After incubation, optical density was measured at 650 nm. Optical density of all the bacteria was maintained to 0.5 either by diluting or concentrating the bacterial suspension separately. All the bacterial suspensions were mixed thoroughly and centrifuged at 10,000 rpm for 30 minutes at 4°C. The pellet of mixed bacterial cultures was washed by dissolving them in small volume of phosphate buffer, 0.05 M, pH 6.8 and recentrifuged at 10,000 rpm for 30 minutes at 4°C. The pellet of microbial consortium prepared as described above was redissolved separately in 2.0 ml of phosphate buffer, 0.05 M, pH 6-8 to obtain cell slurry.
Five different aliquots ranging from 100µl to 1000µl of the prepared

cell slurry were filtered on charged nylon membrane separately under vacuum. The immobilized microbial membranes having different cell density were dried for 4-6 hours. All the dried immobilized microbial membranes were stored in 0.05 M phosphate buffer, pH 6.8 at 4°C. The said immobilized microbial membranes were used for the response study using GGA as a reference-standard. The immobilized microbial membrane of 100µl cell
density of 8 hours grown cells was giving best response and selected for further study.
TABLE 2a
Characterization of selected immobilized microbial membrane with 100µl cell slurry using a range of GGA concentrations
(Table Removed)
TABLE 2b
haracterization of selected immobilized microbial membrane with 500µl cell slurry using a range of GGA concentrations

(Table Removed)
TABLE 2c
Characterization of selected immobilized microbial membrane with 1000µl cell slurry using a range of GGA concentrations

(Table Removed)
Example V
The Visibility study of the selected immobilized microb:al rnambrares of 3- hours grown microbial cells having cell slurry of 100µl scored at different
temperatures ranging from 4oC-37°C, pH 6.8, were carried cut by observing the bacterial growth when the immobilized microbial membrane was placed on tre nutrient agar plate and incubated at 37°C for the desired time period.
Table 3 represents the viability of immobilized microbia membranes stored at different temperatures.
TABLE 3
Viability study of immobilized microbial membrane stored at different temperatures

(Table Removed)
++++ excellent growth
+++ very good growth
++ good growth
+ fair growth
poor growth
On storage, it was observed that the immobilized microbial membrane stored at a temperature of 4°C was viable for the longest time period.
Example VI
The viability study of the selected immobilized microbial membranes having cell slum/ of 100µl of 8 hours grown microbial cells, stored at different pH ranging from 6.4-7.2 and temperature 4°C was carried out by observing the bacterial growth when the immobilized microbial membrane was placed on the nutrient agar plate and incubated at 37°C for the desired time period.
Table 4 represents the viability of microbial consortium immobilized on charged nylon membrane stored at different pH.
TABLE 4
Viability study of immobilized microbia! membrane
(Table Removed)
On storage, it was observed that the immobilized microbial membrane stored in buffer of pH 6.8 was viable for the longest time interval.
Example VII
The electrode assembly was prepared by attaching the selected immobilized microbial membrane to dissolved oxygen probe. An external source of -0.65 V is applied to the system to get the actual reduction of oxygen at cathode. This prepared electrode assembly was used for checking the stability of immobilized microbial membrane.
Example VIII
Table 5 represents the stability study of the selecled microbial membrane immobilized on charged nylon membrane by storing at different temperatures for 180 days. For this, the immobilized microbial membrane of 8 hours grown microbial cells having 100µl cell slurry, stored at a
temperature ranging from 4°C-37°C, pH 6.8 attached with dissolved oxygen probe for the response study using the prepared electrode assembly.
Table 5
Stabilitv study of immobilized microbial membrane stored at different temperatures
(Table Removed)
++++ Eexcellent growth
+++ Very good growth
++ Good growth
+ Fair growth
Poor growth
On storage, it was observed that: the immobilized microbial membrane gave best response when stored at 4°C.
Example IX
The stability studies of the selected immobilized microbiai membrane of 8 hours grown microbial cells having 100µl cell slurry were carried out by
storing in different pH ranging from 6.4-7.2.
Table 6 represents the change in oxygen concentration in terms of current by immobilized microbia! membranes when stored at different pH values.
Table 6 Stability study of immobilized microbial membrane
(Table Removed)
On storage, it was observed that the immobilized microbial membrane stored in pH 6.8 gave best response.
Example X
The prepared electrode assembly was used to observe the change in oxygen concentration in terms of current using GGA , as a reference standard in BOD analysis.
Table 7 represents change in current of GGA concentration ranging between 30-300 mg/l at regular time intervals
Table 7 depicts the change in oxygen concentration in terms of current with increasing GGA concentration. It is observed that higher is the GGA concentration, more is the change in current. This is indicative of the fact that at higher GGA concentration, there is more organic matter, thereby utilizing more oxygen for its oxidation. The utilization of oxygen leads to a decrease in oxygen concentration around the electrode assembly, until a steady state is reached. The steady state shows that the diffusion of oxygen from outside and its utilization are in equilibrium.
Table 7
Change in current with GGA concentrations ranging between 30-300 mg/l
at regular time intervals
(Table Removed)
CHANGE IN CURRENT (AI) OF INDUSTRIAL SAMPLE WITH HIGH Example XI
BIODEGRADABLE ORGANIC LOADThe prepared immobilized microbial membrane of 8 hours grown microbial cells having 100µl cell slurry stored in 0.05 M phosphate buffer, pH
6.8 at a temperature of 4°C attached to the electrode assembly was used to observe the change in oxygen concentration in terms of current of various industrial effluents covering a range from 0.5-20.0% of low, moderate and high biodegradable effluents.
Table 8 represents the change in oxygen concentration in terms of current for rapid and reliable BOD estimation by immobilized microbial membrane of various industrial effluents.
The results indicate that the change in current is linearly proportional to the amount of biodegradabkle organic matter present in the sample.
TABLE 8a
CHANGE IN CURRENT (AI) OF INDUSTRIAL SAMPLE WITH HIGH BIODEGRADABLE ORGANIC LOAD

(Table Removed)
TABLE 8b
CHANGE IN CURRENT (AI) OF INDUSTRIAL SAMPLE WITH MODERATE
BIODEGRADABLE ORGANIC LOAD
(Table Removed)
TABLE 8c
CHANGE IN CURRENT (AI) OF INDUSTRIAL SAMPLE WITH LOW BIODEGRADABLE ORGANIC LOAD

(Table Removed)
Advantages
1. The prepared microbial consortium, acting in a synergistic way is capable of
biodegrading almost all kinds of organic matter present in a wide range of
industrial effluents, thereby giving rapid and reproducible BOD values.
2. The prepared immobilized charged nylon membrane is more stable as
compared to the existing immobilized microbial membranes.
3. The support used for the immobilization is charged nylon membrane
which being positively charged binds specifically to the negatively
charged bacterial cell by adsorption as well as entrapment.
4. The support used for immobilization is non-toxic to the micro-organisms.
5. The support i.e., charged nylon membrane used for the immobilization of
microorganisms is novel for rapid and reliable BOD estimation.

We Claim :
1. A process for the preparation of a novel immobilized microbial membrane comprising a synergistic mixture of the isolated bacterial strains of the kind such as herein described present in equal proportions, the said process comprises:
a) isolating a range of bacterial strains from sewage collected from
sewage treatment plants;
b) culturing the said strains on conventional nutrient media to get pure
cultures;
c) testing the said individual pure bacterial cultures for use as seeding
material in BOD analysis using glucose-glutamic acid (GGA) as a
reference standard by recording BOD values exhibited by individual
strains;
d) comparing the BOD values using the said bacterial strains with the
BOD values using sewage as a seeding material;
e) selecting the bacterial strains giving BOD values equal to or more than
the BOD values using sewage from step (d);
f) formulating the microbial consortium of selected bacterial strains
obtained from step (e);
g) comparing the BOD values using the formulated microbial consortium
of step [f] as seeding material with the BOD values obtained using
sewage as a seeding material;
h) growing the individual bacterial strains comprising the formulated microbial consortium obtained in step [f] by inoculating bacterial strains individually, incubating the said bacterial strains, growing the said incubated strains and mixing them in equal proportions;
i) centrifuging the resultant suspension to obtain pellets, washing the
collected pellet by dissolving in PO4-3 buffer, 0.025 - 0.075 M, pH 6.4 -
7.2 and recentrifuging the pellet; j) dissolving the pellet obtained from step (i) in 2.0 - 4.0 ml PO4-3 buffer,
0.025-0.075 M, pH 6.4 - 7.2, to obtain cell slurry; k) immobilizing the cell slurry obtained from step [j] on commercially
available charged nylon membrane under vacuum; I) drying the immobilized microbial membrane obtained from step (k); m) storing the dried immobilized microbial membrane obtained from step
(I) preferably at 1 - 4°C in said PO4-3 buffer.
2. A process as claimed in claim 1, wherein the formulated microbial
consortium is obtained by inoculating a suspension of the bacteria
selected from a group consisting of Aeromonas hydrophila, Pseudomonas
aeruginosa, Yersinia enterocolitica, Serratia liquefaciens, Pseudomonas
fluorescens, Enterobacter cloaca, Klebsiella oxytoca, Citrobacter
amalonaticus and Enterobacter sakazaki.
3. A process as claimed in claim 1, wherein the individual strains of the
above mentioned bacteria are inoculated separately in a nutrient broth.
4. A process as claimed in claim 1, wherein the incubation of bacterial strains
is carried out by gentle agitation at approximately 75-100 rpm.
5. A process as claimed in claim 1, wherein the incubation of bacterial strains is
carried out at a temperature ranging between 30°C - 37°C for a period of 16-
18 hours.
5. A process as claimed in claim 1, wherein the microbial consortium is
centrifuged at appropriate rpm preferably at 8,000 -12,000 rpm for a period of
approximately 20 - 30 minutes at a temperature of 1 - 4°C.
6. A process as claimed in claim 1, wherein the immobilized microbial
membrane is dried for 4 - 6 hours at a temperature ranging between 25°C -
35°C.
7. A process for the preparation of a novel immobilized microbial membrane
substantially as herein described with reference to the foregoing examples.

Documents:

356-del-2000-abstract.pdf

356-del-2000-claims.pdf

356-del-2000-correspondence-others.pdf

356-del-2000-correspondence-po.pdf

356-del-2000-description (complete).pdf

356-del-2000-form-1.pdf

356-del-2000-form-19.pdf

356-del-2000-form-2.pdf

356-del-2000-form-3.pdf

356-del-2000-petition-137.pdf

« Previous Patent

Next Patent »

Patent Number

238187

Indian Patent Application Number

356/DEL/2000

PG Journal Number

5/2010

Publication Date

29-Jan-2010

Grant Date

22-Jan-2010

Date of Filing

31-Mar-2000

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	DR. (MRS) RITA KUMAR	CENTRE FOR BIOCHEMICAL TECHNOLOGY MALL ROAD, DELHI-7, INDIA
2	DR. SANTOSH DAYARAM MAKHIJANI	CENTRAL POLUTION CONTROL BOARD, PARIVESH BHAWAN, EAST ARJUN NAGAR, DELHI-32
3	MR. A. MANOHARAN	CENTRAL POLUTION CONTROL BOARD, PARIVESH BHAWAN, EAST ARJUN NAGAR, DELHI-32
4	DR. (MRS) ALKA SHARMA	CENTRE FOR BIOCHEMICAL TECHNOLOGY MALL ROAD, DELHI-7, INDIA
5	MRS. SHIKHA RASTOGI	CENTRE FOR BIOCHEMICAL TECHNOLOGY MALL ROAD, DELHI-7, INDIA

PCT International Classification Number

C12P 39/00

PCT International Application Number

N/A

PCT International Filing date

PCT Conventions:

#	PCT Application Number	Date of Convention	Priority Country
1			NA