GB2386124A

GB2386124A - Process for the preparation of reusable immobilised microbial composition in bead form used as ready-to-use seed inoculum in BOD anaysis of waste water

Info

Publication number: GB2386124A
Application number: GB0228840A
Authority: GB
Inventors: Rita Kumar; Anil Kumar; Alka Sharma; Sharad Vishwanath Gangal; Santosh Dayaram Makhijani
Original assignee: Council of Scientific and Industrial Research CSIR
Current assignee: Council of Scientific and Industrial Research CSIR
Priority date: 1998-06-20
Filing date: 1998-06-20
Publication date: 2003-09-10
Anticipated expiration: 2018-06-20
Also published as: GB2386124B; GB0228840D0

Abstract

Preparing ready-to-use reusable immobilised microbial composition comprises: isolating bacteria from sewage, culturing strains on nutrient media to get pure cultures and testing for biological oxygen demand (BOD) analysis, verifying BOD values using Glucose-Glutamic Acid (GGA) as reference standard, comparing those BOD values with domestic sewage collected from treatment plants, selecting those bacterial strains with equal or greater BOD values, preparing mixtures of strains, testing mixtures by BOD comparison with sewage and selecting those with optimum BOD values, inoculating mixture of strains individually and incubating and growing them, mixing strains in equal proportions on basis of optical density, centrifuging resulting suspension to form pellets and washing and suspending in immobilising agent, adding polymerising agent and hardening in calcium chloride and storing formed beads in same, testing beads for BOD values using industrial samples, and washing and storing beads. The beads can be reused up to five times with same efficacy.

Description

A PROCESS FOR THE PREPARATION OF A REUSABLE IMMOBILISED MICROBIAL COMPOSITION USEFUL AS READY-TO-USE SEED INOCULUM IN BOD ANALYSIS The present invention relates to a process for the preparation of a reusable immobilised microbial formulation comprising a synergistic mixture of isolated bacterial strains present in equal proportions.

Biological Oxygen Demand (BOD) is one of the most widely used parameters for estimating the strength of water pollution. The BOD-5 test measures the biodegradable organic matter present in waste-waters. This determination involves the measurement of dissolved oxygen during the biochemical oxidation of organic matter by micro- organisms at a standard duration and temperature. To ensure that meaningful results of BOD values are obtained, the sample must be suitably diluted with water so that adequate nutrients and oxygen are available during the incubation period (APHA, 1995).

Besides this, the test sample in a BOD test is'seeded'with a bacterial culture for biodegradation of organic matter. The conventional BOD test is based on naturally occurring microbes and either raw sewage or treated effuent from a waste-water treatment plant is used as a seeding material, but this does not give reproducible BOD values. The degree of reproducibility of the BOD test cannot be defined precisely because of variations that occur in the bacterial decomposition of various organic substances. This variation is probably due to the source of seeding material (i. e. sewage which is collected from different places) wherein inadequate or variable microbial flora are present. Thus, in BOD analysis, the bacterial seed represents a major variable and it can be controlled both quantitatively and qualitatively by the use of pure cultures (Fitzmaurice and Gray, 1989; Kilroy and Gray, 1995 ; and Hammer, 1975).

The reproducibility of the BOD values can be obtained by formulating a defined microbial composition as seeding material containing a uniform microbial population. Such formulations in the form of dehydrated seeding materials i. e. Polyseed and Bioseed (Fitzmaurice and Gray, 1989) have been developed in the USA and the UK and are available in International markets for use in BOD analysis. Polyseed is a registered trade mark of Polybac Corporation USA, and consists of a blend of specialised dehydrated microbial cultures in capsule form to provide a source of seeding material for BOD analysis. Polyseed is EPA accepted. Bioseed (trade name) is manufactured by International Biochemicals (UK) Limited and is supplied as dehydrated seeding material in capsule form for BOD analysis. Bioseed from International Biochemicals (UK) consists of a range of micro-organisms namely Pseudomonas, Nocardia, Streptomyces, Bacillus, and Micromonospora which are different from those of our composition.

Though these microbial compositions reduce the possibility of controversy in BOD results and make BOD analysis an easy, clean and convenient laboratory test, these microbial compositions need to be revived one day prior to performing the BOD analysis. The other drawbacks of dehydrated seeding materials are that these dehydrated materials are not ready-to-use seeding materials in BOD analysis and are not reusable. Whereas, the advantages of the present invention over and above previous technologies is that it envisages a ready-to-use, and reusable (five times with the same efficacy) seeding material for BOD analysis. Due to this reusable property, such an immobilised microbial composition is more economic than the dehydrated seeding materials.

To avoid the discrepancies in BOD results as well as to get a ready-to-use seeding material in BOD test, in the present invention, a defined microbial composition is formulated as well as immobilised on a support to get a ready-to-use instant seeding material in BOD analysis. Immobilization of micro-organisms leads to a reduction in

cell growth and offers an easy-to-handle and ready-to-use material for many important industrial processes. Immobilised cells also offer a promising potential for the improvement in efficiency of bioprocesses such as BOD analysis and biological wastewater treatment. The advantages of immobilised cells compared to free cells are that the immobilised cells can be used repeatedly and with ease of separation. Immobilised biocatalysts have also been used on an Industrial scale for treating effluent containing phenolic compounds (Anselmo and Novail, 1992) as well as other toxicants (Wong et al., 1993; Gijzen et al, 1988).

Summary of the Invention For solving the aforementioned problems, the inventors have realized that there exists a need to provide a ready-to-use, low cost and reusable seeding material for BOD test.

According to the present invention there is provided a process for the preparation of immobilised microbial formulations which process comprises the steps of : (a) isolating a range of bacterial strains from sewage collected from sewage treatment plants; (b) culturing the said strains on nutrient media to get pure cultures; (c) testing the said individual pure bacterial culture for BOD analysis and recording BOD values obtained from individual strains; (d) verifying the BOD values obtained with glucose-glutamic acid as a reference standard; (e) comparing the BOD values obtained using the said bacterial strains as seeding material with that of the observed BOD values using domestic sewage collected from sewage treatment plants as seeding material;

(f) selecting the bacterial strains which have BOD values equal to or more than the BOD values of sewage treatment plants; (g) preparing mixtures of selected bacterial strains; (h) testing the said mixtures of selected bacterial strains by comparing their BOD values with those of domestic sewage; (i) selecting the said mixtures having optimum BOD values; (j) inoculating the mixture of said bacterial strains obtained in step (i), individually; (k) incubating the said bacterial strains and growing the said incubated strains; (1) mixing the incubated strains from step (k) in equal proportions on the basis of optical density values; (m) centrifuging the resultant suspension to obtain pellets, washing the collected pellet with distilled water and suspending the said pellet in a solution of an appropriate immobilizing agent; (n) adding the resultant slurry to an appropriate polymerising agent to form beads and curing the resultant beads by known methods; (o) hardening the beads by leaving the said beads in 0.1 M to 0.2 M CaCl2 solution for approximately 3 hours; (p) storing the prepared immobilised microbial beads in a concentration range of 0.05 M to 0.2 M CaCl2 solution at a temperature preferably less than or equal to 4 C for longer use; (q) testing formulated beads for BOD analysis using glucose-glutamic acid (GGA) as a reference standard; (r) comparing the BOD values obtained using the formulated beads with those BOD values obtained with sewage as seeding material using synthetic samples; (s) testing the said beads for BOD analysis using different industrial samples ;

(t) washing the used beads with distilled water; and (u) storing at less than or equal to 4 C for further use.

The present invention relates to a process for the preparation of an immobilised microbial composition useful as a ready-to-use seed inoculum for the determination of Biological Oxygen Demand (BOD). More particularly the present invention enhances the reproducibility of results as well as reusability of the immobilised seed inoculum in BOD test. The immobilised formulated microbial mixture is comprised of cultures of the following bacteria, namely: (a) Enterobacter cloaca ; (b) Citrobacter amalonaticus ; (c) Pseudomonas aeruginosa ; (d) Yersinia enterocolitica ; (e) Klebsiella oxytoca ; (f) Enterobacter sakazaki ; and (g) Serratia liquefaciens, which are pre-tested for BOD analysis individually. The formulated microbial composition is obtained by inoculating a suspension of these bacteria individually, incubating at 37 C, and mixing all the bacteria in equal proportions based on optical density.

Accordingly, another object of the present invention, is to provide a process for the production of novel immobilised formulated microbial mixture useful as seed inoculum in BOD analysis, which inoculum is reusable five times with the same efficacy.

The immobilised microbial composition prepared by the present invention is useful in determining the strength of water pollution, waste loadings to treatment plants and evaluating the efficiency of such treatment.

In an embodiment of the invention, the formulated microbial composition is obtained by inoculating a suspension of the bacteria selected from a group consisting of (a) Enterobacter cloaca (b) Citrobacter amalonaticus (c) Pseudomonas aeruginosa (d) Yersinia enterocolitica (e) Klebsiella oxytoca (f) Enterobacter sakazaki (g) Serratia liquefaciens, individually.

The composition produced by a method according to the present invention may contain bacterial such as: 1. Enterobacter cloaca CBTCC/Micro/I (Corresponding ATCC No. 29893) 2. Citrobacter amalonaticus CBTCC/Micro/2 (Corresponding ATCC No. 25406) 3. Pseudomonas aeruginosa CBTCC/Micro/3 (Corresponding ATCC No. 49622) 4. Yersinia enterocolitica CBTCC/Micro/4 (Corresponding ATCC No. 27739) 5. Klebsiella oxytoca CBTCC/Micro/5 (Corresponding ATCC No. 15764) 6. Enterobacter sakazaki CBTCC/Micro/6 (Corresponding ATCC No. 12868) 7. Serratia liquefaciens CBTCC/Micro/7 (Corresponding ATCC No. 25641) as these facilitate obtaining uniform reproducible results in BOD estimations performed at any place. The above micro-organisms are deposited at the Centre for Biochemical Technology Culture Collections (CBTCC) designated as stated above and will be made available to the public on request as per normal official procedures. Preferably, the bacteria are present in uniform amounts and form a synergistic mix. Moreover, the composition of the present invention will be ready more than once for immediate use, instead of each time having to prepare afresh which involves collecting the sewage each time from treatment plants.

In an another embodiment of the invention, the individual strains of the above mentioned bacteria are inoculated separately in a nutrient broth.

In a further embodiment of the invention, the incubation of bacterial strains is carried out by gentle agitation at approximately 75-100 rpm.

In one of the embodiments of the invention, the growth of incubated bacterial strains is carried out at a temperature ranging between 35 C to 40 C for a period of 16-24 hours.

In a further embodiment of the invention, the resultant microbial composition is centrifuged at appropriate rpm preferably at 8, 000 to 12,000 rpm for a period of approximately 20 to 30 minutes.

In another embodiment of the invention, the resultant pellet is washed by dissolving in an appropriate quantity of distilled water and recentrifuged at an appropriate rpm in the range of 8,000 to 12,000 rpm for a period of approximately 20 to 30 minutes at a temperature less than or equal to 4"C.

In an embodiment of the invention, the resultant pellet obtained is immobilised by entrapping it in sodium alginate as an immobilizing agent.

In one of the embodiments of the invention, the resultant slurry used is pumped into a stirred CaCl2 solution with concentration in the range of 0.05 to 0.2 M.

In an embodiment of the invention, the cell slurry is introduced as discrete droplets so as to form beads of appropriate size.

In a further embodiment of the invention, the beads are washed with double distilled water to remove extra CaCI2 solution.

In an embodiment of the invention, the immobilised beads formed may be stored for later use in 0.05 M to 0.2 M Catch solution at a temperature preferably less than or equal to 4 C.

The bacterial cultures of the above composition were isolated from sewage samples collected from Dhirpur Coronation plant near Mukherjee Nagar, Delhi. Sewage is homogenized for 2 minutes and suspended in gram negative broth. Incubation is carried out for 24 hours. Cultures are plated on MacConkey's agar. A mixture of lactose and non-lactose fermentors are isolated. Colonies are mixed on a vortex mixer and all the cultures are isolated in pure cultures after several subcultures.

The pure cultures are checked for the gram reaction. All the gram negative cultures are maintained as stock cultures. The cultures are further identified by the rapid identification schemes.

The cultures which are gram negative bacilli and catalase positive, are included.

As indicated below, seven pure cultures are included in the composition and these are identified by the classification scheme of Edwards and Ewing (1972) along with designated changes by Centre for Disease Control (CDC), Atlanta, 1978.

Cultures Accession number (Corresponding ATCC No.) 1. Enterobacter cloaca CBTCC/Micro/l (29893) 2. Citrobacter amalonaticus CBTCC/Micro/2 (25406) 3. Pseudomonas aeruginosa CBTCC/Micro/3 (49622) 4. Yersinia enterocolitica CBTCC/Micro/4 (27739) 5. Klebsiella oxytoca CBTCC/Micro/5 (15764) 6. Enterobacter sakazaki CBTCC/Micro/6 (12868) 7. Serratia liquefaciens CBTCC/Microl7 (25641)

The immobilization technique of formulated microbial mixture of the present invention may be carried out by inoculating the individual strains of the above mentioned bacteria separately in a nutrient broth, and then undertaking the following steps. All the cultures are incubated preferably at 37 C for approximately 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 the required quantity and then mixed for preparing the microbial composition. The resultant microbial composition is centrifuged at appropriate rpm, preferably at 10,000 rpm for a period of approximately 30 minutes. The resultant pellet is washed by dissolving in a minimum quantity of distilled water and

recentrifuged at appropriate rpm, preferably at 10,000 rpm, for a period of approximately 30 minutes. Preferably during centrifugation, the temperature is maintained at 4 C. The pellets thus obtained, is immobilised by entrapping them in immobilizing agent such as calcium alginate gel. The immobilization can be affected by mixing the pellets using 2% sodium alginate solution and pumping the resultant slurry into a stirred 0.1 MCaCl2 solution. The cell slurry is introduced as discrete droplets so as to form beads of appropriate size. The beads thus formed are left in 0.1 M calcium chloride solution for approximately 3 hours and later washed with double distilled water. The immobilised microbial beads are stored in 0.05 M CaCl2 solution at a temperature preferably below 4 C.

The beads produced by the process of the present invention can be used as seed inoculum in varying quantities for BOD estimation using Glucose-Glutamic Acid (GGA) as a reference standard by the method described in the Standard method for the Examination of Water and Wastewaters 17th edition, American Public Health Association, Washington DC (1989). After optimisation of the quantity of beads in the BOD analysis, the beads were used as seeding material for BOD estimation of various synthetic samples as well as industrial effluents.

This application is a divisional from GB 98 13305 which relates to immobilised reusable microbial formulations.

The examples provided below are given by way of illustration of the invention and therefore, should not be constructed to limit the scope of the invention.

Example I Two loops from agar plates of Enterobacter cloaca, Citrobacter amalonaticus, Pseudomonas aeruginosa, Yersinia enterocolitica, Klebsiella oxytoca, Enterobacter

sakazaki and Serratia liquefaciens were inoculated separately in 500 ml of nutrient broth. All the cultures were incubated at 37 C for 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 bacterial suspensions were mixed thoroughly and centrifuged at 8,000 rpm for 30 minutes at 4 C. The pellet was washed by dissolving it in a small volume of distilled water and recentifuged at 8.000 rpm for 30 minutes at 40 C.

The microbial composition prepared as described above was mixed with 1% sodium alginate solution. The resultant slurry was pumped into a stirred 0.05 M calcium chloride solution. The beads thus formed, were left in 0.1 M calcium chloride solution for 3 hours and later washed with double distilled water. The immobolized microbial beads were stored in 0.05 M calcium chloride solution at 40 C. The beads were nonspherical, less stable.

Example II Two loops from agar plates of Enterobacter cloaca, Citrobacter amalonaticus, Pseudomonas aeruginosa, Yersinia enterocolitica, Klebsiella oxytoca, Enterobacter

sakazaki and Serratia liquefaciens were inoculated separately in 500 ml of nutrient broth.

All the cultures were incubated at 3'r C for 24 hrs. in an incubator shaker at 100 rpm.

After incubation, optical density was measured at 650 nm. Optical density of all the bacteria was maintained to 05 either by diluting or concentrating the bacterial suspension All bacterial suspensions were mixed thoroughly and centrifuged at 10,000 rpm for 30 minutes at 40 C. The pellet was washed by dissolving it in small volume of distilled water and recentifuged at 10. 000 rpm for 30 minutes at 4 C.

The microbial composition was mixed with 1. 5% sodium alginate solution. The resultant alginate bacterial suspension was pumped drop-wise into a stirred 0. 1M calcium chloride solution. The beads thus formed, were left in 0. IM CaCl2 solution for 3 hrs. and later washed with double distilled water. The microbial beads were stored in 0.05 M CaCI solution at 4 C. The beads obtained were slightly spherical and less stable.

Example M

Two loops from agar plates of Enterobacter cloaca, Citrobacter amalonaticus.

Pseudomonas aerugmosa, Yersima enterocolitica, Klebsiella oxytoca, Enterobacter sakazaki and Serratia liquefaciens were inoculated separately in 500 ml of nutrient broth. All the cultures were incubated at 3t C for 24 hrs. in an incubator shaker at 75 rpm.

After incubation, optical density was measured at 650 Dm. Optical density of all the bacteria was maintained to 0. 5 either by diluting or concentrating the bacterial suspension. All bacterial suspensions were mixed thoroughly and centrifuged at 12,000 rpm for 30 minutes at 40 C. The pellet was washed by dissolving it in small volume of distilled water and recentifuged at 12.000 rpm for 30 minutes at 4 C.

The microbial composition was mixed with 2.0% sodium alginate solution.

The resultant alginate bactenal suspension was pumped drop-wise into a stirred 0.15M calcium chloride solution. The beads thus formed, were left in 0.1 M calcium chloride solution for 3 hrs. and later washed with double distilled water. The

immobilized microbial beads were stored in 0 05 M calcium chlonde solution at 40 C.

The beads thus formed were spherical, stable and porous Example IV Two loops from agar plates of Enterobacter cloaca, Citrobacter amalonatlcus, Pseudomonas aeruginosa, Yersima enterocohtica, Klebsiella oxytoca, Enterobacter

All the cultures were incubaied at 37 C for 24 hrs. 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 bacterial suspensions were mixed thoroughly and centrifuged at 10,000 rpm for 30 minutes at 40 C. The pellet was washed by dissolving it in small volume of distilled water and recentifuged at 10. 000 rpm for 30 minutes at 40 C.

The microbial composition was mixed with 2.5% sodium alginate solution.

The microbial beads were prepared by using 0.2 M calcium chloride solution. The beads thus formed, were left in 0.1 M calcium chloride solution for 3 hrs. and later washed with double distilled water. The immobilized microbial beads were stored in 0.05 M calcium chloride solution at 10 C. The beads thus formed were hard and less porous.

Example V Two loops from agar plates of En erobacter cloaca, Citrobacter amalonatlcus, Pseudomonas aerugmosa, Yersmza enterocolitlca, Klebsiella oxytoca, Enterobacter sakazak1 and Serra/ia liquefaciens were inoculated separately in 500 ml of nutrient broth.

All the cultures were incubated at 3'f C for 24 hrs. 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 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 distilled water and recentifuged at 10. 000 rpm for 30 minutes at 4 C The microbial composition was mixed with 3. 0% sodium alginate solution.

The resultant mix was pumped into a stirred 0.1 M calcium chloride solution. The beads thus formed, were left in 0. 1 M calcium chlonde solution for 3 hrs. and later washed with double distilled water. The immobilised microbial beads were stored in 0.2 M calcium chloride solution at 40 C. The beads were more hard and less porous.

Example VI.

Beads prepared from microbial composition using 2.0% sodium alginate (stable and porous beads) were used in BOD analysis using 2 % solution of Glucose-Glutamic Acid (GGA), a reference standard which contains each of 150 mg of Glucose as well as Glutamic Acid. Table 1 shows BOD values of GGA using different quantities of beads as well as sewage as seeding materials. The sample was analysed at five different times.

Standardization of optimum quantity of microbial beads for BOD estimation with 2% GGA

BOD value (02mg/1) using different quantities of beads and sewage Replicates 2 beads 4 beads 8 beads 12 beads Sewage 1 170 194 195 195 188 2 173 199 198 201 190 3 176 190 189 191 187 4 179 196 196 198 191 5 172 189 191 190 193 Mean 174 193.6 193.8 195 189.8 Range 9 10 9 10 6 S. D. 3. 535 4.159 3.701 4.636 2. 387 % CV 2.03 2.14 1.9 2.3 1.2 BOD values of GGA using four or more than four beads were comparable with the values obtained with those by sewage. Based on this observation, four microbial beads were used for further BOD analysis.

Example VU.

Microbial beads in parallel with sewage were used for BOD determination of different synthetic samples namely Peptone, Glucose, L-Glutamic acid & Citric acid.

A stock solution (0.03%) of each of these synthetic samples was prepared. For BOD estimation, 2% solutions of these stocks were used.

Table 2 represents BOD values of all the above four synthetic samples. The BOD analysis was carried out at five different times and results were compared with those obtained with sewage as seeding material

Table 2 BOD estimation of. synthetic samples with immobilized beads as well as sewage.

Replicates BOD values (02 mg/l) of synthetic samples Glucose Peptone Glutamic Citric acid acid Beads Sewage Beads Sewage Beads Sewage Beads Sewage 1. 195 190 215 182 168 164 153 156 2.190 181 194 171 151 152 160 168 3. 185 175 185 180 176 157 170 166 4.182 179 194 175 159 157 155 152 5.195 184 191 178 172 161 173 178 Mean 189 182 196 177 165 158 166 164 Range 20 15 30 15 30 15 25 25 (Max.Min.) S. D. 5. 9 5.6 11.3 4. 3 10. 1 4.5 8.9 10. 3 % CV 3. 1 3. 1 5.8 2.4 6.1 2.9 5.5 6.3 Fairly comparable BOD values were obtained by using immobilized microbial beads as well as sewage as seeding materials

Example VU Table 3 & 4 represents the BOD values of various industrial effluents namely paper & pulp, bioproducts, distillery, pharmaceutical, soft drink and vegetable oil industries. The samples were collected at five different times for BOD analysis in order to check the reproducibility and repeatability. For statistical analysis of the data, five replicates of each sample were analyzed.

Table 3 represents the BOD analysis of paper & pulp, bioproducts and distillery industrial effluents by using immobilized microbial beads as well as sewage as a seeding materials.

Table 3 BOD values of three different industrial samples using beads as well as sewage as a seeding materials

Replicates BOD values (02 mg/l) of industrial samples Paper & Pulp Bio-products Distillery Beads Sewage Beads Sewage Beads Sewage 1. 1, 900 1,820 9,900 8,300 22,200 20,100 2. 1,840 1,700 10,900 6,700 22,850 19,320 3. 1,808 1,790 10,830 7,790 23,720 20,250 4. 1, 970 1,840 10,200 8,100 23,630 19,180 5. 1, 910 1,880 10,640 8,280 22,470 20,870 Mean 1,886 1,806 10,429 7,834 23,174 19,944 Range 162 180 1,000 1,600 1,250 1,690 S. D. 63.3 67.3 429.6 666.1 526.1 697.9 % C. V. 3. 4 3.7 4.1 8. 5 2.3 3. 5 BOD values of paper & pulp industrial effluent by using immobilized microbial beads as well as sewage were found to be reasonably comparable. On the other hand, BOD values of bio-products and distillery industrial effluents by using immobilized beads were higher than those obtained with sewage which indicates better performance of immobilized microbial beads as seeding material in BOD analysis. BOD values of pharmaceutical, soft dnnk and vegetable oil industrial effluents using immobilized microbial beads and sewage as seeding material are presented in Table 4.

Table 4 BOD values of three different industrial samples using beads as well as sewage as a seeding materials

Replicates BOD values (02 mg/l) of industrial samples Pharmaceutical Soft dnnk Vegetable Oil Beads Sewage Beads Sewage Beads Sewage 1. 1,600 1, 250 3,740 3,740 3,470 3,280 2.1, 670 1,430 3, 620 3, 660 3,320 3,330 3.1, 630 1,310 3,780 3, 520 3,410 3,370 4.1, 680 1,400 3,720 3,600 3,390 3,170 5. 1,670 1,360 3,700 3,430 3,510 3,210 Mean 1,650 1,350 3,712 3,590 3,420 3,264 Range 80 150 200 350 210 250 S. D. 33. 9 71.8 59.3 120. 4 73.5 95.8 % C. V. 2.1 5. 3 1.9 3.4 2.1 2.9 Table indicates that BOD values of soft drink as well as vegetable oil industrial effluent by using immobilized microbial beads and sewage as seeding materials were comparable while BOD values of pharmaceutical industrial effluent by using immobilized microbial beads were higher than those obtained using sewage as seeding material.

Example IX To check the reusability of prepared microbial beads, BOD analysis of reference GGA solution was carried out by using the same set of beads for five different times as shown in Table 5. On the other hand, fresh sewage was used for each BOD estimation for the comparison of the results. BOD values of reused beads thus obtained were compared with the sewage. The results with reused beads as seeding material in BOD analysis were found to be comparable with those of sewage when beads were used five times.

Table 5 Reusability studies of beads using GGA

BOD values (02 mg/ !) of beads reused upto 5 times I Time II Time III Time IV Time V Time (After I week) (After 2 weeks) (After 3 weeks) (After 4 weeks) Beads Sewage Beads Sewage Beads Sewage Beads Sewage Beads Sewage 1. 185 186 189 177 184 184 179 186 182 181 2.208 191 213 187 203 194 204 193 202 174 3.210 197 213 177 196 176 197 179 199 186 4.200 185 206 182 187 169 191 190 190 179 5.195 198 210 187 193 178 190 187 193 178 Mean 200 190 206 182 193 180 192 187 193 180 Range 25 20 30 15 25 30 25 14 20 12 S. D. 10.2 7. 7 10 5 6.9 9. 4 9. 3 52 7.9 4. 4 % C. V 5.1 4.1 4.9 2. 7 36 52 4.8 2.8 4. 1 2. 4 As per the results it was observed that same beads are reusable for five times with almost same efficacy.

Example X Stability studies of immobilized microbial beads were carried out by storing the beads in different solutions. The results are presented in Table 6.

Table 6 Stability study of immobilized beads stored at different conditions

Storage BOD values (O2 mg/1) of GGA Time usmg beads stored in different solutions usmg Sewage (days) Distilled 0.1 M 005 M GGA Water CaC12 CaC12 0 178 186 189 171 193 30 178 184 187 168 190 60 165 182 185 160 185 90 166 180 184 161 182 120 165 175 181 160 180 150 162 170 174 158 171 180 155 163 169 149 185

their almost full metabolic On storage, it was observed that the beads retained their almost full metabolic activity in 0. 05 M Caca2 solution.

Example XI Stability studies of immobilized microbIal beads were carried out by storing the beads at different temperatures. The results are presented in Table 7.

TABLE 7 Stability study of immobilized beads stored at different temperatures

Storage BOD values (O2 mg/1) of GGA Time using beads stored at defferent temperatures using Sewage (days) 40C 250C c 450C 0 200 200 200 200 196 15 196 198 194 193 193 30 198 196 190 191 195 60 195 195 187 189 191 90 199 193 185 184 194 120 196 189 183 181 191 150 201 187 180 177 196 180 198 185 178 172 189 On storage it was observed that the beads retained their full metabolic activity at 4 C.

From the examples explained above it is clear that BOD values by using immobilized microbial beads were comparable with domestic sewage used as seeding matenal in conventional BOD test.

ADVANTAGES 1 The prepared immobilized microbial composition (beads) is ready-to-use seed inoculum in BOD test as compared with free microbial composition which needs to be revived one day before the start of BOD test.

2 This immobilised composition is stable and can be reused in BOD analysis.

3. The immobilized microbial composition is more economic than the dehydrated seeding materials due to reusable property.

4 Immobilization of microorganism leads to a reduction in cell growth and offers a easy to handle and ready-to-use material for many important industrial. processes.

5. The advantages of immobilized cells compared to free cells are, that the immobilized cells can be used repeatedly and with ease of separation.

Claims

CLAIMS 1. A process for the preparation of immobilised microbial formulations which process comprises the steps of : (a) isolating a range of bacterial strains from sewage collected from sewage treatment plants; (b) culturing the said strains on nutrient media to get pure cultures; (c) testing the said individual pure bacterial culture for BOD analysis and recording BOD values obtained from individual strains; (d) verifying the BOD values obtained with glucose-glutamic acid as a reference standard ; (e) comparing the BOD values obtained using the said bacterial strains as seeding material with that of the observed BOD values using domestic sewage collected from sewage treatment plants as seeding material; (f) selecting the bacterial strains which have BOD values equal to or more than the BOD values of sewage treatment plants; (g) preparing mixtures of selected bacterial strains; (h) testing the said mixtures of selected bacterial strains by comparing their BOD values with those of domestic sewage; (i) selecting the said mixtures having optimum BOD values ; (j) inoculating the mixture of said bacterial strains obtained in step (i), individually ; (k) incubating the said bacterial strains and growing the said incubated strains; (1) mixing the incubated strains from step (k) in equal proportions on the basis of optical density values;

<Desc/Clms Page number 28>

(m) centrifuging the resultant suspension to obtain pellets, washing the collected pellet with distilled water and suspending the said pellet in a solution of an appropriate immobilizing agent; (n) adding the resultant slurry to an appropriate polymerising agent to form beads and curing the resultant beads by known methods; (o) hardening the beads by leaving the said beads in 0.1 M to 0.2 M CaCIs solution for approximately 3 hours; (p) storing the prepared immobilised microbial beads in a concentration range of 0.05 M to 0.2 M CaCl2 solution at a temperature preferably less than or equal to 4 C for longer use; (q) testing formulated beads for BOD analysis using glucose-glutamic acid (GGA) as a reference standard ; (r) comparing the BOD values obtained using the formulated beads with those BOD values obtained with sewage as seeding material using synthetic samples; (s) testing the said beads for BOD analysis using different industrial samples; (t) washing the used beads with distilled water; and (u) storing at less than or equal to 4 C for further use.

2. A process as claimed in claim 1, wherein the formulated microbial composition is obtained by individually inoculating with a suspension of the bacteria selected from the group consisting of (a) Enterobacter cloaca ; (b) Citrobacter amalonaticus ; (c) Pseudomonas aeruginosa ; (d) Yersinia enterocolitica ; (e) Klebsiella oxytoca ; (f) Enterobacter sakazaki ; and (g) Serratia liquefaciens.

3. A process as claimed in claim 2, wherein the individual strains of the above mentioned bacteria are inoculated separately in a nutrient broth.

<Desc/Clms Page number 29>

4. A process as claimed in claim 1, wherein the incubation of bacterial strains is carried out by gentle agitation at 75-100 rpm.

5. A process as claimed in claim 1, wherein the growth of incubated bacterial strains is carried out at a temperature in the range of 35 C to 40 C for a period of 16-24 hours.

6. A process as claimed in claim 1, wherein the resultant microbial composition is centrifuged for a period of 20 to 30 minutes.

7. A process as claimed in claim 1, wherein the resultant pellet is washed by dissolving in an appropriate quantity of distilled water and recentrifuging for a period of 20 to 30 minutes at a temperature less than or equal to 4 C.

8. A process as claimed in claim 6 or claim 7, wherein centrifugation is carried out at 8,000 to 12,000 rpm.

10. A process as claimed in claim 1, wherein the immobilizing agent introduced at step (m) is sodium alginate.

11. A process as claimed in claim 10, wherein the resultant slurry formed in step (m) is pumped into a polymerising agent being a stirred CaClz solution with a concentration.. in the range of 0.05 to 0.2 M.

12. A process as claimed in claim 10 or claim 11, wherein the resultant slurry formed in step (m) is introduced into the polymerising agent as discrete droplets so as to form beads of appropriate size.

13. A process as claimed in claim 1, wherein the beads are washed with double distilled water to remove extra CaCl2 solution.

14. A process as claimed in claim 1, wherein the immobilised microbial beads formed are stored in 0.05 M CaCl2 solution at a temperature less than or equal to 4 C.

15. A process for the preparation of a microbial formulation for BOD analysis substantially as hereinbefore described.

<Desc/Clms Page number 30>

Amendment to the claims have been filed as follows CLAIMS

1. A process for the preparation of immobilised microbial formulations which process comprises the steps of : (a) isolating a range of bacterial strains from sewage collected from sewage treatment plants; (b) culturing the said strains on nutrient media to get pure cultures; (c) testing the said individual pure bacterial culture for BOD analysis and recording BOD values obtained from individual strains; (d) verifying the BOD values obtained with glucose-glutamic acid as a reference standard; (e) comparing the BOD values obtained using the said bacterial strains as seeding material with that of the observed BOD values using domestic sewage collected from sewage treatment plants as seeding material; (f) selecting the bacterial strains which have BOD values equal to or more than the BOD values of sewage treatment plants; (g) preparing mixtures of selected bacterial strains; (h) testing the said mixtures of selected bacterial strains by comparing their BOD values with those of domestic sewage; (i) selecting the said mixtures having optimum BOD values; (j) inoculating the mixture of said bacterial strains obtained in step (i), individually; (k) incubating the said bacterial strains and growing the said incubated strains; (1) mixing the incubated strains from step (k) in equal proportions on the basis of optical density values;

<Desc/Clms Page number 31>

(m) centrifuging the resultant suspension to obtain pellets, washing the collected pellet with distilled water and suspending the said pellet in a solution of an appropriate immobilizing agent; (n) adding the resultant slurry to an appropriate polymerising agent to form beads and curing the resultant beads by known methods; (o) hardening the beads by leaving the said beads in 0.1 M to 0.2 M CaCl2 solution for approximately 3 hours; (p) storing the prepared immobilised microbial beads in a concentration range of 0.05 M to 0.2 M CaCl2 solution at a temperature less than or equal to 4 C for longer use; (q) testing formulated beads for BOD analysis using glucose-glutamic acid (GGA) as a reference standard; (r) comparing the BOD values obtained using the formulated beads with those BOD values obtained with sewage as seeding material using synthetic samples; (s) testing the said beads for BOD analysis using different industrial samples; (t) washing the used beads thrice with distilled water; and (u) storing at less than or equal to 4 C for further use.

2. A process as claimed in claim 1, wherein the formulated microbial composition is obtained by individually inoculating with a suspension of the bacteria selected from the group consisting of (a) Enterobacter cloaca ; (b) Citrobacter amalonaticus ; (c)

Pseudomonas aeruginosa ; (d) Yersinia enterocolitica ; (e) Klebsiella oxytoca ; (f) Enterobacter sakazaki ; and (g) Serratia liquefaciens.

3. A process as claimed in claim 2, 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 75-100 rpm.

<Desc/Clms Page number 32>

5. A process as claimed in claim 1, wherein the growth of incubated bacterial strains is carried out at a temperature in the range of 35'C to 40 C for a period of 16-24 hours.

6. A process as claimed in claim 1, wherein the resultant microbial composition is centrifuged for a period of 20 to 30 minutes.

7. A process as claimed in claim 1, wherein the resultant pellet is washed by dissolving in an appropriate quantity of distilled water and recentrifuging for a period of 20 to 30 minutes at a temperature less than or equal to 4 C.

8. A process as claimed in claim 6 or claim 7, wherein centrifugation is carried out at 8,000 to 12,000 rpm.

9. A process as claimed in claim 1, wherein the immobilizing agent introduced at step (m) is sodium alginate.

10. A process as claimed in claim 9, wherein the resultant slurry formed in step (m) is pumped into a polymerising agent being a stirred CaCl2 solution with a concentration in the range of 0.05 to 0.2 M.

11. A process as claimed in claim 9 or claim 10, wherein the resultant slurry formed in step (m) is introduced into the polymerising agent as discrete droplets so as to form beads of appropriate size.

12. A process as claimed in claim 1, wherein the beads are washed with double distilled water to remove extra CaCl2 solution.

13. A process as claimed in claim 1, wherein the immobilised microbial beads formed are stored in 0.05 M CaCIs solution at a temperature less than or equal to 4 C.

14. A process for the preparation of a microbial formulation for BOD analysis as claimed in claim 1 and substantially as hereinbefore described.