Process for preparing microbial consortium useful as seeding material for BOD analysis of pulp and paper industrial wastewater

Abstract

The present invention provides a seeding material for accurate and reproducible monitoring of BOD load of pulp and paper wastewater, wherein the bacteria are isolated from various locations in India and comprise Micrococcus sp., Staphylococcus sp., Kurthia zopfii, Alcaligenes faecalis, and Pseudomonas aeruginosa, the microbial composition being capable of exerting an appropriate BOD for pulp and paper industrial wastewater due to the specificity of bacterial stows present therein towards the refractory organic compounds found in these effluents.

Description

FIELD OF THE INVENTION

The present invention relates to a process for preparing a specifically designed seeding material for BOD analysis of pulp and paper wastewater. The seeding material is a uniformly formulated microbial consortium comprising of isolated, acclimatized and synergistic bacterial rains.

BACKGROUND AND PRIOR ART

Most water analysis methods quantify individual components. However, some, such as oxygen demand tests quantify an aggregate amount of constituents with a common characteristic. Broadly speaking, BOD and COD quantify amount of oxygen required to oxidize organic matter in water/wastewater to indicate amount of organic material present. BOD utilizes microorganisms to oxidize organic material, while COD uses inorganic chemical oxidant. BOD measurement is the most fundamental way of determining water pollution levels and of predicting possible effects of waste discharge. Organic matter that is present in water can be from plants, sugars, proteins or other substances that enter water film natural sources or pollution. This matter is broken down biochemically by organisms such as bacteria, which can multiply as long as organic matter is present as food and oxygen is available for respiration. If high population of bacteria continuously consume dissolved oxygen in water at an accelerated rate, atmospheric air will not be able to replenish it. This situation can create a lack of dissolved oxygen in water, threatening and destroying many forms of aquatic life. Oxygen depletion in receiving waters has been regarded as an important negative effect of water pollution. Depletion takes place due to microbes consuming organic matter in water via aerobic respiration. This type of respiration uses oxygen as an electron accepter and organic material being consumed provides the energy source. Since O₂ content is important for may biological and chemical processes, measurements of amount of O₂ actually dissolved in a water sample is of great importance. BOD test relates to amount of O₂ that would be required to stabilize waste after discharging to a receiving water body. BOD is a parameter of great concern. Failing to realize importance of BOD in wastewater/effluent treatment systems can lead to devastating effects on local aquatic ecology and quality of underlying groundwater.

Monitoring BOD removal a treatment plant is necessary to verify proper operation. BOD test is typically performed in municipal or industrial wastewater plant. The results of BOD analysis are used to calculate the degree of pollution and to determine the effectiveness of water treatment by waste water and sewage plant. Different organic compounds show different oxygen demand (mg/l), thus the BOD test only gives an approximate idea of the weight of oxidizable organic matter. BOD test is carried out using standard methods as prescribed in APHA (Standard Method for the Examination of Water and Wastewater, 20^th edn. 5-1-5-6. Baltimore). Test substances and standard substances are dissolved in BOD dilution water. Standard substrate is made of 0.15 mg/l glucose and 0.15 mg/l glutamic acid, which has a calculated BOD of 220 mgL⁻¹. It is expressed in terms of Dissolved Oxygen (DO), which microorganisms, mainly bacteria will consume while degrading organic material in a sample of water under standardized conditions of pH nutrient and microorganisms. The amount of oxygen that dissolves in the water depends on many factors: whether there is adequate time and adequate mixing to fully saturate the water, the water temperature, the air pressure, the salt content of the water, and whether there are substances in the water which consume the O₂

Microorganisms either are present in the water sample or are introduced by taking a small quantity of a suitable microbial source such as settled sewage. The inoculant is called BOD ‘seed’ and the process, ‘seeding’. Since BOD analysis relies on a biological process, there is a greater variance in results then would normally be expected in a strictly chemical assay. The Standard methods for the Examination of Water and Wastewater indicated an acceptable range of ±15% at the 200 mg/l level for the reference GGA (Glucose-Glutamic Acid) solution; using the results from a series of Interlaboratory studies.

For all sources of seed, the possibility exists that some wastes will cause poisoning of the microorganisms. Some wastes will have develop3ed microorganisms adapted to the toxic conditions and hence give expected BOD results. But, in other wastes the microorganisms will adapt over the period of the BOD test. Because of the lag time involved in adaptation, a lower BOD is obtained than might be excepted. If the toxicity is sufficiently acute, a zero or close to zero result is obtained. Further, the ratio of various species of bacteria normally added can change in a 5-day period. BOD is the result of a summation of the oxygen demand of these microorganisms, whose contribution to the oxygen demand will change with time because of the changing population and changing feedstock.

Among the major industries in India, Pulp and Paper is one of those that contribute heavily to water pollution. Pollutants that generally arise from the industry include wood sugars, cellulose, fiber, lignin and other spent chemicals, which impart high BOD, COD, colour, etc. to the effluent. Thus, there arises a need to develop specific seeding (comprising of selected, acclimatized and autochthonous bacterial strains) for analyzing the BOD load of these industrial wastewaters.

In addition, some of these compounds are refractory to biodegradation because of high molecular weight coupled with lesser bioavailability. The BOD analysis of such types of wastewaters poses acute problems because of many reasons, which include the heterogeneity of the samples from time to time, non-specific microorganisms present in general seeding material and lower biodegradation rate of the organic constituents present therein.

The aforesaid problems can be overcome by formulating a uniform microbial composition comprising selected isolated bacterial strains, acclimatized to pulp and paper wastewater. Further, these bacterial isolates must be specific for biodegradation of organic compounds present in these kinds of wastewater. General seeding materials viz., sewage, Polyseed, Bioseed and BODSEED™, when used for BOD analysis of above said wastewater does not work efficiently because of non-specificity of bacterial strains present therein. This leads to erroneous results, which differ from time to time. On the other hand, if specifically designed formulated microbial consortium comprising selected bacterial strains are used as seed for BOD analysis of above said effluents, it may yield reproducible and reliable results.

Thus, for solving the aforementioned problems, the applicants have realized that there exists a need to provide a process for the preparation of a microbial consortium, specifically formulated for use as seeding material for the BOD analysis of Pulp and paper wastewater.

OBJECT OF THE INVENTION

The main object of the invention is to provide a specific seeding mated for the BOD estimation of pulp and paper effluents.

Another object of the invention is to provide a microbial consortium exerting a synergistic effect for the assimilation of pulp and paper wastewater.

Another object of the invention is to provide a process for preparing a microbial consortium specifically formulated for use as seeding material in BOD analysis of pulp and paper wastewater.

SUMMARY OF THE INVENTION

The present invention relates to a process for development of a microbial composition consortium selected acclimatize bacterial Us, isolated from source habitat. The bacterial strains constituting THE microbial consortium are synergistic and exert an exact and reproducible oxygen demand for pulp and paper wastewater. The present invention provides a formulated microbial consortium and a process for the preparation of the said consortium, useful for reliable and reproducible BOD estimation of pulp and paper industrial wastewater.

Accordingly the present invention provides a synergistic microbial consortium for use as a seeding material for estimation of accurate and reproducible biochemical oxygen demand of pulp and paper industrial waste water, the composition comprising five bacterial strains, Micrococcus sp. (MTCC 6602), Staphylococcus sp. (MTCC 6603), Kurthia zopfii (MTCC 6604), Alcaligenes faecalis (MTCC 6719) and Pseudomonas aeruginosa (MTCC 6605) deposited at International Depository at IMTECH, Sector 39A, Chandigarh, India.

In one embodiment of the invention, bacterial strains are taken in equal proportions.

In another embodiment of the invention, the bacteria are isolated from activated sludge and soil samples collected from the vicinity of a selected pulp and paper mill.

In another embodiment of the invention, the characteristics of Micrococcus sp (MTCC 6602) are as follows: Gram—positive, Shape—cocci,

In another embodiment of the invention, the characteristics of Staphylococcus sp. (MTCC 6603) are as follows: Gram—positive, Shape—rods.

In another embodiment of the invention, the characteristics of Kurthia zopfii MTCC 6604) are as follows: Gram—Negative, Shape—rods.

In another embodiment of the invention, the characteristics of Alcaligenes faecalis (MTCC 6719) are as follows: Gram—Negative, Shape—rods,

In another embodiment of the invention, the characteristics of Pseudomonas aeruginosa (MTCC 6605) are as follows: Gram—Negative, Shape—rods.

The present invention also relates to a process for preparing a microbial consortium for use as a seeding material for estimation of accurate and reproducible biochemical oxygen demand of pulp and paper industrial waste water, the consortium comprising five bacterial strains, Micrococcus sp. (MTCC 6602), Staphylococcus sp. (MTCC 6603), Kurthia zopfii (MTCC 6604), Alcaligenes faecalis (MTCC 6719) and Pseudomonas aeruginosa (MTCC 6605) deposited at International Depository at IMTECH, Sector 39A, Chandigarh, India, the process comprising inoculating individual bacterial strains of bacteria separately in a nutrient broth, incubating the cultures, measuring cell colony growth at periodic intervals till required growth is attained, mixing individual cultures in desired proportions to form cell suspension, centrifuging cell suspension to obtain a cell pellet and washing the cell pellet, re-centrifuging the cell pellet to obtain a final cell pellet and suspending final cell pellet in phosphate buffer.

In yet another embodiment of the invention, cell colony growth is determined by measuring optical density of all cultures at 620 nm after stipulated time intervals till required growth where optical density is in the range of 1.0 to 1.2 at 650 nm is attained.

In another embodiment of the invention, cell cultures are incubated at a temperature in the range of 30-37° C. for approximately 12-18 hours and under gentle shaking.

In one embodiment of the invention, the nutrient broth contains per litre, 5.0 g of Peptic digest of a tissue; 5.0 g of Sodium chloride; 1.5 g of Beef extract; 1.5 g of Yeast extract and 0.1 ml Tween-80.

In another embodiment of the invention, the resultant cell suspension is centrifuged at an rpm in the range of 6000-8000 and for a period in the range of 15-25 minutes and at a temperature between 4-12° C.

In another embodiment of the invention, the cell pellet is washed by dissolving in 10-100 mM phosphate buffer having a pH in the range of 6.5-7.5 followed by vortexing for 35-45 seconds, followed by recentrifugation at in the range of 5000-8000 rpm for 15-25 minutes and at a temperature ranging between 4-10° C.

In another embodiment of the invention, the cell pellet washed once is given a second wash with 10-100 mM phosphate buffer having a pH in the range of 6.5-7.5.

In another embodiment of the invention, the cell pellet is suspended in 5.0-10.0 ml of 10-100 mM phosphate buffer having a pH in the range of 6.5-7.5.

In another embodiment of the invention, the cell pellet is washed twice by dissolving in minimum quantity of 50 mM phosphate buffer having a pH 6.8.

In another embodiment of the invention, the re-centrifugation of the washed cell pellet is carried out at a rpm of about 5000 rpm and for a period of about 20 minutes and at a temperature ranging between 4-10° C.

In a further embodiment of the invention, the recentrifuged cell pellet is suspended in desired volume of phosphate buffer and stored at a temperature of about 4° C., till use.

In one embodiment of the invention, bacterial strains are taken in equal proportions.

In another embodiment of the invention, the bacteria are isolated from activated sludge and soil samples collected from the vicinity of a selected pulp and paper mill.

In another embodiment of the invention, the characteristics of Micrococcus sp., (MTCC 6602) are as follows: Gram—positive, Shape—cocci.

In another embodiment of the invention, the characteristics of Staphylococcus sp. (MTCC 6603) are as follows: Gram—positive, Shape—rods.

In another embodiment of the invention, the characteristics of Kurthia zopfii (MTCC 6604) are as follows: Gram—Negative, Shape—rods.

In another embodiment of the invention, the characteristics of Alcaligenes faecalis (MTCC 6719) are as follows; Gram—Negative, Shape—rods.

In another embodiment of the invention, the characteristics of Pseudomonas aeruginosa (MTCC 6605) are as follows: Gram—Negative, Shape—rods.

The present invention also provides a method for the estimation of biochemical oxygen demand of pulp and paper industrial wastewater using a consortium comprising five bacterial strains, Micrococcus sp. (MTCC 6602), Staphylococcus sp. (MTCC 6603), Kurthia zopfii (MTCC 6604), Alcaligenes faecalis (MTCC 6719) and Pseudomonas aeruginosa (MTCC 6605) deposited at International Depository at IMTECH, Sector 39A, Chandigarh, India, the process comprising treating pulp and paper industrial wastewater with the microbial consortium and analysing the biochemical oxygen demand thereof.

In one embodiment of the invention, the bacterial strains are taken in equal proportions.

In another embodiment of the invention, the bacteria are isolated from activated sludge and soil samples collected from the vicinity of a selected pulp and paper mill.

In another embodiment of the invention, the characteristics of Micrococcus sp. (MTCC 6602) are as follows: Gram—positive, Shape—cocci.

In another embodiment of the invention, the characteristics of Staphylococcus sp. (MTCC 6603) are as follows: Gram—positive, Shape—rods.

In another embodiment of the invention, the characteristics of Kurthia zopfii (MTCC 6604) are as follows: Gram—Negative, shape—rods,

In another embodiment of the invention, the characteristics of Alcaligenes faecalis (MTCC 6719) are as follows, Gram—Negative, Shape—rods.

In another embodiment of the invention, the characteristics of Pseudomonas aeruginosa (MTCC 6605) are as follows: Gram—Negative, Shape—rods,

The present invention also provides a method for eating biochemical oxygen demand of pulp and paper industrial wastewater using a consortium comprising five bacterial strains, Micrococcus sp. (MTCC 6602), Staphylococcus sp. (MTCC 6603), Kurthia zopfii (MTCC 6604), Alcaligenes faecalis (MTCC 6719) and Pseudomonas aeruginosa (MTCC 6605), the process comprising:

• • a) isolating a range of bacterial strains from soil near selected pulp and paper industries;
  • b) culturing the strains on a nutrient medium to obtain pure cultures;
  • c) inoculating isolated bacterial strains individually in nutrient medium and incubating them at ambient temperature under gentle shaking for a period of 12-18 hrs;
  • d) observing optical density of the cultures at 620-650 nm till desired growth is attained;
  • e) harvesting grown cells obtained in step (d) by centrifuging for 15-25 min at a temperature in the range of 4 to 10° C. to obtain a cell pellet;
  • f) washing cell pellet obtained in step (e) by dissolving in 10-100 mM phosphate buffer having pH in the range of 6.5-7.5 followed by recentrifugation;
  • g) repeating step (f) for giving a second washing to the obtained cell pellet;
  • h) dissolving pellet obtained from step (g) in desired volume of 10-100 mM phosphate buffer with pH in range of 6.5-7.5 to obtain a seed for BOD analysis of pulp and paper wastewater;
  • i) testing individual pure cultures of isolated bacterial strains obtained in step (h) for analyzing BOD of pulp and paper industrial wastewater;
  • j) verifying BOD values obtained in step (i) using a reference standard;
  • k) comparing the BOD values obtained in step (i) and (j) with values obtained using a synthetic seed;
  • l) select bacterial strains which have BOD values equal to or more than BOD values obtained using the synthetic seed as seeding material observed in step (k);
  • m) bacterial strains selected in step (l) in various combinations to obtain different microbial consortia;
  • n) obtaining cell pellet of each microbial consortium following the method of steps c to h;
  • o) testing seeds as obtained in step (n) for BOD analysis of pulp and paper wastewater,
  • p) selecting the optimal seed from results as obtained in step (o).

In one embodiment of the invention, the cell slurry of individual isolated bacterial strains is tested as seed for BOD analysis of pulp and paper wastewater.

In another embodiment of the invention, the BOD values obtained using seeds are verified using Glucose-Glutamic acid (GGA) as a reference standard.

In one embodiment of the invention, the bacterial strains are taken in equal proportions.

In another embodiment of the invention, the bacteria are isolated from activated sludge and soil samples collected from the vicinity of a selected pulp and paper mill.

In another embodiment of the invention, the characteristics of Micrococcus sp. (MTCC 6602) are as follows: Gram—positive, Shape—cocci.

In another embodiment of the invention, the characteristics of Staphylococcus sp. (MTCC 6603) are as follows: Gram—positive, Shape—rods.

In another embodiment of the invention, the characteristics of Kurthia zopfii MTCC 6604) are as follows: Gram—Negative, Shape—rods.

In another embodiment of the invention, the characteristics of Alcaligenes faecalis (MTCC 6719) are as follows: Gram—Negative, Shape—rods.

In another embodiment of the invention, the characteristics of Pseudomonas aeruginosa (MTCC 6605) are as follows: Gram—Negative, Shape—rods,

DETAILED DESCRIPTION OF THE INVENTION

The specific seeding material provided according to the present invention is a formulated microbial consortium, which contains bacteria consisting of:

Sl. No.	Culture	Identification no.	Deposition No.
1	Micrococcus sp.	MTCC 6602	Awaited
2	Staphylococcus sp	MTCC 6603	Awaited
3	Kurthia zopfii	MTCC 6604	Awaited
4	Alcaligenes faecalis	MTCC 6719	Awaited
5	Pseudomonas aeruginosa	MTCC 6605	Awaited

and which facilitate BOD analysis of pulp and paper wastewater, giving accurate and reproducible BOD values of these wastewaters, performed at any place. The above mentioned bacterial strains are deposited at the International Depository at IMTECH, Sector 39A, Chandigarh, India recognized by Budapest Treaty and will be available to public on request as per the normal official procedures. The main characteristic features of all bacterial isolates used for the present invention are given below: Micrococcus sp. (MTCC 6602)

Micrococcus sp. (MTCC 6602) is aerobic in nature, is gram positive, motile, capable of growth at pH 8.00 and in NaCl (8.5%) and shows opium growth at 37° C. and is also capable of hydrolyzing urea and starch.

Staphylococcus sp. (MTCC 6603)

Staphylococcus sp. (MTCC 6603) is facultative aerobic in nature, is gram positive, non-motile, shows optimum growth at 42° C., is also capable of growth at a pH of up to 11.00 and is capable of utilizing cellobiose and salicin.

Kurthia Zopfli (MTCC 6604)

Kurthia zopfii (MTCC 6604) is facultative aerobic in nature, is gram positive, motile, shows optimum growth at 30° C. and is capable of growth at high pH (11.00) and is capable of utilizing cellobiose and raffinose.

Alcaligenes faecalis (MTCC 6719)

Alcaligenes faecalis (MTCC 6719) is aerobic in nature, is gram negative, motile, shows optimum growth at 37° C. and is positive for cytochrome oxidase and catalase test and is also capable of utilizing dextrose and galactose as carbon source.

Pseudomonas aeruginosa (MTCC 6605)

Pseudomonas aeruginosa (MTCC 6605) is aerobic in nature, is gram negative, motile, fluorescent, shows optimum growth at 40° C., is capable of utilizing arabinose, dextrose, fructose, galactose, mannitol, mannose and xylose.

The formulated microbial consortium preferably contains the bacteria in uniform amounts. The microbial consortium of the invention is useful as seeding material for the BOD analysis of especially, pulp and paper wastewater. The bacterial cultures of the above seeding material are isolated from selected source habitats. The source habitats are soils near pulp and paper industry, which have accumulated industrial wastewater for several years.

The specifically formulated seeding material is prepared by inoculating individual bacterial strains of the above mentioned bacteria separately in nutrient broth containing (per litre), 5.0 g of Peptic digest of animal tissue; 5.0 g of Sodium chloride; 1.5 g of Beef extract; 1.5 g of Yeast extract and 0.1 ml Tween-80. Incubation of all cultures is done preferably at a temperature of 37° C. for approximately 12-18 hours, under gentle shaking. Optical density of all cultures is measured at 620 nm after stipulated time intervals. After attaining required growth (O.D.=1.0-1.2 at 650 nm) in terms of optical density, individual cultures are mixed in desired proportions to formulate different microbial consortia.

The resultant cell suspension is centrifuged at an appropriate rpm (6000-8000) for a period of 20 minutes at a temperature between 4-12° C. The obtained cell pellet is washed twice by dissolving in minimum quantity of 50 mM phosphate buffer, pH 6.8 and recentrifuged at an appropriate rpm, preferably at 5000 rpm for a period of approximately 20 min at a temperature ranging between 4-10° C. The final cell pellet thus obtained is suspended n desired volume of phosphate buffer and stored at a temperature, preferably 4° C., till used.

The formulated microbial consort prepared in the above manner can be used as seed inoculum for the BOD analysis of specifically pulp and paper wastewater, using Glucose-Glutamic acid (GGA) as a pence standard. For this, desired aliquots of the prepared cell suspension, was added as seeding material in the dilution water for BOD. In the invention, BOD analysis was performed as per the method described in the Standard Method for the Examination of Water and wastewater (APHA, 1998).

The invention provides a microbial consortium comprising of a synergistic mixture of at least the following isolated bacterial strains present in equal proportions useful for analyzing the biochemical oxygen demand of especially, pulp and paper wastewater. The invention also provides a process for preparing the microbial formulation, which comprises:

• • a) isolating a range of bacterial isolates from soils near selected pulp and paper industries;
  • b) culturing the isolated bacterial isolates on nutrient medium (Ingredients: peptic digest of animal tissue −5 g/l; Sodium Chloride −5 g/l ; Beef extract −1.5 g/l and Yeast extract −1.5 g/l) to obtain pure cultures;
  • c) inoculating the isolated bacterial isolates individually in nutrient medium (Ingredients: peptic digest of animal tissue −5 g/l; Sodium Chloride −5 g/l; Beef extract −1.5 g/l and Yeast extract −1.5 g/l) and incubating them at an ambient temperature under gene shaking for a period of 12-18 hrs;
  • d) observing the optical density of the grown cultures at 620 nm;
  • e) harvesting the cells obtained in step (d) by centrifuging for 15-25 min at a temperature preferably at 4° C.;
  • f) washing the cell pellet obtained in step (e) by dissolving in 10-100 mM phosphate buffer, pH 6.5-7.5 followed by recentrifugation;
  • g) repeating step (f) for giving a second washing to the obtained cell;
  • h) dissolving the pellet obtained from step (g) in desired volume of 10-100 mM phosphate buffer, pH 6.5-7.5 to obtain the desired seed for BOD analysis of Pulp aid paper wastewater;
  • i) testing the individual pure cultures of the isolated bacterial strains as obtained in step (h) for analyzing the BOD of Pulp and paper industrial wastewater;
  • j) BOD estimation of pulp and paper industrial waster using specific bacterial isolates and bacteria present in BODEED
  • k) comparing the BOD values obtained in step (i) and (j) with those obtained using the synthetic seed viz., BODSEED™;
  • l) selecting the bacterial strains which have BOD values equal to more than the BOD values obtained using BODSEED as seeding material, as observed in step (k);
  • m) mixing the bacterial strains selected in step (l) in various combinations to obtain different microbial consortia;
  • n) obtaining the cell pellet of each microbial consortium in the manner as described in step c, d, e, f, g and h;
  • o) testing different microbial consortia as seeds as obtained in step (n) for the BOD analysis of pulp and paper industrial wastewater;
  • p) select the best seed from the results as obtained in step (o).

The microbial consortium comprises of a synergistic mixture of Micrococcus sp. (MTCC 6602), Staphylococcus sp. (MTCC 6603), Kurthia zopfii (MTCC 6604), Alcaligenes faecalis (MTCC 6719) and Pseudomonas aeruginosa (MTCC 6605). The bacterial strains are isolated from source habitats, which are soils near selected pulp and paper industry. The isolated bacterial strains were cultured on nutrient medium to obtain pure cultures. The individual bacterial strains isolated as stated above, were inoculated separately in nutrient medium followed by incubation at 30-37° C. for 12-18 hrs at 75-150 rpm. Optical density of the grown bacterial strains is observed at 620-650 nm. The obtained cell suspension having desired optical density is centrifuged at an appropriate rpm, preferably at 5000-8000 rpm for a period of approximately 15-25 min at a temperature ranging between 4-10° C. to harvest the cells. The harvested cell pellet is washed twice by dissolving in appropriate quantity of 10-100 mM phosphate buffer, pH 6.5-7.5, followed by centrifugation at an appropriate rpm in the range of 5000-8000 rpm for 15-25 min at a temperature preferably at 4° C. The washed cell pellet is dissolved in appropriate volume of 10-100 mM phosphate buffer, pH 6.5-7.5 to obtain the seed(s) for BOD analysis. The seeds thus obtained are tested for their efficiency towards BOD analysis of pulp and paper wastewater. BOD values of pulp and paper wastewater obtained using the seeds are compared with those using a synthetic seed viz., BODSEED™. The bacterial strains comprising the seeds, which exert BOD values equal to or more than BOD values observed using BODSEED™ are selected. The bacterial strains selected are then mixed in various combinations to obtain different microbial consortia. The cell pellet of each microbial consortium is obtained by the method described above. The microbial seeds thus obtained are tested for the BOD analysis of pulp and paper wastewater. The formulated microbial consortium exhibiting highest BOD for pulp and paper wastewater is selected for use as specific seeding material in BOD analysis of pulp and paper wastewater.

The invention is described with references to the following examples and should not be construed to limit the scope of the invention.

EXAMPLE 1

Isolation of the Bacterial Strains from the Source Habitat

Soil samples in vicinity of a selected pulp and paper Mill (Century Pulp & Paper Mill, LalKuan, Uttaranchal) were selected for isolation of bacterial strains. Different media were chosen for isolation, and are listed in Table 1. Collected soil samples were enriched for autochthonous bacterial population present therein by adding 10.0 g of collected soil sample in a medium containing 72% soil infusion, 20% nutrient broth, 0.1% lignin, 0.1% vanillin and 0.1% tannin. This suspension was incubated at 37° C. for 48-50 hrs under gentle shaking. The obtained enriched suspension was used for isolating bacterial strains using serial dilution method. Serial dilutions for this purpose were prepared till concentration of 10⁻¹² in 50 mM phosphate buffer, pH 6.8. Appropriate aliquots of each dilution were plated on different media (listed in Table 1) and plates were incubated at 37° C. for 20-24 hrs in upright position. The number of various bacterial isolates appearing as colonies were noted and further purified for use and storage (see Table 2).

TABLE 1
Various Media Used for Isolating Bacterial Strains from Source Habitat
Sl. No.	Medium	Medium Composition
1.	ASM1	sludge infusiona
2.	SM1	soil infusionb
3.	ASM2	sludge infusion + 0.1% lignin
4.	SM2	soil infusion + 0.1% lignin
5.	ASM3	50% sludge infusion + 50% Nutrient broth
6.	SM3	50% soil infusion + 50% Nutrient broth
7.	M4	Pulp and paper wastewater
8.	M5	Nutrient broth + 0.1% lignin
9.	M6	Nutrient Broth
10.	ASM7	Sludge infusion - 400 ml; Yeast extract - 3.0 g;
		Glucose - 7.5 g; K2HPO4 - 0.5 g; KH2PO4 - 0.5 g;
		MgSO4 - 0.5 g; distilled water - 600 ml and
		lignin - 1.0
11.	SM7	Soil infusion - 400 ml; Yeast extract - 3.0 g;
		Glucose - 7.5 g; K2HPO4 - 0.5 g; KH2PO4 - 0.5 g
		MgSO4 - 0.5 g; distilled water - 600 ml and
		lignin - 1.0

TABLE 2
Number of Various Bacterial Isolates that Appeared on Different Nutrient Media
	Number of bacterial strains isolated
	Source: Activated sludge	Source: Soil Sample
	Medium	Dilutions at which bacteria are isolated
Sl. No.	No.	10−2	10−4	10−6	10−7	10−8	10−9	10−2	10−4	10−6	10−7	10−8	10−9
1.	ASM1			4	2
2.	SM1								3			1	1
3.	ASM2		1	3	3
4.	SM2									5	1	3	2
5.	ASM3			1	1	1
6.	SM3									2	5	3
7.	M4			2	2	1				3	2	4
8.	M5		3	3	4					2	3	1
9.	M6		1	1	6	4				4	3		1
10.	ASM7			3	4	1
11.	SM7								2	1	1	3

EXAMPLE 2

Activated sludge samples from a selected pulp and paper mill (Century Pulp & Paper Mill, LalKuan, Uttaranchal) were selected for isolation of bacterial strains. Different media were chosen for isolation and are listed in Table 1. The collected samples were enriched for the autochthonous bacterial population present therein, by adding 10.0 ml of the sludge sample in a medium containing 72% soil infusion, 20% nutrient broth, 0.1% lignin, 0.1% vanillin and 0.1% tannin. This suspension was incubated at 37° C. for 48-50 hrs under gentle shaking. The obtained enriched suspension was used for isolating the bacterial strains using the serial dilution method. Serial dilutions for this purpose were prepared from 10⁻¹ to 10⁻¹² in 50 mM phosphate buffer, pH 6.8, 100 μl of each dilution were plated on different media (as listed in Table 1) and the plates were incubated at 37° C. for 20-24 hrs in an upright position. The number of various bacterial isolates hat appeared as colonies were noted and further purified for use and storage (see Table 2).

EXAMPLE 3

BOD Analysis of Pulp and Paper Wastewater Sample Using Individual Isolated Bacterial Strains

Some of the bacterial strains were chosen randomly from the total isolated strains on the basis of their growth rate for use as seeding material for BOD analysis of pulp and paper wastewater. Those bacterial strains having a comparatively faster growth rate and thus a short generation time were selected for further set of experiments.

		Measurement of bacterial growth in
	Isolate	terms of optical density at 650 nm
	No.	0 hr.	4 hr.	8 hr.	12 hr.	16 hr.
	1.	0.040	0.075	0.232	1.102	1.50
	2.	0.042	0.066	0.183	1.125	1.45
	3.	0.041	0.067	0.154	1.154	1.32
	4.	0.048	0.066	0.212	1.168	1.48
	5.	0.044	0.069	0.163	0.994	1.25
	6.	0.056	0.068	0.178	0.962	1.58
	7.	0.055	0.070	0.118	0.976	1.57
	8.	0.057	0.069	0.142	0.951	1.21
	9.	0.054	0.068	0.107	0.945	1.23
	10.	0.047	0.067	0.189	0.986	1.25
	11.	0.041	0.069	0.227	1.011	1.33
	12.	0.042	0.071	0.257	1.059	1.35
	13.	0.039	0.072	0.109	1.045	1.45
	14.	0.054	0.073	0.127	1.064	1.38
	15.	0.053	0.074	0.147	0.983	1.56
	16.	0.045	0.072	0.106	1.008	1.58
	17.	0.046	0.071	0.208	0.895	1.31
	18.	0.051	0.068	0.155	0.913	1.21
	19.	0.050	0.066	0.126	0.962	1.35
	20.	0.049	0.067	0.135	0.951	1.45
	21.	0.043	0.069	0.168	0.824	1.58
	22.	0.046	0.070	0.154	0.807	1.46
	23.	0.040	0.071	0.173	0.813	1.47
	24.	0.041	0.075	0.152	0.882	1.37
	25.	0.045	0.076	0.202	0.895	1.40
	26.	0.044	0.077	0.214	0.859	1.29
	27.	0.047	0.076	0.192	0.825	1.24
	28.	0.049	0.075	0.182	0.865	1.26
	29.	0.047	0.078	0.114	0.904	1.27
	30.	0.051	0.079	0.124	0.925	1.22
	31.	0.052	0.080	0.133	0.936	1.23
	32.	0.050	0.082	0.152	0.984	1.21
	33.	0.051	0.081	0.141	0.995	1.35
	34.	0.053	0.078	0.163	0.952	1.38
	35.	0.054	0.076	0.201	0.945	1.39
	36.	0.055	0.074	0.152	1.009	1.40
	37.	0.057	0.075	0.174	1.056	1.41
	38.	0.057	0.076	0.164	1.066	1.25
	39.	0.056	0.071	0.136	1.089	1.54
	40.	0.059	0.069	0.153	1.100	157
	41.	0.058	0.068	0.104	1.114	1.59
	42.	0.057	0.067	0.131	1.010	1.51
	43.	0.056	0.066	0.147	1.028	1.49
	44.	0.058	0.069	0.154	1.158	1.45
	45.	0.060	0.070	0.162	1.163	1.43
	46.	0.061	0.071	0.171	1.190	1.53
	47.	0.059	0.073	0.236	1.177	1.32
	48.	0.062	0.0742	0.207	1.184	1.38
	49.	0.063	0.071	0.220	1.203	1.29
	50.	0.064	0.072	0.252	1.152	1.36
	51.	0.061	0.074	0.241	1.098	1.34
	52.	0.063	0.075	0.125	1.087	1.21
	53.	0.059	0.076	0.133	1.029	1.22
	54.	0.058	0.077	0.175	1.088	1.23
	55.	0.056	0.072	0.151	0.996	1.24
	56.	0.057	0.067	0.140	0.975	1.25
	57.	0.055	0.070	0.193	0.936	1.26
	58.	0.053	0.070	0.186	0.826	1.27
	59.	0.050	0.071	0.116	0.916	1.28
	60.	0.048	0.072	0.164	1.158	1.29
	61.	0.045	0.073	0.136	0.989	1.27
	62.	0.047	0.075	0.141	0.879	1.25
	63.	0.048	0.077	0.200	0.817	1.30
	64.	0.043	0.078	0.222	0.984	1.45
	65.	0.042	0.075	0.230	0.911	1.56
	66.	0.045	0.073	0.197	0.895	1.25
	67.	0.041	0.074	0.173	0.888	1.33
	68.	0.040	0.076	0.150	0.994	1.53
	69.	0.039	0.078	0.224	0.851	1.54
	70.	0.042	0.080	0.212	0.818	1.47
	71.	0.043	0.079	0.243	0.874	1.56
	72.	0.045	0.081	0.141	0.981	1.57
	73.	0.042	0.077	0.168	1.016	1.58
	74.	0.041	0.075	0.173	1.035	1.36
	75.	0.043	0.073	0.19	1.027	1.38
	76.	0.045	0.068	0.152	1.048	1.37
	77.	0.044	0.066	0.131	1.058	1.31
	78.	0.048	0.070	0.114	1.064	1.32
	79.	0.047	0.068	0.106	1.099	1.34
	80.	0.046	0.067	0.120	1.111	1.35
	81.	0.045	0.069	0.151	1.050	1.56
	82.	0.049	0.071	0.135	1.077	1.45
	83.	0.050	0.073	0.143	1.088	1.50
	84.	0.052	0.072	0.172	1.098	1.40
	85.	0.053	0.077	0.202	1.100	1.59
	86.	0.054	0.075	0.212	1.119	1.58
	87.	0.051	0.074	0.233	1.126	1.57
	88.	0.049	0.078	0.162	0.838	1.54
	89.	0.053	0.079	0.182	0.869	1.30
	90.	0.054	0.075	0.145	0.876	1.36
	91.	0.055	0.068	0.171	0.899	1.51
	92.	0.057	0.066	0.224	0.950	1.57
	93.	0.058	0.067	0.203	0.965	1.49
	94.	0.059	0.070	0.194	0.977	1.42
	95.	0.060	0.073	0.133	1.153	1.53
	96.	0.061	0.074	0.158	1.146	1.55
	97.	0.062	0.076	0.214	1.176	1.56
	98.	0.063	0.082	0.253	1.184	1.57
	99.	0.064	0.081	0.231	1.120	1.58
	0	0.045	0.060	0.141	1.190	1.30

EXAMPLE 4

The bacterial strains selected as stated above were individually inoculated in 50 ml of nutrient broth having 0.01% Tween-80, All the cultures were incubated at 37° C. for 16-20 hrs in an incubator shaker at 120 rpm. Optical density of all the cultures was maintained to 1.0±0.1 (at 620 nm). Cells were harvested by centrifugation at 5000 rpm for 15-20 min at a temperature ranging between 4° C. The pellet thus obtained was washed twice with 50 mM phosphate buffer, pH 6.8 by suspending it in 5.0-10.0 ml of the same buffer, vortexing for 30-45 sec followed by centrifugation at 5000 rpm for 20 min at 4° C. The cell pellet of individual bacterial isolates thus obtained was resuspended in 5.0 ml of the same buffer and used as seeding material @ 0.1% and 0.2% for the BOD analysis of pulp and paper wastewater using GGA as a reference standard. The results of the study are presented in Table 3.

TABLE 3
BOD analysis of pulp and paper wastewater sample
using individual isolated bacterial strains
	BOD (mg/l)
			Pulp and paper
			wastewater
		GGA: 300 mg/l	(COD: 240 mg/l)
		BOD: 204 mg/l	BOD: 80 mg/l by
	Bacterial	by BODSEED	BODSEED ™
Sl. No.	Isolates	0.1% seed	0.2% seed	0.1% seed	0.2% seed
1.	Isolate 1	175	177	130	132
2.	Isolate 2	169	165	118	116
3.	Isolate 4	179	152	121	121
4.	Isolate 6	182	182	130	108
5.	Isolate 7	160	165	101	112
6.	Isolate 13	184	179	124	126
7.	Isolate 14	171	178	136	130
8.	Isolate 15	178	171	126	120
9.	Isolate 16	166	173	110	118
10.	Isolate 20	204	200	108	112
11.	Isolate 21	157	151	100	108
12.	Isolate 22	87	80	05	10
13.	Isolate 23	90	92	08	24
14.	Isolate 24	95	107	07	21
15.	Isolate 25	110	101	11	30
16.	Isolate 35	108	121	24	41
17.	Isolate 36	125	120	32	52
18.	Isolate 37	112	118	60	64
19.	Isolate 39	186	180	85	87
20.	Isolate 40	181	179	88	80
21.	Isolate 41	185	188	90	95
22.	Isolate 42	192	190	95	97
23.	Isolate 43	190	185	109	101
24.	Isolate 44	70	76	41	43
25.	Isolate 45	195	197	117	120
26.	Isolate 46	190	184	116	120
27.	Isolate 64	171	168	125	115
28.	Isolate 65	78	60	20	30
29.	Isolate 68	75	80	28	32
30.	Isolate 69	168	173	121	128
31.	Isolate 70	171	178	124	121
32.	Isolate 71	180	168	113	121
33.	Isolate 72	171	172	99	108
34.	Isolate 73	177	170	98	108
35.	Isolate 81	140	135	21	28
36.	Isolate 82	175	171	112	113
37.	Isolate 83	168	170	108	109
38.	Isolate 84	131	121	31	39
39.	Isolate 85	127	120	55	50
40.	Isolate 86	105	113	51	59
41.	Isolate 87	101	91	60	68
42.	Isolate 88	170	172	121	124
43.	Isolate 91	173	177	117	117
44.	Isolate 92	168	168	110	108
45.	Isolate 93	92	100	34	36
46.	Isolate 94	97	105	51	55
47.	Isolate 95	68	70	62	38
48.	Isolate 96	71	78	36	46
49.	Isolate 97	82	90	41	34
50.	Isolate 98	87	81	48	50
51.	Isolate 99	101	107	52	52

EXAMPLE 5

BOD Analysis of Pulp and Paper Wastewater Using Different Formulated Microbial Consortium

Out of the total individual bacterial isolates used for BOD analysis, as described in Example 3, 27 isolates were selected for the formulation of 44 microbial consortia (Table 4) Those individual bacterial isolates, which exhibited BOD values higher to or comparable to BODSEED™ were chosen.

TABLE 4
Microbial consortia as made from selected bacterial isolates
Sl. No.	Microbial consortium	Selected Isolates
1.	Consortium 1 (Seed)	Isolate 2, Isolate 69 and Isolate 88
2.	Consortium 2 (Seed)	Isolate 20, Isolate 43 and Isolate 64
3.	Consortium 3 (Seed)	Isolate 71, Isolate 88 and Isolate 92
4.	Consortium 4 (Seed)	Isolate 1, Isolate 64 and Isolate 71
5.	Consortium 5 (Seed)	Isolate 7, Isolate 21 and Isolate 43
6.	Consortium 6 (Seed)	Isolate 6, Isolate 20 and Isolate 66
7.	Consortium 7 (Seed)	Isolate 15, Isolate 21 and Isolate 92
8.	Consortium 8 (Seed)	Isolate 46, Isolate 71 and Isolate 16
9.	Consortium 9 (Seed)	Isolate 13, Isolate 43 and Isolate 43
10.	Consortium 10 (Seed)	Isolate 4, Isolate 15 and Isolate 91
11.	Consortium 11 (Seed)	Isolate 21, Isolate 73 and Isolate 88
12.	Consortium 12 (Seed)	Isolate 13, Isolate 15 and Isolate 91
13.	Consortium 13 (Seed)	Isolate 45, Isolate 46 and Isolate 88
14.	Consortium 14 (Seed)	Isolate 43, Isolate 73 and Isolate 83
15.	Consortium 15 (Seed)	Isolate 6, Isolate 88 and Isolate 92
16.	Consortium 16 (Seed)	Isolate 72, Isolate 91 and Isolate 92
17.	Consortium 17 (Seed)	Isolate 16, Isolate 45 and Isolate 88
18.	Consortium 18 (Seed)	Isolate 6, Isolate 46 and Isolate 82
19.	Consortium 19 (Seed)	Isolate 72, Isolate 92 and Isolate 91
20.	Consortium 20 (Seed)	Isolate 21, Isolate 46 and Isolate 88
21.	Consortium 21 (Seed)	Isolate 20, Isolate 83 and Isolate 91
22.	Consortium 22 (Seed)	Isolate 6, Isolate 64 and Isolate 70
23.	Consortium 23 (Seed)	Isolate 64, Isolate 73 and Isolate 83
24.	Consortium 24 (Seed)	Isolate 20, Isolate 88 and Isolate 92
25.	Consortium 25 (Seed)	Isolate 13, Isolate 45 and Isolate 73
26.	Consortium 26 (Seed)	Isolate 82, Isolate 92 and Isolate 88
27.	Consortium 27 (Seed)	Isolate 2, Isolate 71 and Isolate 92
28.	Consortium 28 (Seed)	Isolate 21, Isolate 69 and Isolate 71
29.	Consortium 29 (Seed)	Isolate 14, Isolate 15 and Isolate 45
30.	Consortium 30 (Seed)	Isolate 1, Isolate 73 and Isolate 83
31.	Consortium 31 (Seed)	Isolate 43, Isolate 71 and Isolate 92
32.	Consortium 32 (Seed)	Isolate 21, Isolate 43 and Isolate 45
33.	Consortium 33 (Seed)	Isolate 21, Isolate 82and Isolate 99
34.	Consortium 34 (Seed)	Isolate 20, Isolate 70 and Isolate 46
35.	Consortium 35 (Seed)	Isolate 64, Isolate 88 and Isolate 92
36.	Consortium 36 (Seed)	Isolate 2, Isolate 70 and Isolate 92
37.	Consortium 37 (Seed)	Isolate 20, Isolate 91 and Isolate 92
38.	Consortium 38 (Seed)	Isolate 46, Isolate 69 and Isolate 83
39.	Consortium 39 (Seed)	Isolate 14, Isolate 64 and Isolate 73
40.	Consortium 40 (Seed)	Isolate 46, Isolate 92 and Isolate 88
41.	Consortium 41 (Seed)	Isolate 2, Isolate 45 and Isolate 82
42.	Consortium 42 (Seed)	Isolate 43, Isolate 83 and Isolate 91
43.	Consortium 43 (Seed)	Isolate 4, Isolate 45 and Isolate 82
44.	Consortium 44 (Seed)	Isolate 73, Isolate 88 and Isolate 92

EXAMPLE 6

The individual bacterial strains comprising the said microbial consortium were inoculated separately in 50 ml of nutrient broth having 0.01% Tween-80. All the cultures were incubated at 37° C. for 16-20 hrs in an incubator shaker at 120 rpm. Optical density of all the cultures was maintained to 1.0±0.1 (at 620 nm). The individual cell suspensions were mixed in desired proportions to obtain different microbial consortia. Cells of each microbial consortium were harvested by centrifugation at 5000-7000 rpm for 15-20 min at a temperature rid between 4-10° C. The pellet thus obtained was washed twice with 50 mM phosphate buffer, pH 6.8 by suspending it in 5.0-10.0 ml of the same buffer, vortexing for 30-45 sec followed by centrifugation at 5000-7000 rpm for 20 min at 4° C. The cell pellet thus obtained was resuspended in 5.0 ml of the same buffer and used as seeding material @ 0.1% for the BOD analysis of pulp and paper wastewater using GGA as a reference standard. The results of the BOD analysis performed using the microbial seeds obtained in the above said manner are illustrated in Table 5.

TABLE 5
BOD analysis of pulp and paper wastewater
using different formulated microbial consortia
	BOD (mg/l)
			Pulp and paper
			wastewater
			(COD: 410 mg/l)
			BOD: 130 mg/l
Sl. No.	Microbial consortium	GGA: 300 mg/l	by BODSEED
1.	Consortium 1 (Seed)	119	77
2.	Consortium 2 (Seed)	189	92
3.	Consortium 3 (Seed)	118	76
4.	Consortium 4 (Seed)	147	54
5.	Consortium 5 (Seed)	182	79
6.	Consortium 6 (Seed)	146	99
7.	Consortium 7 (Seed)	179	99
8.	Consortium 8 (Seed)	150	72
9.	Consortium 9 (Seed)	150	93
10.	Consortium 10 (Seed)	84	45
11.	Consortium 11 (Seed)	124	57
12.	Consortium 12 (Seed)	118	66
13.	Consortium 13 (Seed)	128	60
14.	Consortium 14 (Seed)	128	52
15.	Consortium 15 (Seed)	97	60
16.	Consortium 16 (Seed)	05	03
17.	Consortium 17 (Seed)	131	117
18.	Consortium 18 (Seed)	165	145
19.	Consortium 19 (Seed)	139	103
20.	Consortium 20 (Seed)	131	91
21.	Consortium 21 (Seed)	140	101
22.	Consortium 22 (Seed)	154	78
23.	Consortium 23 (Seed)	172	157
24.	Consortium 24 (Seed)	156	135
25.	Consortium 25 (Seed)	140	193
26.	Consortium 26 (Seed)	147	157
27.	Consortium 27 (Seed)	153	137
28.	Consortium 28 (Seed)	136	131
29.	Consortium 29 (Seed)	154	100
30.	Consortium 30 (Seed)	160	82
31.	Consortium 31 (Seed)	153	107
32.	Consortium 32 (Seed)	158	123
33.	Consortium 33 (Seed)	144	90
34.	Consortium 34 (Seed)	164	73
35.	Consortium 35 (Seed)	111	112
36.	Consortium 36 (Seed)	120	145
37.	Consortium 37 (Seed)	144	128
38.	Consortium 38 (Seed)	150	154
39.	Consortium 39 (Seed)	168	178
40.	Consortium 40 (Seed)	168	135
41.	Consortium 41 (Seed)	170	172
42.	Consortium 42 (Seed)	154	124
43.	Consortium 43 (Seed)	143	120
44.	Consortium 44 (Seed)	158	140

EXAMPLE 7

Some of the microbial consortia used for the BOD analysis (described in Example 6) were selected for further set of experiments on the basis of their ability to biodegrade the constituents of pulp and paper wastewater, thereby exerting an oxygen demand comparable to or higher than that exerted by BODSEED™. The selected microbial consortia were again tested for the BOD analysis of a fresh lot of pulp and paper wastewater. The resulting BOD values are presented in Table 6.

TABLE 6
BOD analysis of pulp and paper wastewater
using selected microbial consortia
	BOD (mg/l)
			Pulp and paper
			wastewater
			(COD: 328 mg/l)
			BOD: 109 mg/l by
Sl. No.	Microbial consortium	GGA: 300 mg/l	BODSEED
1.	Consortium 2 (Seed)	176	66
2.	Consortium 6 (Seed)	168	101
3.	Consortium 7 (Seed)	176	97
4.	Consortium 9 (Seed)	194	94
5.	Consortium 17 (Seed)	142	119
6.	Consortium 18 (Seed)	148	156
7.	Consortium 23 (Seed)	141	153
8.	Consortium 24 (Seed)	127	142
9.	Consortium 25 (Seed)	123	152
10.	Consortium 26 (Seed)	126	147
11.	Consortium 27 (Seed)	128	156
12.	Consortium 28 (Seed)	141	150
13.	Consortium 32 (Seed)	154	152
14.	Consortium 35 (Seed)	146	143
15.	Consortium 36 (Seed)	151	154
16.	Consortium 38 (Seed)	158	159
17.	Consortium 39 (Seed)	161	164
18.	Consortium 41 (Seed)	160	162

EXAMPLE 8

Out of the 14 microbial consortia selected for BOD analysis (described in Example 7), 5 microbial consortia were selected, which exhibited the best values for pulp and paper wastewater. The results were repeated with three samples, collected at different time intervals for authentication (see Table 7).

TABLE 7
BOD analysis of pulp and paper wastewater
using best performing microbial consortia
	BOD (mg/l)*
			Pulp and paper
			wastewater
			(COD: 312 mg/l)
			BOD: 94 mg/l by
Sl. No.	Microbial consortium	GGA: 300 mg/l	BODSEED
1.	Consortium 23 (Seed)	144	150
2.	Consortium 25 (Seed)	134	154
3.	Consortium 38 (Seed)	159	159
4.	Consortium 39 (Seed)	160	162
5.	Consortium 41 (Seed)	168	165
*all values are mean of the analysis done with three different samples

EXAMPLE 9

Out of the best five microbial consortia, the following consortium was selected as an authenticated and best seeding material for the estimation of accurate and reproducible estimation of biochemical oxygen demand of pulp and paper industrial wastewater. The bacterial strains in this consortium were identified as Micrococcus sp. (MTCC 6602), Staphylococcus sp. (MTCC 6603), Kurthia zopfii (MTCC 6604), Alcaligenes faecalis (MTCC 6719) and Pseudomonas aeruginosa (MTCC 6605) which are deposited at International Depository at IMTECH, Sector 39A, Chandigarh, India

TABLE 8
BOD analysis of pulp and paper wastewater
using best performing microbial consortia
	BOD (mg/l)*
			Pulp and paper
			wastewater
			(COD: 312 mg/l)
	Microbial		BOD: 94 mg/l by
Sl. No.	consortium	GGA: 300 mg/l	BODSEED
1.	Consortium 41 (Seed)	168	165
*all values are mean of the analysis done with three different samples

Advantages

• • 1. The selected formulated microbial consortium comprising of the isolated bacterial strains act in a synergistic way and is capable of degrading the easily assimilable as well as the refractory organic compounds present in Pulp and paper wastewater.
  • 2. These types of seeds will detail the quantity of organic compounds contained in these wastes, which is released unnoticed when the wastes are analyzed for their BOD load using the general seeds. This leads to accurate BOD values of these types of wastewaters, which is difficult to achieve with the microorganisms present in general seeding material.
  • 3. The use of such specific seeding material for the BOD analysis of Pulp and paper wastewaters will lead to a better control of the treatment process of these types of wastewaters.

Claims

1. A synergistic microbial consortium for use as a seeding material for estimation of accurate and reproducible biochemical oxygen demand of pulp and paper industrial waste water, the composition comprising five bacterial strains, Micrococcus sp. (MTCC 6602), Staphylococcus sp. (MTCC 6603), Kurthia zopfii (MTCC 6604), Alcaligenes faecalis (MTCC 6719) and Pseudomonas aeruginosa (MTCC 6605) deposited at International Depository at IMTECH Sector 39A, Chandigarh, India.

2. A microbial consortium as claimed in claim 1 wherein the baronial strains are taken in equal proportions.

3. A microbial consortium as claimed in claim 1 wherein the bacteria are isolated from activated sludge and soil samples collected from the vicinity of a selected pulp and paper mill.

4. A microbial consortium as claimed in claim 1 wherein characteristics of Micrococcus sp. (MTCC 6602) are as follows: Gram—positive, Shape—cocci.

5. A microbial consortium as claimed in claim 1 wherein characteristics of Staphylococcus sp. (MTCC 6603) are as follows: Gram—positive, Shape—rods.

6. A microbial consortium as claimed in claim 1 wherein characteristics of Kurthia zopfii (MTCC 6604) are as follows: Gram—Negative, Shape—rods.

7. A microbial consortium as claimed in claim 1 wherein characteristics of Alcaligenes faecalis (MTCC 6719) are as follows: Gram—Negative, Shape—rods.

8. A microbial consortium as claimed in claim 1 wherein characteristics of Pseudomonas aeruginosa (MTCC 6605) are as follows: Gram—Negative, Shape—rods.

9. A process for preparing a microbial consortium for use as a seeding material for estimation of accurate and reproducible biochemical oxygen demand of pulp and paper industrial waste water, the consortium comprising five bacterial strains, Micrococcus sp. (MTCC 6602), Staphylococcus sp. (MTCC 6603), Kurthia zopfii (MTCC 6604), Alcaligenes faecalis (MTCC 6719) and Pseudomonas aeruginosa (MTCC 6605) deposited at International Depository at IMTECH, Sector 39A, Chandigarh, India, the process comprising inoculating individual bacterial strains of the bacteria separately in a nutrient broth incubating the cultures, measuring cell colony growth at periodic intervals till required growth is attained, mixing the individual cultures in desired proportions to form a cell suspension, centrifuging the cell suspension to obtain a cell pellet and washing the cell pellet, re-centrifuging the cell pellet to obtain a final cell pellet and suspending the final cell pellet in phosphate buffer.

10. A process as claimed in claim 9 wherein the nutrient broth contains per litre, 5.0 g of Peptic digest of animal tissue; 5.0 g of Sodium chloride; 1.5 g of Beef extract, 1.5 g of Yeast extract and 0.1 ml Tween-80.

11. A process as claimed in claim 9 wherein the cell cultures are incubated at a temperature in the range of 30 to 37° C. for approximately 12-18 hours and under gentle shaking.

12. A process as claimed in claim 9 wherein cell colony growth is determined by measuring optical density of all cultures at 620-650 nm after stipulated time intervals till required growth where optical density is in the range of 1.0 to 1.2 at 650 nm is attained.

13. A process as claimed in claim 9 wherein the cell suspension is centrifuged at an rpm in the range of 6000-8000 and for a period in the range of 15-25 minutes and at a temperature ranging between 4-12° C.

14. A process as claimed in claim 9 wherein the cell pellet is washed by dissolving in 10-100 mM phosphate buffer having a pH in the range of 6.5-7.5 followed by vortexing for 35-45 seconds, followed by recentrifugation at in the range of 500-8000 rpm for 15-25 minutes and at a temperature ranging between 4-10° C.

15. A process as claimed in claim 14 wherein the cell pellet washed once is given a second wash with 10-100 mM phosphate buffer having a pH in the range of 6.5-7.5.

16. A process as claimed in claim 9 wherein the cell pellet is suspended in 5.0-10.0 ml of 10-100 mM phosphate buffer having a pH in the range of 6.5-7.5.

17. A process as claimed in claim 9 wherein the cell pellet is washed twice by dissolving in minimum quantity of 50 mM phosphate buffer having a pH 6.8.

18. A process as claimed in claim 9 wherein re-centrifugation of the washed cell pellet is carried out at a rpm of about 5000 rpm and for a period of about 20 minutes and at a temperature ranging between 4-10° C.

19. A process as claimed in claim 9 wherein the recentrifuged cell pellet is suspended in desired volume of phosphate buffer and stored at a temperature of about 4° C., till use.

20. A process as claimed in claim 9 wherein the bacterial strains are taken in equal proportions.

21. A process as claimed in claim 9 wherein the bacteria are isolated from activated sludge and soil samples collected from the vicinity of a selected pulp and paper mill located.

22. A process as claimed in claim 9 wherein characteristics of Micrococcus sp. (MTCC 6602) are as follows: Gram—positive, Shape—cocci.

23. A process as claimed in claim 9 wherein characteristics of Staphylococcus sp. (MTCC 6603) are as follows: Gram—positive, Shape—rods.

24. A process as claimed in claim 9 wherein characteristics of Kurthia zopfii (MTCC 6604) are as follows: Gram—Negative, Shape—rods.

25. A process as claimed in claim 9 wherein characteristics of Alcaligenes faecalis (MTCC 6719) are as follows: Gram—Negative, Shape—rods.

26. A process as claimed in claim 9 wherein characteristics of Pseudomonas aeruginosa (MTCC 6605) are as follows: Gram—Negative, Shape—rods.

27. A method for estimating biochemical oxygen demand of pulp and paper industrial wastewater using a consortium comprising five bacterial strains, Micrococcus sp. (MTCC 6602), Staphylococcus sp. (MTCC 6603), Kurthia zopfii (MTCC 6604), Alcaligenes faecalis (MTCC 6719) and Pseudomonas aeruginosa (MTCC 6605) deposited at International Depository at IMTECH, Sector 39A, Chandigarh, India, the process comprising treating pulp and paper industrial wastewater with the microbial consortium and analysing the biochemical oxygen demand thereof.

28. A method as claimed in claim 27 wherein the bacterial strains are taken in equal proportions.

29. A method as claimed in claim 27 wherein the bacteria are isolated from activated sludge and soil samples collected from the vicinity of a selected pulp and paper mill.

30. A method as claimed in claim 27 wherein characteristics of Micrococcus sp. (MTCC 6602) are as follows: Gram—positive, Shape—cocci.

31. A method as claimed in claim 27 wherein characteristics of Staphylococcus sp. (MTCC 6603) are as follows: Gram—positive, Shape—rods.

32. A method as claimed in claim 27 wherein characteristics of Kurthia zopfii (MTCC 6604) are as follows: Gram—Negative, Shape—rods.

33. A method as claimed in claim 27 wherein characteristics of Alcaligenes faecalis (MTCC 6719) are as follows: Gram—Negative, Shape—rods.

34. A method as claimed in claim 27 wherein characteristics of Pseudomonas aeruginosa (MTCC 6605) are as follows: Gram—Negative, Shape—rods.

35. A method for estimating biochemical oxygen demand of pulp and paper industrial wastewater using a consortium comprising five bacterial strains, Micrococcus sp. (MTCC 6602), Staphylococcus sp. (MTCC 6603), Kurthia zopfii (MTCC 6604), Alcaligenes faecalis (MTCC 6719) and Pseudomonas aeruginosa (MTCC 6605) deposited at International Depository at IMTECH, Sector 39A, Chandigarh, India, the process comprising: (a) isolating bacterial strains from soil mu sell pulp and paper industries; (b) culturing the strains on a nutrient medium to obtain pure cultures; (c) inoculating isolated bacterial strains individually in nutrient medium and incubating them at ambient temperature under gentle shaking for a period of 12-18 hrs; (d) observing optical density of the cultures at 620-650 nm till desired growth is attained; (e) harvesting grown cells obtained in step (d) by centrifuging for 15-25 min at a temperature in the range of 4 to 10° C. to obtain a cell pellet; (f) washing cell pellet obtained in step (e) by dissolving in 10-100 mM phosphate buffer having pH in the range of 6.5-7.5 followed by recentrifugation; (g) repeating step (f) for giving a second washing to the obtained cell pellet; (h) dissolving pellet obtained from step (g) in desired volume of 10-100 mM phosphate buffer with pH in range of 6.5-7.5 to obtain a seed for BOD analysis of pulp and paper wastewater; (i) testing individual pure cultures of isolated bacterial strains obtained in step (h) for analyzing BOD of pulp and paper industrial wastewater; (j) verifying BOD values obtained in step (i) using a reference standard; (k) comparing the BOD values obtained in step (i) and (j) with values obtained using a synthetic seed; (l) selecting bacterial strains which have BOD values equal to or more than BOD values obtained using the synthetic seed as seeding material observed in step (k); (m) mixing bacterial strains selected in step (l) in various combinations to obtain different microbial consortia; (n) obtaining cell pellet of each microbial consortium following the method of steps c, d, e, f, g and h; (o) testing seeds as obtained in step (n) for BOD analysis of pulp and paper wastewater; (p) selecting the optimal seed tom results as obtained in step (o).

36. A method as claimed in claim 35 wherein the cell slurry of individual isolated bacterial strains is tested as seed for BOD analysis of pulp and paper wastewater.

37. A method as claimed in claim 35 wherein the BOD values obtained using seeds are verified using Glucose-Glutamic acid (GGA) as a reference standard.

38. A method as claimed in claim 35 wherein the bacterial strains are taken in equal proportions.

39. A method as claimed in claim 35 wherein the bacteria are isolated from activated sludge and soil samples collected from vicinity of a selected pulp and paper mill.

40. A method as claimed in claim 35 wherein characteristics of Micrococcus sp. (MTCC 6602) are as follows: Gram—positive, Shape—cocci.

41. A method claimed in claim 35 wherein characteristics of Staphylococcus sp. (MTCC 6603) are as follows: Gram—positive, Shape—rods.

42. A method as claimed in claim 35 wherein characteristics of Kurthia zopfii (MTCC 6604) are as follows: Gram—Negative, Shape—rods.

43. A method as claimed in claim 35 wherein characteristics of Alcaligenes faecalis (MTCC 6719) are as follows: Gram—Negative, Shape—rods.

44. A method as claimed in claim 35 wherein characteristics of Pseudomonas aeruginosa (MTCC 6605) are as follows: Gram—Negative, Shape—rods.