FIELD OF THE INVENTION

The present invention relates to a microbial consortium comprising a plurality of bacterial strains, capable of sequestering nitrogen and phosphate from biomass, for environmental protection and sustenance. The present invention further provides a method of treatment of wastewater effluent by treating the wastewater effluent with a microbial consortium comprising a plurality of bacterial strains, capable of sequestering nitrogen and phosphate.

BACKGROUND OF THE INVENTION

[0001] Wastewater treatment has grown significantly over the past few decades. Environmental protection regulations require treatment of disposable effluent from wastewater prior to its drainage into natural water system. Waste water is contaminated by both organic and inorganic waste compounds. Organic matter is much easier to decompose and remove from the wastewater as compared to the inorganic waste. Conventionally, wastewater contaminated from domestic and industrial sources is chemically treated prior to being discharged into a natural water system. The chemical treatment is much more expensive and further cause greater water quality problems. So to make the water treatment process more economical and safer for drinking, recently the wastewater is treated biologically. Biological treatment means degradation of the impurities with the help of microorganisms/plants/combination of both.

[0002] Nitrate and phosphate are two major pollutants due to excessive use of fertilizers in agriculture which have emerged as a global problem and its potential threat is marked on the environmental sustenance as well as on the public health. Nitrates are highly water soluble, and so move readily with surface runoff into rivers or with water percolating through the soil profile into the groundwater below. However, phosphate is not water soluble, so it moves only with soil movement, as it adheres to soil particles. [0003] The runoff of nitrate and phosphate into lakes and streams causes accelerated eutrophication. Eutrophication is a natural process that typically occurs as lakes age. However, human-caused, accelerated eutrophication occurs more rapidly, and causes problems in natural water bodies. Eutrophication stimulates the growth of algae causing depletion of oxygen and diminishing the penetration of light which in turn harms the aquatic life in water.

[0004] Nitrate is also a potential human health threat especially to infants, causing the condition known as methemoglobinemia, also commonly known as "blue baby syndrome". Central Nervous System and Cardiovascular System may also be affected as it can be carcinogenic. Nitrate pollution is also reported from paddy as well as tea cultivation where the nitrate leaching causes acidification of soil and increases intensity of nitrate level in ground water. Thus nitrate could be called as a necessary evil, which is extremely essential for plant growth but when leached into water bodies, causes major disaster for environmental as well as human health.

[0005] Moreover phosphate, the limiting nutrient, being extensively used in agricultural practices, is of high concern and due to its finite reserve it is at the verge of extinction. Recovery and reuse of this valued nutrient would render a sustainable management of phosphorous reserve and check the depletion of this invaluable resource in an alarming rate

[0006] Therefore there is a continuous need to provide a method to prevent eutrophication of water bodies and also to ensure that phosphate and nitrate which are used extensively in the fertilizers in agriculture practices are recovered back and reused for the sustainable management.

[0007] Biological phosphorus removal processes are well known in the art and have been applied to the wastewater for the removal of phosphate. Enhanced biological phosphorus removal (EBPR), is one such process which is a key player in recovering phosphate from waste stream and curbing down the possibilities of eutrophication. EBPR is an essential cost effective step in phosphate removal and recovery compared to the traditional process of phosphate removal by chemical precipitation. The phosphate removing organisms responsible for EBPR have been seen to possess the unique property of polyphosphate accumulation. Previous studies have demonstrated that microorganisms like Aerobacter spp., Pseudomonas spp. , Moraxella spp., Escherichia coli, Mycobacterium spp., Beggiatoa spp., Neisseria meningitides, Klebsiella spp., Azotobacter vinelandii, Neurospora crassa are efficient in accumulating and releasing phosphorous. (Tsai et al, 1979; Cramer et al, 1980, Bitton, 1994)

[0008] Denitrification of simulated municipal wastewater treatment plant effluent using a three-dimensional biofilm-electrode reactor: operating performance and bacterial community demonstrated a nitrate removal of 98.3 with C/N ratio of 3.0 and HRT of 7h while 85.0- 90.0% of nitrate removal with a C/N ratio of 1.5 and HRT of lOh due to cooperative heterotrophic and autotrophic denitrification.

[0009] Phosphorus removal is effected through its assimilation as microorganisms grow and uptake the inorganic phosphorus in excess which become stored within the cells in the form of polyphosphate granules (volutin). Poly phosphate kinase (PPK) gene, exopolyphosphatase (PPX) gene along with a regulatory site constituting ppk/ppx operon, are primarily responsible for this synthesis of polyphosphate which is a key of phosphate removal and recovery from waste water. Inorganic polyphosphate (polyP) is a rich source of energy. These internal reserves support the prolonged growth and survival of the organism in adversity. PolyP compounds are linear polymers containing tens to hundreds of phosphate residues linked by energy-rich phosphoanhydride bonds. PolyP helps the microorganisms to adapt to extremes of salinity, osmolality, desiccation, UV radiation, pH, and temperature in the environment. In bacteria, polyP levels are controlled by several polyphosphatases as well as by two families of polyP kinases, PPKl and PPK2. The sequences of these enzymes are not related. PPKl synthesizes most of the polyP in the cell, using ATP as a substrate, whereas the only example of PPK2 characterized up to now generates GTP from GDP.

[00010] Over the last few decades, microalgal and cynobacterial species are applied intensively to perform the biological treatment of the effluents for remediation of phosphorous from the wastewater. To improve the phosphorous removal from wastewater, many genetically modified high phosphate accumulating microorganisms are also developed. By adopting the phosphorus-accumulating microorganisms to treat waste water, the cost is low, the efficiency is stable and no secondary pollutants are produced, thereby effectively treating phosphorous-containing waste water and protecting the environment. There are many biological solutions available in the market like Nitrex or Phosphex but they consume much longer time for the removal of phosphate and nitrate.

[00011] Phosphate is obtained from rock phosphate whose reserves are limited while nitrate production is an energy intense process. Thus, sequestration of nitrate along with phosphate instead of losing them as nitrogen gas and reutilizing them as biofertilizer would be the desirable alternative.

[00012] The present invention satisfies these needs, as well as others, and efficiently overcomes the deficiencies found in the background art.

OBJECTS OF THE INVENTION

[00013] It is an object of the present invention to provide a microbial consortium, capable of sequestering phosphate and nitrate from biomass.

[00014] It is another object of the present invention to provide a phosphate and nitrate removing microbial consortium, wherein the consortium works better than individual strains in removal of phosphate and nitrate.

[00015] It is another object of the present invention to provide a microbial consortium to sequester phosphate and nitrate from waste water effluent.

[00016] It is another object of the present invention to reutilize the biomass comprising sequestered phosphate and nitrate as biofertilizer.

[00017] It is another object of the present invention to reuse treated effluent wastewater for non-potable purposes.

[00018] It is a yet another object of the present invention to promote plant growth and maintains fertility of soil in terms of nitrate and phosphate concentration.

[00019] It is still another object of the present invention to provide a microbial consortium wherein the consortium provides better nutritional quality of seeds as compared to chemical fertilizer treated seeds.

[00020] It is another object of the present invention to provide a simple and inexpensive method for removal of phosphate and nitrate from wastewater effluent using a microbial consortium comprising of phosphate and nitrate accumulators.. [00021] It is still another object of the present invention to provide an efficient and cost effective method that is an alternative to an expensive chemical approach or a less efficient biological approach.

[00022] Other objects of the present invention will be apparent from the description of the invention herein below.

SUMMARY OF THE INVENTION

[00023] The present invention relates to a microbial consortium, capable of sequestering phosphate and nitrate from biomass, comprising bacterial strains selected from the group consisting of Bacillus sp MCC0008, Bacillus sp MCC2071 and Bacillus sp MCC2059.

[00024] In an embodiment of the present invention, the microbial consortium sequesters phosphate and nitrate from wastewater effluent.

[00025] In an embodiment of the present invention, Bacillus sp MCC0008, Bacillus sp MCC2071 and Bacillus sp MCC2059 are present in equal ratio.

[00026] In one embodiment, microbial consortium of the present invention sequesters 62 to 66% phosphate and sequesters and removes 94.6 to 98.98% nitrate from waste water effluent.

[00027] The present invention also relates to a method of treatment of wastewater effluent, comprising the steps of :

a) providing a biofilm of microbial consortium comprising bacterial strains selected from a group consisting of Bacillus sp MCC0008, Bacillus sp MCC2071 and Bacillus sp MCC2059 on a solid support in a bioreactor,

b) incubating the biofilm of the microbial consortium to remove phosphate and nitrate from the wastewater effluent.

[00028] In one embodiment of the present invention, the solid support is a steel mesh.

[00029] In another embodiment of the present invention, the bioreactor is a packed bed bioreactor.

[00030] In another embodiment of the present invention, the microbial consortium is capable of preventing eutrophication of water bodies.

[00031] In another embodiment of the present invention, the biomass with sequestered phosphate and nitrate is used as biofertilizer. [00032] In another embodiment, microbial consortium of the present invention is capable to fasten initiation of flowering in plants.

[00033] In a yet another embodiment, microbial consortium of the present invention is capable to promote plant growth and maintain fertility of soil.

BRIEF DESCRIPTION OF THE DRAWINGS

These and other features, aspects and advantages of the invention will become better understood when the description is read with reference to the accompanying drawings, wherein:

[00034] Figure 1 illustrates a growth curve for a microbial consortium of the present invention consisting of a combination of MCC0008, MCC2059, and MCC2071, in a 5 litre (L) suspended bed bioreactor.

[00035] Figure 2a illustrates reduction of concentration of nitrate in media with time.

[00036] Figure 2b illustrates reduction of concentration of phosphate in media with time.

[00037] Figure 3a illustrates reduction kinetics of nitrate for a microbial consortium of the present invention consisting of a combination of MCC0008, MCC2059, and MCC2071, in a 5L suspended bed bioreactor.

[00038] Figure 3b illustrates reduction kinetics of phosphate for a microbial consortium of the present invention consisting of a combination of MCC0008, MCC2059, and MCC2071, in a 5L suspended bed bioreactor.

[00039] Figure 4a illustrates percentage reduction of nitrate with a reducing concentration of nitrate broth in 9.0 L packed bed reactor

[00040] Figure 4b illustrates percentage reduction of phosphate with a reducing concentration of nitrate broth in 9.0 L packed bed reactor.

[00041] Figure 5a illustrates percent reduction in nitrate concentration in 9L bioreactor in batch mode.

[00042] Figure 5b illustrates percent reduction in phosphate concentration in 9L bioreactor in batch mode.

[00043] Figure 6a illustrates percent reduction in nitrate concentration in 9L bioreactor in continuous mode.

[00044] Figure 6b illustrates percent reduction in phosphate concentration in 9L bioreactor in continuous mode. [00045] Figure 7a illustrates percent reduction in nitrate concentration in 9L bioreactor in continuous mode with changing flowrate.

[00046] Figure 7b illustrates percent reduction in phosphate concentration in 9L bioreactor in continuous mode with changing flowrate.

[00047] Figure 8 illustrates time course of nitrate reduction in a 9L packed bed bioreactor in presence of different concentrations of nitrate in an influent.

[00048] Figure 9 illustrates TEM images of strains showing vacuoles upon accumulation of nitrate (MCC0008) and phosphate (MCC0008, MCC2071 and MCC2059).

[00049] Figure 10 illustrates comparison of different parameters reflecting enhanced yield with a microbial consortium of the present invention consisting of a combination of MCC0008, MCC2059, and MCC2071, as compared to Control (no fertilizer) but less than chemical fertilizer application.

DETAILED DESCRIPTION OF THE INVENTION

[00050] The embodiments herein and the various features and advantageous details thereof are explained more fully with reference to the non-limiting embodiments that are illustrated in the accompanying figures and detailed in the following description. Descriptions of well-known components and processing techniques are omitted so as to not unnecessarily obscure the embodiments herein.

[00051] The present invention relates to a microbial consortium, capable of sequestering phosphate and nitrate from biomass, comprising bacterial strains selected from the group consisting of Bacillus sp MCC0008, Bacillus sp MCC2071 and Bacillus sp MCC2059.

[00052] In accordance with the present invention, the bacterial strains comprising Bacillus sp MCC0008, Bacillus sp MCC2071 and Bacillus sp MCC2059 are deposited at the Microbial Culture Collection, Pune, India. In an embodiment of the present invention, the three bacterial strains are studied at genomic level with draft genome sequence available at GenBank with accession number ANAU00000000, ANFK00000000, ANGA00000000 and are found to be novel.

[00053] In an embodiment of the present invention, the microbial consortium sequesters phosphate and nitrate from wastewater effluent. [00054] The microbial consortium of the present invention can be used to prevent eutrophication of water bodies. Furthermore, the microbial consortium of the present invention can also be used to reduce phosphate and nitrate concentration from agricultural runoff, which promotes plant growth and increase soil fertility.

[00055] The bacterial strains comprising Bacillus sp MCC0008, Bacillus sp

MCC2071 and Bacillus sp MCC2059 are phosphate and nitrate accumulators and reducers. Bacillus sp MCC0008 also has an ability to accumulate nitrate intracellular. The extent of nitrate accumulation by MCC0008 increases with an increase in external concentration of nitrate. The capability of MCC0008 to accumulate nitrate is evident from Example 10 described herein below.

[00056] The microbial consortium of the present invention facilitates removal of phosphate and nitrate from wastewater effluent, lowering levels to limits which are not harmful to environment. The wastewater effluent treated with the microbial consortium of the present invention, comprising of sequestered nitrate and phosphate, can be used as biofertilizer.

[00057] In an embodiment of the present invention, Bacillus sp MCC0008, Bacillus sp MCC2071 and Bacillus sp MCC2059 are present in equal ratio (1 : 1 : 1).

[00058] The microbial consortium of the present invention can be grown in nitrate broth under optimum conditions. The microbial consortium can be mixed into wastewater effluent, which further results in decrease in eutrophication phenomenon. In one embodiment of the present invention, a plant grown in a soil mixed with the microbial consortium obtained after treatment of wastewater effluent can result into voluminous yield and growth of the plant with better quality.

[00059] The microbial consortium prepared in the above defined ratio is used as an inoculum for treating wastewater effluent, specifically for removal of phosphate and nitrate.

[00060] The present invention also relates to a method of treatment of wastewater effluent, comprising the steps of :

a) providing a biofilm of a microbial consortium comprising bacterial strains selected from a group consisting of Bacillus sp MCC0008, Bacillus sp MCC2071 and Bacillus sp MCC2059 on a solid support in a bioreactor, and

b) incubating the biofilm of the microbial consortium to remove phosphate and nitrate from the wastewater effluent.

[00061] In one embodiment of the present invention, the solid support is a steel mesh. [00062] In another embodiment of the present invention, the bioreactor is a packed bed bioreactor. The bioreactor of the present invention is adapted to support a biofilm and to enable the biofilm to be brought into contact with the wastewater effluent.

[00063] According to an exemplary embodiment of the present invention, the microbial consortium can sequester 62 to 66% phosphate and can remove 94.6 to 98.98% nitrate simultaneously in a biofilm based bioreactor system operating in a continuous mode at 65/75/84 revolutions per minute speed of peristaltic pump within 2 hours.

[00064] In another embodiment of the present invention, the bacterial strains consisting of MCC0008, MCC2071 and MCC2059 can be mixed together in preferably 1 : 1 : 1 ratio to form the consortium of the present invention. In a further embodiment, the microbial consortia can be mixed and grown in nitrate broth under optimum condition resulting into phosphate and nitrate sequestering consortium of the present invention.

[00065] According to one embodiment, the consortium of the present invention can be mixed into soil resulting into removal of nitrate and phosphate from wastewater, which further results in a decrease in eutrophication phenomenon. A plant grown in soil mixed with the consortium of the present invention can result into voluminous yield and plant growth with better quality.

[00066] In another embodiment, the microbial consortium of the present invention is used for plant growth promoting activity. The term "plant growth-promoting activity", as used herein, encompasses a wide range of improved plant properties, including, for example without limitation, improved nitrogen fixation, improved root development, increased leaf area, increased plant yield, rapid seed germination, increased photosynthesis, or an increased in accumulated biomass of the plant. A microbial consortium of the present invention, supply a plant with nutrients and thereby can promote plant growth.

[00067] In one exemplary embodiment of the present invention, biomass comprising sequestered nitrate and phosphate can work as biofertilizer in case of Mung bean (Vigna radiata) resulting in a 10.21% increase in yield per hectare as compared to control set (without fertilizer). The yield in case of chemical fertilizer was 23.02% higher than control. However, elemental as well as nutritional quality of the seeds was better in case of biofertilizer treated samples as compared to chemical fertilizer treated seed samples. EXAMPLES

[00068] The invention is illustrated by the following examples which are only meant to illustrate the invention and not to act as limitations. The examples used herein are intended merely to facilitate an understanding of ways in which the embodiments herein may be practiced and to further enable those of skill in the art to practice the embodiments herein. Accordingly, the examples should not be construed as limiting the scope of the embodiments herein.

Example 1: Ratio of the three bacterial strains

[00069] The three isolates (MCC 2071, MCC 2059 and MCC 0008) were mixed in varying ratios as represented in Table 1 and checked for the percentage reduction of nitrate and phosphate in nitrate broth for selection of an optimum combination. The highest percentage reduction of nitrate and phosphate was reported using Combination 7 (MCC 2071 : MCC 2059: MCC 0008 in the ratio of 1 : 1 : 1) as indicated in the Table 2a and Table 2b. The growth of cell, nitrate and phosphate measurements were made using spectrophotometer (Biochrome Libra S70).

Table 1: Ratio of MCC 2071, MCC 2059 and MCC 0008 in the combinations

Table 2a: Percentage reduction of nitrate by individual strains and combinations

MCC 2071 77.92 84.38

MCC 2059 74.68 85.03

MCC 0008 46.53 90.67

COMBI 8 78.38 84.28

COMBI 9 56.16 88.74

COMBI 10 108.66 78.21

COMBI 1 57.92 88.39

COMBI 2 47.82 90.41

COMBI 3 42.36 91.51

COMBI 4 67.27 86.51

COMBI 5 57.92 88.39

COMBI 6 55.14 88.94

COMBI 7(control) 51.06 89.76

Table 2b: Percentage reduction of Phosphate by individual strains and combinations

Individual % reduction of phosphate strains/Combinations I ppm

Media 47.9

MCC 2071 14.40 69.94

MCC 2059 15.44 67.77

MCC 0008 30.59 36.13

COMBI 8 15.53 67.57

COMBI 9 28.75 39.97

COMBI 10 33.89 29.23

COMBI 7(control) 16.99 64.52

COMBI 1 16.45 65.66

COMBI 2 11.73 75.51

COMBI 3 6.43 86.58

COMBI 4 6.16 87.14

[00070] The 1: 1 : 1 combination of the three isolates was picked up for further studies with the objective of getting maximum nitrate and phosphate accumulation simultaneously. Example 3: Time course of removal of phosphate and nitrate

[00071] The time course of removal of nitrate and phosphate from medium was checked in a Slitre (lit) suspended bed reactor (IIC Industrial Corporation, Kolkata, India) for 10 hours at an agitation speed of 150 rpm at 37°C temperature with aeration at 21pm following 1% inoculation. Samples were collected at 60 minutes interval. The growth as well as nitrate and phosphate removal with time by the consortium in the suspended bed bioreactor is represented in Figures 1, 2a, 2b. The kinetics of reduction for nitrate and phosphate was determined as per the method of Nasipuri P et al. [American journal of microbiology. 1(2): 23-29, 2010] and represented in Figures 3a, 3b.

[00072] In Figure 3a, Rm (maximum rate of nitrate reduction) = slope = 34.016; and Km (half saturation constant) = y intercept = -4.0152. In Figure 3b, Rm (maximum rate of phosphate reduction) = slope =18.637; and Km (half saturation constant) = y intercept = - 0.8992.

[00073] However, suspended bed reactor requires high energy and cost. Henceforth the combination was tested in a packed bed reactor (91it under unadjusted/ anaerobic condition) using steel mesh as immobilization matrix.

Example 4: Evaluation of the microbial preparation in bioreactor

[00074] In order to make the application sustainable for effluent treatment, the packed bed bioreactor system was selected. 1% innoculum was used to allow biofilm development on a steel mesh in a packed bed bioreactor (2.51it and 91it) as per reported protocol. The consortium once developed into a biomass was acclimatized to waste water with a carbon source (0.05% citric acid) selected depending on substrate utilization test performed for the isolates. The column was recharged with fresh medium every 24 hours and the differences in concentration of nitrate and phosphate measured using spectrophotometer (Biochrome Libra S70) between 0 hour and after 24 hours were used to calculate the percentage reduction every 24 hours with or without external aeration. In order to study the time course of removal of nutrients, the extent of reduction of nitrate and phosphate per hour was monitored for the packed bed reactor as per the method of Cataldo (Cataldo et al. (1975) Rapid colorimetric determination of nitrate in plant tissues by nitration of salicylic acid. Commun. Soil Science and Plant Analysis 6(1) 71-80. DOI: 10.1080/00103627509366547) and Denige's method (Denige 1920) respectively. The data was statistically validated using paired t-test. The number of days the consortium was acclimatized under immobilized condition in different concentrations of nitrate broth (NB) for the two packed bed is provided in Table 3.

Table 3: Number of days the consortium has been acclimatized to the different concentration of medium

[00075] Combination 7 was tested in the packed bed reactor (91it under unadjusted/ anaerobic condition) for reduction of nitrate and phosphate as depicted in Figures 4a, 4b. The nitrate and phosphate reduction in this packed bed reactor turned out to be quite satisfactory. The statistical validation of the data was carried out using t-test as depicted in Table 4a and 4b.

Table 4a: Table representing statistical validation of the extent of nitrate reduction by the microbial consortium, different within the bioreactor with variable composition of the solution undergoing treatment. The t-test was conducted with following null and alternative hypothesis:

Ho= No significant variation in the nitrate concentration upon 24hrs incubation.

Hi=Variation is significant 100 34 99% 86.179 2.728 significant

75 8 99% 42.803 3.355 significant

50 8 99% 24.338 3.355 significant

25 5 99% 21.655 4.032 significant

10 11 99% 34.037 3.106 significant

5 5 99% 62.183 4.032 significant

2 8 99% 29.676 3.355 significant

2% with waste 8 99% 33.457 3.355 significant water

1% with waste 23 99% 10.721 2.807 significant water l%waste 23 99% 17.612 2.807 significant water +Citric

acid

Waste 17 99% 18.079 2.898 significant water+citric

acid

[00076] At 99% confidence level, all values for each treatment is significant. Hence the null hypothesis is rejected.

Table 4b: Table representing statistical validation of the extent of phosphate reduction by the microbial consortium, different within the bioreactor with variable composition of the solution undergoing treatment. The t-test was conducted with following null and alternative hypothesis:

Ho= No significant variation in the phosphate concentration upon 24hrs incubation.

Hi=Variation is significant Nitrate Degree of Confidence T value Critical value Significance broth(%) freedom level

100 32 98% 2.760 2.449 Significant

75 8 Nil -8.374 Nil Insignificant

50 8 80% 1.738 1.397 Significant

25 8 90% 2.222 1.86 Significant

10 11 Nil -4.680 Nil Insignificant

5 8 Nil -1.169 Nil Insignificant

2 8 Nil -3.581 Nil Insignificant

2% with 17 Nil -0.131 Nil Insignificant waste water

l% with 23 99% 3.605 2.807 Significant waste water

1%+citric 32 99% 6.861 2.738 Significant acid

Only citric 31 99% 12.424 2.728 Significant acid Treatment for which t value is insignificant, null hypothesis is accepted.

Treatment for which t value is significant, null hypothesis is rejected.

[00077] The removal of nitrate and phosphate from the charged waste water in presence of 0.05% citric acid was significant. The 9L bioreactor was run in batch mode (agricultural effluent + 0.05% citric acid) for treatment of agricultural effluent with respect to reduction of nitrate and phosphate from July 22, 2013 to August 5, 2013. The representative graph for the same is given in Figures 5a, 5b, where approximately 92% reduction in nitrate concentration and 71% reduction in phosphate concentration can be observed with 24 hrs incubation.

Example 5: Operation of 9L bioreactor in continuous mode

[00078] As the consortium developed was able to successfully treat agricultural runoff in the packed bed 9L bioreactor in batch mode, the aim was to scale up the process to continuous mode treatment. The 9L bioreactor was operated in continuous mode with canal water containing 0.05% citric acid for 19 days at a flow rate of 40.5 ml/min or 2.43 1/hour (inflow) and an outflow of effluent (treated wastewater with respect to nitrate and phosphate) at the rate of 34 ml/min or 2.041itre/hour with a retention time of 1.93 hours.

Example 6: Optimization of the flowrate

[00079] To determine the optimum flowrate for the most efficient treatment of the agricultural effluent, the speed, i.e., the revolution per minute of the peristaltic pump was varied from between 65 to 105 revolutions per minute. The flow rate at different speeds is represented in Table 5.

Table 5: The Flow rate of the influent and effluent as a result of variation in the speed of the peristaltic pump.

84 40.5 ml/min; 2.43 34 ml/min; 2.04 1.93 hours

1/hour 1/hour

95 46 ml/min; 2.76 1/hour 40 ml/min; 2.4 1/hour 1.70 hours

105 54 ml/min; 3.24 1/hour 47 ml/min; 2.82 1.45 hours

1/hour

[00080] The nitrate and phosphate reduction were estimated and compared as depicted in Figure 7a, 7b. It was seen that the flowrate of 40.5 ml/min; 2.43 1/hour (inflow) gave the best reduction within short time (within 30min to 2hours) for both nitrate and phosphate. On increasing the speed further the extent of nitrate and phosphate reduction decreases.

[00081] Enhancement of reduction was evident with increase in concentration of nitrate in the solution to be treated as evident in Figure 8.

Example 7: Evidence for intracellular accumulation

[00082] The reduction/removal of nitrate and phosphate from the solution was monitored spectrophotometrically in the above studies. It is already known in case of bacteria that intracellular accumulation of polyphosphate is as granules within the cytoplasm. Similarly nitrate accumulation is also within vacuoles. In order to localize for accumulation of the same, Transmission electron microscopy (TEM) of unstained cells was carried for all the three isolates separately post fixation using standard procedure. The formation of vacuoles was seen in case of all three strains (Figures 9a, 9b, 9c, 9d, 9e, 9f). The intracellular accumulation was reconfirmed through spectroscopy of cell lysate.

Example 8: Plant growth promotion study

[00083] Since the consortium of the present invention could be used for both nitrate and phosphate sequestration from waste water, it was tested for PGPB traits and vigour index calculation. The formulation was used for germination trail, pot trial as well as field trial for Vigna radiata cultivation by adding consortium to soil during sowing. Vigna radiata var Samrat (developed by IIPR, Kanpur) was used for the trail from Feb 2013 to May 2013 (Spring Summer Planting). For germination trial, 14 days experiment was carried out in triplicate. The soaked seeds were sown, followed by formulation application in the soil. The setup was maintained for 14 days while monitoring germination and other growth parameters. The germination index and vigour index were calculated from the data. Pot trial and field trial were carried out. For field trial randomized block design with four replicates were carried out. The sowing was done in the north south orientation. The seeds post germination was subjected to thinning such that each lm ² area contains a total of 40 plants (4 rows of 10 plants each). The inoculum for the germination trial was 4.2xl0 ⁶ cells per 125gm soil in a thermo coal glass/ germination tray, 1.39xl0 ⁷ cells per 8kgs soil in each pot and 3.68xl0 ⁹ cells per lm ² plot for field trial. The yield per plot as well as per Hectare of land was calculated for the consortium when compared with control and chemical fertilizer application.

[00084] During 14 days germination trial with direct application to soil, the shoot and root weight increase with the consortium application was 1.8 and 0.88 folds higher than control un-inoculated sample. The germination energy index and the vigour index for the consortium was 64.18% and 30.27% higher when compared with the control set respectively. Thus the germination percentage of the seeds treated with the consortium was higher than the untreated seeds. It is expected since these isolates produce plant growth hormones.

Example 9: Elemental and Nutritional Quality Analysis of the seeds

[00085] The seeds were lyophilized for 24 hours, manually ground in the mortar and passel, 0.2gm ground material was pelleted using Pelletizer (Technolab, Kbr Press) at 110 Kg/cm ² . The mineral content of the pellets were assessed using Energy Dispersive X ray fluorescence (Jordan Valley EX-3600) analysis as per reported protocol. The nutritional quality analysis like moisture [IS:4333(Part-II):2002], total protein (AOAC 920.87), available carbohydrate (AOAC 986.25), fat (AOAC 963.15), energy (Analytical Chemistry of Food by CS James: 1995), ash content (AOAC 941.12), sugar (AOAC 923.09) and fibre (AOAC 985.29) content were carried out at SGS India Private Limited, Kolkata as per standard protocol.

Table 6: Elemental Analysis of the seeds

Mn 12.07 7.08

Cu 14.51 8.97

P 5.89 -66.60

K 2.55 -19.16

S 6.05 -26.24

Ca 8.81 -12.59

[00086] The elemental content has improved post application as compared to control as measured through EDXRF Analysis. This shows that though the yield with chemical fertilizer is higher but the elemental content as compared to control is better in consortium treated seeds while poor following chemical treatment.

Table 7: Nutritional Quality Analysis of the seeds

[00087] As per the nutritional parameter they are comparable to the seeds produced through chemically fertilizer application.

[00088] Thus, it can be concluded from this study that the microbial consortium of the present invention is by far the fastest nitrate and phosphate sequestering consortium which also has plant growth promoting activity utilizing the sequestered nutrients as fertilizer in case of Mung bean (Vigna radiata). Example 10: Nitrate Accumulation by MCC0008

Nitrate accumulation was checked by cell lysis method using sonication. The extent of accumulation per gm of wet weight of pellet was found to be varing between 1278.66 to 1302.12ppm/gm of wet weight (0.021M) of Nitrate using sonication followed by spectrophotometric analysis as per the method of Cataldo as reported in the literature (Cataldo et al. (1975) Rapid colorimetric determination of nitrate in plant tissues by nitration of salicylic acid. Commun. Soil Science and Plant Analysis 6(1) 71-80. DOI: 10.1080/00103627509366547).

The strain MCC0008 was subjected to elevated concentrations of nitrate and the corresponding nitrate accumulation was monitored (Table 8). The cell shows higher accumulation when grown in presence of higher concentrations of nitrate.

Table 8: Table representing an extent of nitrate accumulation in ppm per gram of wet weight of cells when grown in presence of different concentrations of nitrate in the growth medium

ADVANTAGES OF THE INVENTION

[00089] The present invention provides a microbial consortium, capable of removing phosphate and nitrate simultaneously by a biomass, comprising of bacterial strains, for environmental protection and agricultural sustenance.

[00090] The present invention provides a phosphate and nitrate removing microbial consortium wherein the consortium has a capability of plant growth promotion.

[00091] The present invention provides a phosphate and nitrate removing microbial consortium wherein the consortium fastens initiation of flowering in plants.

[00092] The present invention provides a phosphate and nitrate removing microbial consortium wherein the consortium prevents eutrophication of water bodies. [00093] The present invention provides a phosphate and nitrate removing microbial consortium, wherein the consortium removes as well as sequesters phosphate and nitrate from waste water and biomass and in turn is used as a source of phosphate and nitrate for plant growth promotion.

[00094] The present invention provides a phosphate and nitrate removing microbial consortium, wherein the biomass comprising sequestered phosphate and nitrate can be used as biofertilizer.

[00095] The present invention provides a phosphate and nitrate removing microbial consortium wherein the consortium promotes plant growth while constantly maintaining fertility of soil in terms of phosphate and nitrate concentration.

[00096] The present invention provides a phosphate and nitrate removing microbial consortium wherein the consortium provides better nutritional quality of seeds as compared to chemical fertilizer treated seeds.

[00097] Other advantages of the present invention will be apparent from the description of the invention hereinabove.