DESC:TECHNICAL FIELD OF THE INVENTION:
The present invention relates to a sustained release formulation comprising at least one bioactive agent and suitable binders appropriate for liquid waste management, bioremediation of waterbodies and as a deodorizer and/or a de-colorizer. Further, the present invention provides a process for the preparation of the said sustained release formulation.

BACKGROUND AND PRIOR ART OF THE INVENTION:
Since the advent of industrial civilization, water bodies have been incessantly used as sites of disposal for domestic and industrial wastes. This has led to an indiscriminate use of water and unchecked dumping of waste in natural water bodies in the form of sewage or effluents. Due to this, both surface and groundwater tables in metropolitan cities across the world face an imminent threat of being destroyed beyond repair if left unchecked. Recently, the authorities of various countries and environmental organizations have been advocating the importance of conscious use of water as well as have initiated active efforts on recycling and remediating water.
The main sources of water pollution are domestic sewage and effluents, agricultural run-off and industrial effluents. The facilities to treat waste water are not adequate. In a country like India, only about 10% of the waste water generated is treated; the rest is discharged as it is into water bodies. Due to this, pollutants enter groundwater, rivers, and other water bodies. Such water eventually ends up being used for household activities and is often highly contaminated with disease causing microbes. Another problem faced in under-developed and developing countries is open sewer lines or nullahs which are perceived as a first sight of poor hygiene. The sewage flowing through open sewer lines comprises domestic/ household waste which poses a serious threat to the environment and human health. The inherent microflora of sewage can infect humans and animals.
Human excretory waste and its degradation are of major concern as it inherently has a high organic load accompanied with potentially pathogenic microbes. Such wastewaters with microbes can possibly harm humans, livestock and environment when discharged into water streams or on land. Biological degradation of sanitary wastewaters is a clever solution to the problem but not many advances are being done in this sector.
Conventional technologies for bio-remediation of water bodies employ aquatic plants and microbial formulations in powdered form. Aquatic plants are susceptible to pollutant stress and can also reduce the dissolved oxygen levels of the water bodies at night, which can hamper the aquatic life in it. The use of targeted microbes in remediation of water may help in reducing pollution but have a temporary effect due to loss of microbes during run off. Repeated inoculation of measured dosage of microbial formulation cannot be easily managed owing to large amounts of wastes to be handled.
In industrial wastewater plants, effluents are treated using microbes either in aerated or non-aerated/anaerobic systems. In most cases, the source of microbes used is cow dung; which does not exclusively contain the micro-organisms necessary for efficient degradation of organic and inorganic wastes present in the effluent. Moreover, use of cow dung in effluent treatment may disturb the treatment plant by causing sludge bulking or generate a foul odor.
Industrial wastewater do not carry high organic load but are rich in recalcitrant compounds. The trend of using microbial consortia for degradation of such waste is flourishing considering the efficiency and environmental compatibility of this approach. Natural or artificial water-bodies such as lakes/ponds are more prone to a condition called eutrophication which is a result of altered nutrient content. Such a situation can be brought under control by use of microbes or their formulations. Microbial methods offer an advantage of being more sustainable and eco-friendly. However, delivering the measured dosage, retention of microbial activity in the formulation and its repeated application is cumbersome while treating industrial effluents and water bodies in general. Therefore, treatment systems have been employing chemical agents rather than microbial formulations to degrade and reduce organic wastes and hazardous compounds that constitute a large portion of industrial and sewage make up. This has led to a build-up of toxic chemical agents in the food chain.
In light of water conservation, waterless or water free urinals were conceptualized to counter the problem of excessive water wastage. The major drawback of water less urinals is generation of foul odour as a result of incomplete microbial degradation of urine components. Moreover, accumulation of urine residue causes corrosion of urinal pipes. Bacteria can also use trace amounts of protein and carbohydrates present in urine as a food source and produce other odour causing compounds.
Currently, a few products are available in the market to combat the issue of odour generation in urinals viz. para-dichlorobenzene (PDB) and naphthalene balls or urinal mats with fragrance. However, naphthalene and PDB balls are carcinogenic and highly toxic to the environment. The urinal mats contain biocides to restrict microbial degradation that can disturb the microbial flora which is essential for efficient functioning of septic tanks. Moreover, the mat matrix is non-biodegradable in nature. There also exist enzyme based formulations called bio-tabs which claim to deodorize waterless urinals. Though these products provide a temporary solution of eliminating or diminishing odour during active usage, they are ineffective in the long run as enzymes are highly unstable and fail to sustain at conditions in urinals. Micro-organisms have been isolated and have been noted for their ability to degrade organic waste and as deodorizers, however, efficient application and accurate dosages for treatment of sewage plants have not been disclosed. The PCT Publication No.WO2007/128897 has disclosed a novel micro-organism ‘Alcaligenes faecalis’ for treating wastewater, however, the inventors have failed to demonstrate compositions comprising the said strain with complete retention of its ‘deodorizing or biodegrading’ activity.
US Patent No. 4,722,801 discloses a toilet bowl cleaner in cake form containing a polyethyleneglycol distearate. However, the cleaner does not comprise biological ingredients possessing deodorising/bioremediation activities having sustained release activity over a protracted period of time to alleviate issues encountered in the art.
Keeping in mind the lacunae presented by prior art techniques, the present inventors have attempted to provide an efficient, environmentally friendly, biodegradable sustained release formulation comprising bioactive agents having usage in bioremediation of waste water bodies, to facilitate biodegradation of organic waste and in minimizing odour generation.

OBJECT OF THE INVENTION:
The primary object of the present invention is to provide a sustained release formulation comprising at least one bioactive agent and a mixture of binders to be used for remediation of waste waters and fresh water bodies, to minimize odour generation, to mitigate development of undesirable conditions in water bodies and to facilitate biological waste degradation ensuring its safe disposal in the environment.

SUMMARY OF THE INVENTION:
The present invention provides a sustained release formulation comprising at least one bioactive agent and suitable binders for bioremediation of waste water bodies and for use in urinals.
In an aspect, the present invention provides a sustained release formulation comprising at least one micro-organism or a consortium of micro-organisms and suitable binders.
In another aspect, the present invention provides a process for the preparation of the sustained release formulation, the said process comprising;
(i) providing at least one micro-organism or a consortium of micro-organisms in a concentration ranging from of 1 x 102 CFU/gm to 1 x 107 CFU/gm;
(ii) supplementing the dried powder of step (i) with binders selected from the group comprising glycerol monostearate, stearic acid and polyethylene glycol in a concentration ranging from about 1:200 to about 1:1, respectively, to provide a mixture comprising micro-organisms and a binder in a ratio ranging from 1:10 to 10:1;
(iii) supplementing the mixture of step (ii) with a salt of fatty acid in a ratio of 20:1, respectively;
(iv) compressing the mixture obtained in step (iii) in a hydraulic press at 1 ton to 5 ton pressure to obtain the said sustained release formulation.
In yet another aspect, the present invention provides a sustained release formulation having an extended release profile ranging from about 3 days to about 3 months.
In one aspect, the present invention provides sustained release formulations comprising at least one bioactive agent and suitable binders for use in bio-digestor or septic tanks to facilitate degradation of organic waste; in remediation of fresh water bodies and waste waters and in deodorization and reduction in pathogenic load of urinals, sewage present in septic tank, sewage treatment plants and open sewer systems or open channels leading to waste disposal sites.

In a further aspect, the present invention provides a tablet formulation of the sustained release formulation comprising;
(i) a bioactive agent in a concentration ranging from 9 gm% to 91 gm%;
(ii) binders selected from the group comprising glycerol monostearate, stearic acid and polyethylene glycol in a concentration ranging from 16 gm% to 67 gm%;
(iii) a salt of fatty acid in a concentration ranging from 3 gm% to 50 gm% .
Alternatively, the said formulation can be used in aquaculture for decontamination and/or remediation of water wherein aquatic life forms nurture.

DETAILED DESCRIPTION OF THE INVENTION:
The invention will now be described in detail in connection with certain preferred and optional embodiments, so that various aspects thereof may be more fully understood and appreciated.
In the most preferred embodiment, the present invention provides a sustained release formulation comprising at least one bioactive agent or sources thereof and suitable binders appropriate for bioremediation of wastewater and fresh water bodies and/or as a deodorizer in urinals.
More particularly, the present invention provides a sustained release formulation comprising at least one bioactive agent and binders selected from the group comprising Glycerol ester of fatty acids (such as glycerol monostearate Monostearin, 2,3-Dihydroxypropyl octadecanoate), stearic acid and polyethylene glycol or combinations thereof in a ratio ranging from 1:10 to 10:1 for liquid waste management and bioremediation of water-bodies.
The bioactive agent is selected from the group comprising of micro-organisms, herbal ingredients, biosurfactants, biopolymers, chelators, enzymes, natural, synthetic or semisynthetic substances for potential use in water bodies or by-products thereof.
Further, the combinations of binders selected from the group comprising glycerol monostearate, stearic acid and polyethylene glycol is in a ratio ranging from 1:1:1 to 1:4:1 respectively.
In a preferred embodiment, the present invention provides a sustained release formulation comprising at least one microorganism or a consortium of micro-organisms and suitable blend of binders. The formulation can also be provided with one or more sources of nutrients to facilitate growth of microorganisms.
Amongst the micro-organisms, bacteria, fungi and actinomycetes can be used in the present formulation to achieve degradation of organic waste and remediation of water bodies.

The micro-organism or a consortium of micro-organisms used in the present invention is selected from bacterial species. The bacteria used in the present invention is preferably selected from the Bacillus species particularly selected from the group comprising Bacillus subtilis ATCC 11774, Bacillus licheniformis ATCC 14580 and Bacillus megaterium ATCC 9885.
The bacterial species may be spray dried, tray dried, oven dried or freeze dried powders used in the preparation of the sustained release formulation.
In an embodiment, the present invention provides a sustained release formulation comprising bacteria either individually or in a consortium in a concentration ranging from about 1 x 102 cfu/gm to about 1 x 107 cfu/gm.
Further, the dried powder comprising micro-organisms along with binders is in a ratio ranging from about 10:1 to about 1:10, respectively. The binders mixed with the dried powdered mixture of micro-organisms are selected from the group comprising of glycerol monostearate, stearic acid, polyethylene glycol, glycerol esters of long chain fatty acids, long chain pyrrolidones and their derivatives, long chain fatty acids and their salts & derivatives, glycolic compounds, hydrates of inorganic salts or any other hydrophobic or hydrophilic material.
Salts of long chain fatty acids in concentration ranging from 1:30 to 1:1 and excipients in the concentrations ranging from 1:5 to 9:1 is then added to the mixture comprising the bacterial consortium and the binders to obtain the sustained release formulation.
The mixture comprising micro-organisms and a binder in a ratio ranging from 1:10 to 10:1 is further supplemented with a salt of fatty acid in a ratio of 1:1 to 30:1. Fatty acids are additional excipients used as lubricants.
In a further embodiment, the present sustained release formulation comprises suitable excipients selected from the group comprising talc, husks of grains or fruits, chelating agents, disintegrants, fragrances, dyes and natural, synthetic or bio-organic chemicals.
In another preferred embodiment, the present invention provides a process for the preparation of the sustained release formulation, the said process comprising;
(i) providing at least one micro-organism or a consortium of micro-organisms in a concentration ranging from of 1 x 102 CFU/gm to 1 x 107 CFU/gm;
(ii) supplementing the dried powder of step (i) with binders selected from the group comprising glycerol monostearate, stearic acid and polyethylene glycol in a concentration ranging from about 1:200 to about 1:1, respectively, to provide a mixture comprising micro-organisms and a binder in a ratio ranging from 1:10 to 10:1;
(iii) supplementing the mixture of step (ii) with a salt of fatty acid in a ratio of 20:1, respectively; and
(iv) compressing the mixture obtained in step (iii) in a hydraulic press at 1 ton to 5 ton pressure to obtain the said sustained release formulation.
The dried powdered mixture comprises micro-organisms selected from the group comprising Bacillus subtilis ATCC 11774, Bacillus licheniformis ATCC 14580 and Bacillus megaterium ATCC 9885.
The binder(s) employed in step (a) of the process are selected from the group comprising glycerol monostearate, stearic acid, polyethylene glycol, long chain pyrrolidones and their derivatives, long chain fatty acids and their salts and derivatives, glycolic compounds, hydrates of inorganic salts or any other hydrophobic or hydrophilic material or combinations thereof.
The disintegration of the pellet comprising the present sustained release formulation was studied under simulated conditions which involved placing the tablet on a wired mesh and intermittent pouring of water over it.

In yet another preferred embodiment, the present invention provides a sustained release formulation for an extended period of time ranging from about 3 days to about 3 months.
The present formulation was evaluated for its dissolution profile and was found to take around 3 days to 3 months to completely dissolve. The formulation was subjected to definite volumes of urine aspirated every hour for consequent 12 hours. Evaluation of the dissolution of the present formulation in water bodies showed an extended release profile from 3 days to over 3 months.
In another embodiment, the present invention provides a sustained release formulation in the form of pellets, balls, granules, blocks, tablets, bricks, slabs, bullets and the like.
In a further preferred embodiment, the present invention provides a tablet formulation of the sustained release formulation comprising;
(i) a bioactive agent in a concentration ranging from 9 gm% to 91 gm%;
(ii) binders selected from the group comprising glycerol monostearate, stearic acid and polyethylene glycol in a concentration ranging from 16 gm% to 67 gm%; and
(iii) a salt of fatty acid in a concentration ranging from 3 gm% to 50 gm% .

More preferably, the present invention provides a tablet formulation of the sustained release formulation comprising;
(i) a bioactive agent in a concentration ranging from 10 gm% to 45 gm%;
(ii) binders selected from the group comprising glycerol monostearate, stearic acid and polyethylene glycol in a concentration ranging from 25 gm% to 50 gm%;
(iii) a salt of fatty acid in a concentration ranging from 3 gm% to 10 gm%.

In yet another embodiment, the tablet formulation comprising the sustained release formulation can be prepared by the compression method, while brick/or blocks are prepared by compression or moulding/casting methods.
The concentration of the binder can be varied depending on the application area/volume, size of the formulation and desired sustained release time by varying the combination as well as quantity of the binders used. Source of nutrients to support the growth of bacteria can also be supplied along with the formulation.
In one embodiment, the present invention provides the use of the sustained release formulation in degradation of organic wastes in bio-digestor and septic tanks. The present sustained release formulation in the form of pellets, balls, granules, blocks, tablets, bricks, slabs or bullets is required to be introduced in septic/ bio-digester tanks. Accordingly, the sustained release formulations may be introduced in the tank in an amount ranging from about 1gm/L to about 100gm/L.
In one more embodiment, the present invention provides the use of the sustained release formulations in deodorization of septic tanks, urinals and open sewage systems. The sustained release formulations may be introduced in the aforesaid systems in an amount ranging from about 1gm/L to about 100gm/L.
Further, the present sustained release formulation is used in the remediation of ponds or lakes having high levels of eutrophication. The formulations so prepared can also be used in the wastewater treatment sector to treat industrial effluents with microbial consortia/microbes individually contained in the said formulations.
The present sustained release formulation helps to provide an optimum dosage of microbial population for efficient bioremediation without the need of frequent dosing of microbial formulations. Thus, the study of sustained release of microbial formulation was carried out in in-vitro conditions.
The efficiency of the present formulation in water treatment processes is estimated using measurement of water quality parameters such as chemical oxygen demand (COD), phosphate content and ammonia content, nitrate content and others chemical parameter tests. Accordingly, the bioremediation efficiency of the sustained release formulation was determined by monitoring the change in COD and phosphate levels in the in-vitro effluent studies performed. The reduction in the said parameters is an indication of positive bioremediation activity.
Examples: Following examples are given by way of illustration therefore should not be construed to limit the scope of the invention.
Example 1: Selection of binders for sustained release formulation
Different binders were tested for preparation of sustained release formulations. These included sugars such as dextrose, sucrose and lactose polymers such as starch, dextrin, o-polyethylene glycol 6000 and microcrystalline cellulose 105 fatty acid salts such as palmitic acid, stearic acid, glycerol monostearate, magnesium stearate and sodium oleate and inert insoluble material such as talc and bentonite. 5 gm of individual binder was compressed at 3 ton pressure using a hydraulic press to form a single unit compacted form. Each compacted binder was dispensed in 1 litre of water and disintegration pattern was observed. The observations are recorded in the following table:
Table 1: Disintegration pattern of different binders
Binder Disintegration pattern Disintegration time
Dextrose Slow and gradual disintegration without forming cracks 1 hour 15 minutes
Sucrose Fast disintegration without forming cracks 16 minutes
Lactose Very immediate break down and disintegration into fine particles 2 minutes
Starch Irregular disintegration into large layers of matrix due to capping 8 hours
Dextrin Irregular disintegration into large lumps at the bottom of the flask 3 hours
O-polyethylene glycol 6000 Slow and gradual disintegration without forming cracks 4 hours
Microcrystalline cellulose 105 Very immediate break down and disintegration into large layers of matrix due to capping 2 minutes
Palmitic acid No change in formulation structure Above 5 days
Stearic acid Slow release with crack formation (about 10% reduction occurred at day 3) Above 5 days
Glycerol monostearate Slow release of material with large cracks formed due to swelling of matrix 2 days
Sodium oleate Release is slow and gradual but creates a milky emulsion in water 2 days
Talc No change in formulation structure Above 5 days
magnesium stearate No change in formulation structure Above 5 days
bentonite Irregular disintegration into large particles forming a lump at the bottom of the flask 2 hours

From the above table it was found that compacted forms of microcrystalline cellulose, dextrose, sucrose and lactose had a very quick complete disintegration or quick release of binder material which is not ideal for application in sustained release. The disintegration pattern of palmitic acid, magnesium stearate and talc were also found to be unfavourable due to practically no disintegration over a duration of 5 days. The disintegration pattern of starch, dextrin and bentonite was irregular and immediate. The disintegration pattern of sodium oleate was found to be slow and gradual. Sodium oleate also showed prolonged disintegration but resulted in emulsion formation and thus was considered not apt to be used in a sustained release formulation.
Conclusively, from the above disintegration pattern study stearic acid, o-polyethylene glycol 6000 and glycerol monostearate were selected to be used in preparation of the present sustained release formulation. The use of combination of these binders provides a prolonged release of material as each component provides a distinct disintegration pattern. Also, as all the three ingredients are biodegradable the use of these binders does not impose an adverse environmental impact.
Example 2: Preparation of formulations
Formulation A:
0.53 gm of mixture of formulation A containing spray dried / tray dried / oven dried or freeze dried powders of three of the bacteria used in remediation of wastewater and waterbodies (viz. B subtilis ATCC 11774, Bacillus licheniformis ATCC 14580 and Bacillus megaterium ATCC 9885) was mixed with 3.121 gm of binder mixture containing 1.41gm of each of o-polyethylene glycol 6000, glycerol monostearate and stearic acid. This was then supplemented with 0.25gm of magnesium stearate. It was supplemented with 0.25 ml of commercially available fragrance. This mixture was then compressed in a hydraulic press at 3 ton pressure.
Table 1(A): Weight of each component in 5.01 gm of formulation.
Components Weight in grams Weight Percentage in %
Culture consortium powder 0.53 10.579
o-polyethylene glycol 6000 1.41 28.144
glycerol monostearate 1.41 28.144
stearic acid 1.41 28.144
magnesium stearate 0.25 4.99
Total weight 5.01 100%

Example 3: Study of disintegration potential of formulations containing selected binders
Each of the three binders shortlisted in Example 1 was mixed with bacterial consortium (useful in remediation of waste water and water-bodies) in a 1:9 proportion. Additionally, equi-proportional mixture of all three binders was also mixed with bacterial consortium in 1:9 proportions. All the formulations were supplemented with magnesium stearate in a ratio of 20:1. The four mixtures were individually compressed using a hydraulic press at 3 ton pressure to form a formulation. Each compressed formulation was dispensed in 500 ml water. The disintegration patterns of the tablets were also observed over time. The observations are noted in the table herein below.
Table 2: Disintegration pattern of different binder formulation
Binder Disintegration after 3 hours of soaking Disintegration after 24 hours of soaking Disintegration after 72 hours of soaking
o-polyethylene glycol 6000 100% disintegration with no residue left over - -
glycerol monostearate Partial disintegration 100% disintegration of tablet and residue material observed -
stearic acid No disintegration was observed No disintegration was observed Tablet structure retained with few shredded residue material
glycerol monostearate : stearic acid : o-polyethylene glycol 6000 (1:1:1) 20-30% disintegration 60% disintegration 100% disintegration

As observed from the above table, it is observed that the rate of disintegration of o-polyethylene glycol 6000 formulation is the highest between the three binders tested. Glycerol monostearate formulation showed moderate disintegration and release. The stearic acid formulation showed poor disintegration. The equi-proportional mixtures comprising o-polyethylene glycol 6000, glycerol monostearate and stearic acid disintegrated in a gradual pattern which resulted in better release after three days. Thus, to obtain a gradual and slow release of active ingredients in the formulation, mixture of materials like o-polyethylene glycol 6000 and glycerol monostearate were used to create hydrophilic channels in between a hydrophobic matrix like stearic acid. Thus, to obtain a longer, rather differential sustained release the combination of the above binders needs to be carried out.
Example 4: Selection of binder proportions
The selected binders were combined in different proportions such as 1:1:1, 4:1:1, 1:4:1 and 1:1:4 of glycerol monostearate, stearic acid and o-polyethylene glycol 6000 respectively. The different binder combinations were mixed with bacterial consortium in the ratio 9:1. This was then supplemented with magnesium stearate in the ratio of 20:1 and compressed in a hydraulic press at 3-ton pressure. Formulations so prepared were placed in 1 litre sterile water suspended in a muslin cloth mesh bag and incubated at RT under static conditions. After every 24 hour of incubation, the tablet was aseptically shifted to another flask containing 1 litre sterile water. This assembly was again kept under static condition for another 24 hours. The process was repeated till complete disintegration of each formulation. The disintegration pattern was observed by intermittently drawing samples and viable count was estimated for the same. The observation of release pattern of different combination is as follows:
Table 3: Disintegration pattern for formulation for different binder proportions in a formulation weighing 5.01gm.
Binder Proportions Weight of Glycerol monostearate Weight of Stearic acid in Weight of o-polyethylene glycol 6000 Disintegration percentage
Day 1 Day 2 Day 3 Day 4 Day 5
1:1:1 (G:S:P) 1.67 gm 1.67 gm 1.67 gm 20 % disintegration 60 % disintegration 100% disintegration - -
4:1:1 (G:S:P) 3.33 gm 0.83 gm 0.83 gm 80% disintegration 100% disintegration - - -
1:4:1 (G:S:P) 0.83 gm 3.33 gm 0.83 gm No change No change No change No change No disintegration even above day 5
1:1:4 (G:S:P) 0.83 gm 0.83 gm 3.33 gm 50% disintegration 70% disintegration 100% disintegration - -
(G- glycerol monostearate, S – stearic acid, P – o-polyethylene glycol 6000)

Observations from the above table indicate that at equi-proportional concentrations, the release of microbial population is at a slow constant rate of disintegration and release. Increasing the concentration of glycerol monostearate and o- polyethylene glycol resulted in faster disintegration of tablet. Increasing the concentration of stearic acid resulted in poor disintegration and release of material. Thus, the proportion of different binders can be varied in the formulation to obtain desired duration of complete disintegration and sustained release of active ingredients.

Example 5(A): Effect of different compression pressure on sustained release

A formulation with glycerol monostearate, stearic acid and O-polyethylene glycol 6000 in 1:1:1 was mixed with microbial consortium in 9:1 proportion. This was then supplemented with magnesium stearate in a ratio of 20:1 and compressed at different pressure. The compression was carried out at pressures of 1 ton, 2 ton, 3 ton, 4 ton and 5 ton pressure using a hydraulic press. The formulations so prepared were placed in 1 litre sterile water suspended in muslin cloth mesh bag and incubated at RT under static conditions. After every 24 hour of incubation the tablet was shifted into a litre sterile water flask under sterile conditions. The observation of release pattern of different combination is as follows:
Table 4(A): Disintegration patterns for different compression pressures
Different compression pressures Disintegration
Day 1 Day 2 Day 3
1 ton 10 % disintegration 40% disintegration 100% disintegration
2 ton 10 % disintegration 40% disintegration 100% disintegration
3 ton 10 % disintegration 40% disintegration 100% disintegration
4 ton 10 % disintegration 40% disintegration 100% disintegration
5 ton 10 % disintegration 40% disintegration 100% disintegration

The above table indicates that there is no significance of pressure in the disintegration pattern of the different tablets. Hence an intermediate pressure of 3 ton was used during the preparation of formulation.

Example 5(B): Effect of compression on bacteria
The bacteria used in the formulation were evaluated for their robustness under conditions of pelleting/moulding/casting. Pressure used for pelleting was 3 ton. The stability of bacteria at this pressure was checked by counting the number of bacteria in terms of CFU/gm before pelleting (i.e. in the powdered form) and after pelleting. The study was conducted individually for each culture and for the consortium as well.
Results of the study are documented in the table below:
Table 4(B):
Culture/consortium name CFU/gm of bacteria in powder (before compression) CFU/gm of bacteria in pellet (after compression)
Bacillus subtilis 1.9 x 107 1.2 x 107
Bacillus megaterium 3.2 x 107 2.8 x 107
Bacillus licheniformis 1.7 x 107 1.1 x 107
Consortium 6.0 x 107 5.4 x 107

Example 6: Study of different combinations of Magnesium Stearate
Glycerol monostearate, Stearic acid and O-polyethylene glycol 6000 in a 1:1:1 ratio was mixed with a microbial carrier consortium in a 9:1 proportion. This mixture was then mixed with magnesium stearate in different ratios of 30:1, 20:1, 10:1 and 1:1 respectively. The compression of all the formulations was carried out at 3 ton pressure using a hydraulic press. The compressed combinations were placed in 1litre sterile water suspended in a muslin cloth mesh bag and incubated at RT under static conditions. The observation of release pattern of different combinations is as follows:
Table 5: Disintegration pattern observations for different combinations of magnesium stearate in 5 gm of formulation
Different concentrations of magnesium stearate. Disintegration pattern
Weight of magnesium stearate Day 1 Day 2 Day 3 Day 4 Day 5
30:1 0.167 gm 50% disintegration 100% disintegration - - -
20:1 0.25 gm 30% disintegration 60% disintegration 100% disintegration - -
10:1 0.5 gm 20% disintegration 30% disintegration 60% disintegration 70% disintegration No change
1:1 5 gm No change No change No change No change No change

The above observations indicate that increasing the concentration of magnesium stearate delays the disintegration of the formulation. The combination of 20:1 showed better and consistent release of material intermediary to all the formulation used. The combination 1:1 showed very poor release of material indicating that an optimum concentration (1:20) of magnesium stearate in the formulation is a necessity for efficient release of material.

Example 7: Effect of different proportions of culture consortium
An equi-proportionate mixture of glycerol monostearate: stearic acid : O-polyethylene glycol 6000 was mixed with microbial consortium in different proportions such as 1:10, 1:9, 2:8, 5:5, 8:2, 9:1 and10:1. All the formulations were supplemented with magnesium stearate in the ratio 20:1 and compressed in a hydraulic press at 3 ton pressure. The compressed combinations were placed in 1 litre sterile water suspended in a muslin cloth mesh bag and incubated at RT under static conditions.

After every 24 hour of incubation the tablet was shifted into another flask of 1 litre sterile water and incubated under sterile conditions. The disintegration pattern was observed by intermittently drawing samples and viable count was estimated for the same. The observation of release pattern of different combination is as follows:Table 5: Disintegration pattern and microbial release observations for different binder to culture consortium proportions
Culture to Binder consortium Proportions Weight of culture consortium in 5 gm of formulation Weight of binder in 5 gm of formulation Disintegration
Day 1 Day 2 Day 3 Day 4 Day 5
10:1 0.5 gm 4.5 gm 10% disintegration 20% disintegration No further change No further change No further change9
9:1 0.55 gm 4.45 gm 10% disintegration 10% disintegration 20% disintegration No further change No further change
8:2 1.25 gm 3.75 gm 10% disintegration 10% Reduction 10% disintegration 20% disintegration No further change
5:5 2.5 gm 2.5 gm 20% disintegration 20% disintegration 40% disintegration 50% disintegration No further change
2:8 3.75 gm 1.25 gm 30% disintegration 30% disintegration 50% disintegration 60% disintegration No further change
1:9 4.45 gm 0.55 gm 30% disintegration 60% disintegration 100% disintegration --- ---
1:10 4.5 gm 0.5 gm 20% disintegration 30% disintegration 50% disintegration 60% disintegration No further change

The observations of the above table indicate that increasing the concentration of carrier based culture consortium had slow disintegration of the tablet. Thus addition of minimum binder formulation to culture consortium in the ratio 9:1 was preferred over other proportions. The concentration of binder mixture however can be changed to obtain desired duration of sustained release.

Example 8: Study of release patterns
(i) Release pattern studies for urinals: The release pattern for both the formulation A was studied under simulated conditions for urinals. The set up for the same consisted of a funnel with metal mesh above which an aspirated bottle with artificial urine was fixed in an inverted position. The formulation to be evaluated for its release pattern is placed in the funnel. To simulate the liquid flow in the urinal, definite volumes of artificial urine was aspirated every hour for consequent 12 hours. The time taken for complete dissolution of the formulation was recorded for the formulation is recorded in the table below.
Table 7: Release pattern for different formulations in urinals

Sr No Formulation code Time taken for complete dissolution
1 Formulation A 7 days

It was observed that changing nature and concentration of the binders could results in differential dissolution times under simulated conditions. Different values of dissolution time may be achieved by slight modifications in the formulation.
(ii) Release pattern studies for static water bodies: The release pattern for both the formulation A was studied under simulated conditions for urinals. To do so, formulations A was submerged in a flask containing 1 Litre of water and were kept at static condition. The time taken for complete dissolution of the formulation was recorded for the formulation and recorded in table below:
Table 8: Release pattern for formulation in static water bodies
Sr No Formulation code Time taken for complete dissolution
1 Formulation A 3 days

Example 9: Compatibility testing of the cultures used in the consortium

Compatibility of both the microbes used in the consortium was studies by cross streaking both the culture on an agar plate in triplicates. The plates were incubated at two different temperatures viz 25°C and 37°C. No growth inhibition was observed for any of the cultures under any incubation temperatures. Thus, it can be concluded that Bacillus subtilis ATCC 11774 and B. megaterium ATCC 9885, and B. licheniformis ATCC 14580 are compatible with each other and do not pose any inhibitory action on each other at any incubation temperature. Hence, they can safely be contained in the bacterial consortium of the present formulation.
Example 10: Enzyme activity of the cultures used in consortium
Enzymes play a crucial role in bioremediation of wastewater or any water body. The enzyme assay testing of the microbes used in the consortium was performed using agar plug method. The detection of cellulase activity was carried out on cellulase agar containing carboxy methyl cellulose 1.5g/L and agar 10 g/L in 1 litre of water. The detection of amylase activity was carried out using amylase agar containing soluble starch 15 g/L and agar 1g/L in 1 litre of water. The detection of lipase activity was carried out using lipase agar containing glycerol tributyrate 15 g/L and agar 1g/L in 1 litre of water. The detection of pectinase activity was carried out using pectinase agar containing pectin 15 g/L and agar 1g/L in 1 litre of water.

The detection of protease activity was carried out using protease agar containing sodium caseinate 15 g/L and agar 1g/L in 1 litre of water. The 72 hour old culture plugs of individual strain was placed on specific enzyme substrate agar and incubated for 24 hours. After completion of the incubation period, the staining of cellulase enzyme plates and pectinase agar plates was performed by flooding agar plates with 0.3 % Congo red solution for 5 minutes followed by washing/de-staining with 0.584 % sodium chloride for 5 minutes. The observation of colourless zone of clearance is an indication of positive enzyme activity. For lipase and protease agar the observation of zone of clearance or halo on respective agar plates around the plugs is a confirmation of enzyme activity. The detection of amylase activity is carried out by staining the amylase agar plate with staining solution containing iodine 3.3gm/L, potassium iodide 6.7g/L in 1 litre of water.

The observation of unstained zones around the plug indicates positive amylase activity. The detection of uricase activity was performed by streaking the culture on uricase agar containing uric acid 2.5 g/L, magnesium sulphate 0.200 g/L, calcium chloride 0.020 g/L, monopotassium phosphate 1.000 g/L, dipotassium phosphate 1.000 g/L, ammonium nitrate 1.000 g/L, ferric chloride 0.050 g/L, agar 20.000 g/L, final pH 7.0±0.2. The results of the enzyme assay performed for the strains are as follows:

Table 9: Enzyme activity of test strains on different enzyme substrate media
Strains Cellulase Amylase Lipase Pectinase Protease Uricase
Bacillus licheniformis ATCC 14580 ++ + ++ ++ ++ +++
Bacillus subtilis ATCC 11774 + +++ ++ ++ ++ +++
Bacillus megaterium ATCC 9885 + + ++ ++ + +
Key: + - slight activity, ++ - moderate activity, +++ - high activity

As shown in the table above, each member of the bacterial consortium have enzyme production of some degree. Thus, making use of these bacteria in the formulation will be effective in bioremediation of water bodies.

Example 11: Bioremediation potential of type strains
The COD reduction activity of the strains were individually tested in a synthetic effluent, with composition urea 1.5g/L, Ammonium Chloride 0.22 g/L, Sodium acetate 1.368g/L, Peptone 0.3 g/L, Magnesium sulphate 0.5g/L Potassium dihydrogen orthophosphate 0.4 g/L, Ferrous sulphate 0.1 g/L in 1 litre of water. The culture suspension of respective Bacillus subtilis ATCC 11774 and B. megaterium ATCC 9885, and B. licheniformis ATCC 14580 were prepared and inoculated in a sterile synthetic effluent at 1% inoculum density. The COD reduction of the test culture in the synthetic effluent was recorded after 72 hours of incubation under shake flask condition.
The COD reduction was observed as below:
Table 10: COD reduction of different bacteria in synthetic effluent
Samples COD (in ppm) COD Reduction %
Blank Control 2635 -
B. licheniformis ATCC 14580 595 77.00%
Bacillus subtilis ATCC 11774 310 88.00%
B. megaterium ATCC 9885 750 71.00%
Consortium 80 97.00%

The above table indicates that the strains Bacillus subtilis ATCC 11774 and B. megaterium ATCC 9885 and B. licheniformis ATCC 14580 have a significant COD reduction activity. Hence, utilization of these strains in the given formulation will impart the bioremediation capacity to the formulation.

Example 12: Sustained release of microbes from tablets
Formulation A comprising of spray dried / tray dried / oven dried or freeze dried powders of three of the bacteria used in remediation of wastewater and waterbodies (viz. B subtilis ATCC 11774, Bacillus licheniformis ATCC 14580) was mixed with equi-proportional mixture of o-polyethylene glycol 6000, glycerol monostearate and stearic acid in the proportion of 1:9. This was then supplemented with magnesium stearate in a ratio of 20:1. 5 gm of mixture was then compressed in a hydraulic press at 3 ton pressure. Formulation was suspended in 1 litre sterile water suspended in muslin cloth mesh bag and incubated at RT under static conditions in 1 litre of sterile water and incubated at room temperature under static condition. The initial count was enumerated followed by the count of the 24 hour incubated sample. Subsequently after 24 hour of incubation the tablet was removed from the initial suspension and transferred to a separate flask containing sterile water. All transfers were conducted under sterile condition of a laminar flow hood.

Table 11: Sustained release of bacteria under static condition
Sample Count (CFU/ml)
Bacterial consortium
(powder form) Formulation A
Day 0 3x 105 -
Day 1 - 2 x 103
Day 2 - 2 x 103
Day 3 - 4.8 x 103

Thus, by observing the results of tablet discharge it is indicated that the release pattern observed for Formulation A indicates a sustained release of microbial population as compared to of bacterial consortium upto day 3.

Example 13: Bioremediation potential (COD reduction activity) of sustained release formulations
Formulation A comprising spray dried / tray dried / oven dried or freeze dried powders of three of the bacteria used in remediation of wastewater and waterbodies (viz. B subtilis ATCC 11774, Bacillus licheniformis ATCC 14580) was mixed with equi-proportional mixture of o-polyethylene glycol 6000, glycerol monostearate and stearic acid in the proportion of 1:9. This was then supplemented with magnesium stearate in a ratio of 20:1. 5 gm of mixture was then compressed in a hydraulic press at 3 ton pressure. The COD reduction activity of compressed Formulation A tablet was tested in synthetic effluent with composition glycerol 1 g/L, ammonium chloride 0.5g/L and sodium phosphate 0.3 g/L in 1 litre of water. 5 gm tablet of Formulation A were respectively placed in 200 ml of synthetic effluent in 500ml volumetric flask suspended in muslin cloth mesh bag. After 24 hours of incubation and the tablet was transferred into sterile synthetic effluent flask. The COD, ammonia and phosphate reduction in the samples was estimated for all the samples.

Table 12: COD reduction of different formulations in Synthetic effluent
Samples COD (in ppm)
Blank Broth 11750

Day 1 Day 2 Day 3
Formulation A powder 10580 - -
Formulation A tablet 270 5380 2370
The results of COD reduction test performed indicate positive reduction in phosphate concentration in tablet formulation as compared to powder.

Example 14: Bioremediation phosphate reduction activity of sustained release formulations
Higher amount of phosphate is one of the major problems in wastewater. The phosphate reduction activity of Formulation A tablet was tested in synthetic effluent, with composition glycerol 1 g/L, ammonium chloride 0.5g/L and sodium phosphate 0.3 g/L in 1 litre of water. 5 gm tablet of Formulation A were respectively placed in 200 ml of synthetic effluent in 500ml volumetric flask suspended in muslin cloth mesh bag. After 24 hours of incubation, the tablet was transferred into sterile synthetic effluent flask. The phosphate reduction in the samples was estimated for all the samples.

Table 13: Phosphate reduction of different formulations in Synthetic effluent
Samples Phosphate (in ppm)
Blank broth 260
Day 1 Day 2 Day 3
Formulation A powder 210 - -
Formulation A tablet 90 182 211

The results of phosphate reduction test performed indicate positive reduction in phosphate concentration in tablet formulation as compared to powder.

Example 15: Study of sustained release of bioactive compounds viz., enzymes
Enzymes play a crucial role in remediation of water bodies. Owing to this, the formulation containing enzymes (protease) in place of bacteria were prepared and studied for their sustained release pattern. Glycerol monostearate : stearic acid : O-polyethylene glycol 6000 in a 1:1:1 ratio, was mixed with an enzyme mix powder in a 9:1 proportion, respectively. The formulation was then mixed with magnesium stearate at 20:1 proportion and compressed at 3 ton pressure using a hydraulic press. The compressed combinations were placed in 1 litre sterile water suspended in muslin cloth mesh bag and incubated at RT under static conditions. After every 24 hour of incubation the formulation was shifted into a 200 ml water flask under sterile conditions. The enzyme mix powder was diluted in the same ratio with binder matrix in 9:1 ratio material and added to 200ml of sterile water as control. The enzyme assay was performed for all the samples collected at 24 hour interval. The enzyme assay was performed by loading 20 microlitres of each sample in agar wells in protease agar containing sodium caseinate 15 g/L and agar 1g/L in 1 litre of water. The agar plates were incubated for 24 hours and zone sizes were measured as follows:

Table 14: Sustained release of enzymes from the formulation
Day 0 Day 1 Day 2 Day 3
Powder 22mm 22 mm 0 mm 0 mm
Formulation 0 mm 20mm 16mm 16mm

The diameter of the zones represented the amount of enzyme released. Thus from the above observations, it is evident that the given fromulation was able to release same quantities of enzyme at regular interval of time upto its complete disintegration.

Example 16: Study of sustained release of bioactive compounds, viz., Sodium alginate
Sodium alginate is a common chemical used for flocculation of the liquid waste and can be used as bioactive agent in the present sustained release formulation. Glycerol monostearate : stearic acid : O-polyethylene glycol 6000 in a 1:4:1 ratio, was mixed with sodium alginate powder in a 9:1 proportion. The formulation was then mixed with magnesium stearate at 20:1 proportion and compressed at 3 ton pressure using hydraulic press. The compressed formulation was placed in 1 litre sterile water suspended in muslin cloth mesh bag and incubated at RT under static conditions. After every 24 hours of incubation, the tablet was shifted into a 1 litre sterile water flask under sterile conditions. The sodium alginate powder was diluted in the same ratio with a binder matrix in a 9:1 ratio material and added to 200ml of sterile water as a control. Samples from the flask were collected after every 24 hour incubation and flocculation test was performed for the same. The flocculation test was carried out on filtered samples by adding Kaolin at 4g /L concentration and 0.25g/L Calcium chloride followed by thorough mixing of the resultant suspension. The absorbance at 550nm of the supernatant after flocculation was measured for all samples after an incubation time of 2 min at 550nm. The flocculation percentage was estimated by measuring the percentage difference between the absorbance of kaolin solution in plain water and test samples.The observation of release pattern of different combination is as follows: Table 15: Sustained release of sodium alginate from the formulation
Day 0 Day 1 Day 2 Day 3
Flocculation % Flocculation % Flocculation % Flocculation %
Powder 87% - - -
Tablet 2.44% 90.28% 97.2% 96.73%
(Abs – Abosrbance at 550nm, Floc% -Flocculation %)

Thus from the above observations it was concluded that the release of sodium alginate was achieved in a sustained pattern to achieve flocculation activity. The flocculation potential of sustained release formulation was much higher than the control. Such sustained release formulation is of tremendous potential in remediation of bodies where there is a regular influx of flocculating or sedimentary waste.

Advantages of the invention:
• The formulations of the present invention provide a sustained release of consortium of microbes having degenerative potential that help in efficient removal of organic and recalcitrant waste generated during sanitary and industrial practices.
• The formulations can be used for remediation of waste water and water bodies.
• The formulation can even be used in a bio-digestor tank or septic tank for degradation of organics waste, removal of odour and reduction in pathogenic load of the sewage present in septic tank, sewage treatment plants and open sewer systems or open channels leading to waste disposal sites.
• In case of urinals, the bacteria contained in the present formulations have the ability to flow through urinal pipes and colonize walls of the pipes to prevent ammonia generation and pipe corrosion. The run off from these formulations helps to fortify the microbial flora in septic tanks and helps in efficient remediation of the biological sewage. While in case of digestor tanks, ponds, lakes, industrial wastewater treatment tanks, the accurately weighed formulation can be directly applied to achieve bacterial action over a desired period of time.
• The formulation is easy for application and can deliver accurate amount of dosage.
• Source of nutrients can also be introduced in the formulation to facilitate the growth of microbes and enhance the efficiency of the formulation.
,CLAIMS:1. A sustained release formulation comprising at least one bioactive agent and a combination of binders selected from the group comprising glycerol monostearate, stearic acid and polyethylene glycol in a ratio ranging from 1:10 to 10:1 for liquid waste management and bioremediation of water-bodies.
2. The sustained release formulation as claimed in claim 1, wherein the said formulation is further supplemented with a salt of fatty acid in a ratio ranging from 30:1 to 1:1, respectively.
3. The sustained release formulation as claimed in claim 1, wherein the said salt of fatty acid is selected from the group comprising magnesium stearate, calcium stearate and zinc stearate.
4. The sustained release formulation as claimed in claim 1, wherein the said bioactive agent is selected from the group comprising micro-organisms, enzymes, herbal ingredients, bio-surfactants, biopolymers, chelators, natural, synthetic or semisynthetic substances or combinations thereof.
5. The sustained release formulation as claimed in claim 4, wherein the said micro-organism is selected from Bacillus species which is further selected from the group comprising Bacillus subtilis, Bacillus licheniformis and Bacillus megaterium.
6. The sustained release formulation as claimed in claim 5, wherein the concentration of the said Bacillus species is ranging from 1 x 102 CFU/gm to 1 x 107 CFU/gm.
7. The sustained release formulation as claimed in claim 1, wherein the combinations of binders selected from the group comprising glycerol monostearate, stearic acid and polyethylene glycol is in ratio ranging from 1:1:1 to 1:4:1 respectively.
8. The sustained release formulation as claimed in claim 1, wherein the said formulation is in the form of tablets, pellets, balls, granules, blocks, bricks, slabs or bullets.
9. The sustained release formulation as claimed in claim 1, wherein the said formulation comprises excipients selected from the group comprising talc, husks of grains or fruits, chelating agents, disintegrants, fragrances, dyes and natural, synthetic or bio-organic chemicals.
10. A tablet formulation of the sustained release formulation as claimed in claim 1 comprising;
(i) a bioactive agent in a concentration ranging from 9 gm% to 91% gm%;
(ii) binders selected from the group comprising glycerol monostearate, stearic acid and polyethylene glycol in a concentration ranging from 16 gm% to 67 gm%; and
(iii) a salt of fatty acid in a concentration ranging from 3 gm% to 50 gm% .
11. The tablet formulation as claimed in claim 10, wherein the bioactive agent is selected from the group comprising micro-organisms, enzymes, herbal ingredients, bio-surfactants, biopolymers, chelators, natural, synthetic or semisynthetic substances or combinations thereof
12. A process for preparing a sustained release formulation as claimed in claim 1, the said process comprising;
(i) providing at least one micro-organism or a consortium of micro-organisms in a concentration ranging from of 1 x 102 CFU/gm to 1 x 107 CFU/gm;
(ii) supplementing the dried powder of step (i) with binders selected from the group comprising glycerol monostearate, stearic acid and polyethylene glycol in a concentration ranging from about 1:200 to about 1:1, respectively, to provide a mixture comprising micro-organisms and a binder in a ratio ranging from 1:10 to 10:1;
(iii) supplementing the mixture of step (ii) with a salt of fatty acid in a ratio of 20:1, respectively; and
(iv) compressing the mixture obtained in step (iii) in a hydraulic press at 1 ton to 5 ton pressure to obtain the said sustained release formulation.