DESC:Field of the invention:
The present invention relates to the field of environmental biotechnology. More particularly, the present invention relates to field of wastewater treatment.

The present invention relates to a microbial formulation for biodegradation of wastewater, dealing with common problem faced in sewage treatment plants like poor sludge settling, odour, and inadequate nutrient removal and improper sludge volume. The present invention also relates to a novel submerged technology to obtain biological formulation for wastewater treatment. The microbial formulation of the present invention is capable of achieving recommended discharge norms of wastewater, recovery and reuse of treated wastewater and stabilizes plant performances to handle shock load and process fluctuation.

Background of the invention:
Due to population growth, accelerated urbanization and economic development, the quantity of wastewater generated and diversity of pollution load are increasing globally. This ultimately puts increasing pressure on natural water resources. The availability of safe and sufficient water supplies is inextricably linked to wastewater management. Increased amounts of untreated sewage, combined with agricultural runoff and industrial discharge, have degraded water quality and contaminated water resources around the world. Globally, 80% of wastewater flows back into the ecosystem without being treated or reused, contributing to a situation where around 1.8 billion people use a source of drinking water contaminated with faeces, putting them at risk. In developing countries, a large proportion of wastewater is discharged directly into the surface water drain or informal drainage channel, sometime without or with very little treatment. The growth of urban demand for water will require new approaches to wastewater treatment and management. The stench produced during Sewage Treatment Plant (STP) operation is carried by air to the nearby residence causing nuisance. A non-performing STP is a potential hazard to the environment, discharge of untreated wastewater led to surface and ground water contamination. The cost of operating/maintenance and performance of STP’s are prime problem in treating wastewater.

Wastewater treatment technologies depend on combination of physical, chemical and biological factors. Improving the treatment process by altering physical/chemical factors needs a huge capital investment, whereas altering/engineering biological factor is cost effective.

CN102863122B discloses a method and apparatus for sewage treatment. The method includes precipitation digestion, anaerobic reactor, aerobic biodegradation, re-precipitation process. The anaerobic reaction is segmented into hydrolytic acidification and two methanogenesis process. This setup facilitates appropriate environment for different microbes to thrive in the suitable environment and increases pollutant degradation capabilities. In aerobic process, biological membrane is attached to a thin layer, where water comes in contact with air and high oxygen transferred is achieved. In large-scale the biofilm contacts both air and sewage alternatively. Compared with the prior art, this setup provides optimized spatial arrangement of functional units, compact structure, better sewage treatment effect, no energy, no personnel to operate and high utilization of the process space.

JP2002263684A teaches continuous and efficient waste water treatment by treating waste water of high concentration by using microorganisms which are mainly and further subjecting the waste water with lower concentration obtained in the above treatment to biofilm treatment. The method for treating wastewater by microorganisms has the following features. A culture tank of microorganisms is charged with the waste water, microorganisms which are mainly bacillus and an activating agent for the microorganisms so as to breed and culture the microorganisms under aerobic conditions. Then the treated water is subjected to biofilm treatment and sent to a precipitation tank, while a part of the sludge of microorganisms produced by the biofilm treatment is returned to one of or both of the culture tank of microorganisms and the biofilm treating tank.

US 6383797 discloses bacterial consortium EBC1000 and a method using the bacterial consortium EBC1000 for remedying biologically recalcitrant toxic chemicals contained in industrial wastewater, waste materials and soils”. The consortium uses genetically modified organisms.

IN201821033799 discloses a process for bio-sludge reduction in hydrocarbon refinery effluent treatment plant through microbial interventions. The process provides utilizing microbial consortia comprising at least one species of Pseudomonas and at least one species of Bacillus in a ratio of 10:1 to 1:10. The species of Pseudomonas and species of Bacillus have constitutive expression of at least one hydrocarbon degrading gene. The species of Pseudomonas are selected from the group consisting of Pseudomonas stutzeri (MTCC 25027), Pseudomonas aeruginosa (MTCC 5389), Pseudomonas aeruginosa strain IOC DHT (MTCC, 5385), Pseudomonas putida IOCR1 (MTCC 5387), Pseudomonas putida IOC5a1 (MTCC 5388) and a mutant thereof. The species of Bacillus are selected from the group consisting of Bacillus subtilis (MTCC 25026), Bacillus subtilis (MTCC 5386), Bacillus thermoleovorans (MTCC 25023), Bacillus stearothermophilus (MTCC 25030), Lysinibacillus sp. (MTCC 25029), Lysinibacillus sp. (MTCC 5666) and a mutant thereof. The microbial consortia is used in concentration of at least 102 CFU/ml.

CN102604865B discloses pseudomonas putida CT2025 with preservation department of CGMCC (China General Microbiological Culture Collection Center) and preservation address of No.3, No.1 Institution, Beichen West Road, Chaoyang District, Beijing, Chinese Academy of Sciences Institute of Microbiology, and preservation date of February 17th, 2012 and preservation number of CGMCC No.5767. The invention also discloses a harmless treatment method of caffeine-containing waste water. The method comprises the following steps of: inoculating 0.8-1.2kg of pseudomonas putida in every 100ton of caffeine-containing waste water, and intervening and applying in any one of the following waste water treatment stages: aerobic treatment stage after anaerobic fermentation and application after the aerobic treatment on the waste water is finished.

EP2890646A2 discloses a bio-inoculant composition to efficiently treat waste water of effluent treatment plants (ETP), comprising a consortia of microorganisms selected from group of Bacillus sp. (IOC-EP-1), Pseudomonas putida (IOC-EP2), Bacillus subtilis (IOC-EP-3) and Pseudomonas putida (IOC-EP4). The microbes have high ability of self-propagating at concentration of 102 cfu/ml in ETP waste water.

CN113403235A discloses a high-temperature-resistant petroleum hydrocarbon degrading bacterium, a degrading bacterium composition, a degrading bacterium agent and application thereof, belonging to the technical field of microorganisms and environmental protection. The invention obtains two new strains of Bacillus (Bacillus paramycoides) A4-1 and P4-3 through screening, wherein the Bacillus A4-1 has the capability of efficiently degrading crude oil in a high-temperature environment, and the Bacillus (Bacillus paramycoides) P4-3 has an efficient demulsification effect on O/W emulsion and O/W emulsion, so that oil-water separation is rapidly realized, and the two strains are mixed for use, so that oil-water separation and petroleum degradation can be synchronously realized, and the invention has good practical application value.

However, technology in the existing state of the art is costly, time consuming and shows low viable active ingredient. Therefore, there is an urgent need of environmental friendly and cost effective wastewater treatment technology without using genetically modified organisms.

Object of the invention:
To obviate the drawbacks in the existing state of the art, the main object of the present invention is to provide a biological formulation for wastewater treatment.

Another object of the present invention is to provide a biological formulation of microbial strains of desired enzymatic activity.

Yet another object of the present invention is to provide a biological formulation of consortium of microbial strain of desired enzymatic activity.

Yet another object of the present invention is to provide a stable, non-toxic and environmentally harmonious biological formulation capable to restore water quality and remove odour.

Yet another object of the present invention is to provide a process of obtaining a biological formulation for wastewater treatment.

Yet another object of the present invention is to provide a process of obtaining a biological formulation for wastewater treatment based on a novel submerged fermentation technology.

Yet another object of the present invention is to provide a biological formulation for wastewater treatment and a process to obtain thereof to reduce operational and maintenance cost of wastewater treatment.

Summary of the invention:
Accordingly, the present invention provides a novel submerged fermentation technology based biological formulation for enhanced biodegradation of waste water. The present invention provides a viable biological formulation that works in synergistic manner that helps to achieve recommended discharge norms, considerable reduction in operational and maintenance cost, faster settling of sludge and develop stable, non-toxic and environmentally harmonious biological formulation. The present invention relates to a composition of a microbial formulation promoting faster biological degradation of contaminants in wastewater. The present invention also provides a formulation with micronutrients that enhances the microbial activity in STPs and their mass multiplication.

It should be noted that the particular description with features, designs, components, construction, working and embodiments set forth in the specification below are merely exemplary of the wide variety and arrangement of instructions which can be employed with the present invention. The present invention maybe embodied in other specific forms without departing from the spirit or essential characteristics thereof. All the features disclosed in this specification may be replaced by similar other or alternative features performing similar or same or equivalent purposes. Thus, unless expressly stated otherwise, they all are within the scope of present invention. Various modifications or substitutions are also possible without departing from the scope or spirit of the present invention. Therefore, it is to be understood that this specification has been described by way of the most preferred embodiments and for the purposes of illustration and not limitation.

The present invention provides a novel submerged fermentation technology based biological formulation for enhanced biodegradation of contaminants of waste water and reuse of the treated water. The present invention enhances the process of biodegradation of domestic wastewater and reuse of the treated water by developing a stable, non-toxic, environmentally harmonious and viable biological formulation that works in synergistic manner together with achieving recommended discharge norms.

The biological formulation of the present invention comprises of microbial strains selected from, but not limited to, Bacillus paramycoides, Acidovorax temperans, Pseudomonas putida, Bacillus flexus, Bacillus megaterium, with desired enzymatic activity. The selected microbial strains are used alone or in various combinations as a consortium.

The process to obtain the biological formulation includes media optimization for said selected microbial strain or consortium of said selected microbial strains. The process is optimized by determining the optimized process parameters such as, but not limited to, temperature, pH etc.

In an embodiment, microorganism play an essential role in degradation of organic pollutant. The present invention reduces the energy required for operation/treatment process significantly and increases the average life span of the filters. The developed microbial consortium helps to achieve pollution control discharge norms in a shorter batch time with lesser intervention/alternation in the existing wastewater treatment process.

In another embodiment, microorganism plays an essential role in degradation of organic pollutant. The present invention utilizes a defined mixed microbial population so that the efficacy of the wastewater treatment plant can be increased and the load on subsequent filters namely multigrade filter/ activated carbon filter (MGF/ACF) can be minimized.

In an embodiment, the developed microbial consortium of the present invention is an autochthonous microbial population that exhibits synergistic effect. The present invention ultimately increases the rate of degradation of organic pollutants in the wastewater.

In an embodiment, the present invention describes development of robust, highly economical and efficacious formulation consisting of microbial strain or a consortium of microbial strains with compatible carrier or adjuvants or emulsifiers or surfactants etc. or a combination thereof.

In an embodiment, the developed microbial consortium helps to achieve pollution control discharge norms in a shorter batch time with lesser intervention/alternation in the existing wastewater treatment process and the performance of an operating/existing sewage treatment plant (STP) can be increased considerably.

In a first embodiment, the process for the production of biological formulation consists of a microbial consortium as an active ingredient to be used for promoting faster biodegradation of pollutants in wastewater.

In a second embodiment, the present invention describes the validated and standardized protocols for mass multiplication of active microbes in formulation by state of the art and novel submerged fermentation technology.

The present invention helps in accelerating nutrient removal in terms of Biological Oxygen Demand (BOD), Chemical Oxygen Demand (COD), and Total Dissolved Solids (TDS)) from wastewater. The present invention stabilizes the plant performance and restore water quality, results in faster settling of sludge, maintains optimal sludge volume index (SVI) without usage of chemical additives, works effectively in all aerobic wastewater treatment process (sequencing batch reactor (SBR), membrane reactor (MBR), moving bed biofilm reactor (MBBR), aerated lagoon), handles shock load and process fluctuations of a typical wastewater plant.

The present invention does not require structural changes in existing systems to implement the technology and is effective under wide range of conditions.

The microbial formulation of the present invention is capable of achieving recommended discharge norms of wastewater, recovery and reuse of treated wastewater and stabilizes plant performances to handle shock load and process fluctuation.

DETAILED DESCRIPTION OF INVENTION WITH ILLUSTRATIVE EXAMPLES AND NON-LIMITING EMBODIMENTS:
It should be noted that the particular description and embodiments set forth in the specification below are merely exemplary of the wide variety and arrangement of instructions which can be employed with the present invention. The present inventions may be embodied in the other specific forms without departing from the spirit or essentials characteristics thereof. All the features disclosed in this specification may be replaced by similar other or alternative features performing similar same or equivalent purposes. Thus, unless expressly stated otherwise, they all are within the scope of present invention. Various modifications or substitutions are also possible without departing from the scope or spirit of the present invention. Therefore, it is to be understood that this specification has been described by way of most preferred embodiment and for the purpose of illustration and not limitation.

The present invention provides bacterial formulation for biological nutrient removal in aerobic wastewater treatment. The bacterial formulation comprises microbial strains of Pseudomonas putida and Bacillus paramycoides with desired enzymatic activity. The microbial strains are isolated from the sludge of in-house wastewater plant.

The formulation comprises the bacterial strains of Pseudomonas putida and Bacillus paramycoides in ratio of 3:1. The viable cell count of Pseudomonas putida and Bacillus paramycoides is at about 1-5x1010 CFU per ml and 1-2x1010 CFU per ml.

The microbial formulation has shelf life of more than 24 months. The formulation is in the form of liquid, powder and granules.

The invention also provides process to obtain the bacterial formulation for biological nutrient removal in aerobic wastewater treatment. The process comprises the optimization of growth parameters of consortium of microbial strains in the formulation on laboratory scale to pilot plant scale. The efficacy of formulation is analysed for biological nutrient removal from waste water of treatment plants.

The desired microbial strains of Pseudomonas putida and Bacillus paramycoidesare screened and identified from the sludge. Each of the microbial strains is cultured separately in optimized culture medium in shake flask to obtain pre-inoculum of the microbial strains of Pseudomonas putida and Bacillus paramycoides. The media components used in the optimized culture medium are added in water and sterilized before inoculating with desired microbial strain to obtain pre-inoculum of optimized growth of each of the microbial strain of Pseudomonas putida and Bacillus paramycoides.

The lab scale optimization of culture medium is carried out under shake flask condition with various volume of 100 ml, 200 ml, 500 ml, 1000 ml capacity. Several organic and inorganic media components are used in the fermentation and optimized production of Pseudomonas putida and Bacillus paramycoides. The shake flask containing sterilized culture medium for submerged fermentation for each of the microbial strain separately are inoculated with pre-inoculum of Pseudomonas putida and Bacillus paramycoidescultures in the petri dishes. The inoculated fermenter medium in the shake flask is maintained under pre-determined agitation rate and aeration at specific temperature for pre-determined process time to attain optimum growth of the microbial strains in the culture medium in the shake flask. The constant pre-determined agitation rate and aeration at specific temperature for specific time are controlled/maintained by orbital shaker.

The pre-determined agitation rate ranges from 210 RPM to 300 RPM (Rotation per minute) and aeration ranges from 0.2 VVM to 1.0 VVM (Vessel Volume per minute). The pre-determined temperature ranges from 25° C to 37° C. The pre-determined process time ranges from 48 hr to 72 hr.

The fermented cultures of Pseudomonas putida and Bacillus paramycoides from respective shake flask are taken in ratio of 3:1 to inoculate fermenter culture medium in fermenters (RO2 media) of 10 L to 6000 L capacity. The inoculum is placed into a sufficient volume of a suitable liquid culture medium to permit large scale production. The inoculum size of 1-20% (in 1:1 ratio) is used for the inoculation of fermenter. The fermenter medium contains water as solvent or base in which previously optimized media components are added sequentially and mixed uniformly.

The pH of the medium is not monitored during the incubation period. The initial pH was kept at 6.0-7.0 for RO2 media while final pH after harvest is around 6.0-9.0. The inoculated fermenter medium is incubated until the density of the cells of the microbial strains reaches at about 1-5x1010 and 1-2x1010 CFU per ml for Pseudomonas putida and Bacillus paramycoides, respectively. The optimum density of the cells of the microbial strains is attained at incubation period of 48 hours. The fermented product containing optimum density of the microbial strains is harvested. Liquid, powder and granules formulation can be prepared from the harvested liquid. The shelf life of the formulation is more than 24 months.

The harvested liquid is mixed with silicate in the ratio of 1:4 (v/w) with suitable water soluble binding and thickening agent, such as carboxymethyl cellulose powder (CMC), Polyvinylpyrrolidone (PVP) to prepare powder formulation of the harvested liquid. The harvested liquid can also be formulation as granules. Therefore, the formulation of present invention is in the form of liquid, powder or granules.

The culture medium components and their respective concentration is same in the media for pre-inoculum, shake flask and fermenters. The culture media suitable for growth of both the microbial strains of the present invention comprises a carbon source, a nitrogen source, and micronutrients. The carbon source is selected from jaggery or glycerol or a mixture thereof. The nitrogen source is selected from yeast extract or peptone or a mixture thereof. The micronutrients are selected from dipotassium hydrogen phosphate, ferrous sulphate, magnesium sulphate, triammonium citrate, manganese (II) sulphate, calcium carbonate, sodium chloride, ammonium chloride, mannitol, succinic acid and sodium nitrate.
Example 1:
The culture media comprises of:
Jaggery: 5% to 10 %
Yeast extract: 1% to 2 %
Peptone: 2% to 3 %
Dipotassium Hydrogen phosphate: 1% to 4 %
Magnesium Sulphate: 1% to 2 %
Sodium Chloride: 1% to 2 %
Ammonium chloride: 1% to 2 %
Mannitol: 5 %
Succinic acid: 2 %
Sodium nitrate: 2 %

Validation studies have been conducted on powder formulation. Experiment were carried out in shake flask with wastewater obtained from inhouse wastewater treatment plant. Preliminary studies were conducted in lab scale with synthetic wastewater and actual hotel wastewater. The results depicted 77% improvement in non-performing STP setup (no sludge) and 20% improvement in performing STP (with sludge). The formulation of the present invention helps in degrading fat oil and grease (FOG). About 10% increase in FOG degradation was achieved when compared to control experiment.

After several lab scale validation with different wastewater samples collected from local streams, drain and from hotels in and around Delhi. Field trials were conducted to further validate the product in actual treatment facility at JW Marriot hotel, Aerocity, New Delhi, Shangrila hotel, New Delhi Shanti palace, STP plant M2K Corporate Park, Gurugram, Kisan Sahkari Chini Mills Ltd, Najibabad, U. P., India. The formulation reduced chemical oxygen demand (COD) from 1493 mg/l to 177.6 mg/l in aeration tank.

For a 1 megaliter per day (MLD), non performing plant, formulation dose is 50 to 300kg (two dosage in a month). For a 1 MLD, performing plant, the dose is 50 to 150 kg (two dosage in a month). On a health plant to enhance the performance, a single dose of 0.025 % is sufficient, i.e. 50 to 100 kg/250 KL of aeration tank with capacity of 1 MLD.

The chemical oxygen demand (COD) test has been performed on wastewater using 1% inoculum of Bacillus paramycoides, Pseudomonas putida and formulation comprising combination of Bacillus paramycoides, Pseudomonas putida of the present invention. The COD in case of Bacillus paramycoides was 949.44 mg/L, Pseudomonas putida 186.24 mg/L and in case of the formulation of present invention 141.12 mg/L (Fig. 1). The data shows the synergistic effect of the formulation of the present invention.

The formulation improves filter life of wastewater treatment plants, thereby requiring less frequency of backwash. The formulation also reduces wastewater plant commissioning cost.

Thus, the present invention provides a synergistic microbial formulation of bacterial consortium of in liquid, powder and granules forms. The formulation contains density of the cells of about 1-2x1010 and 1-5×1010 CFU per ml for Bacillus paramycoides and Pseudomonas putida, respectively. The formulation is capable of accelerated biological nutrient removal from wastewater and faster settling of sludge while maintaining optimal SVI without usage of chemical additives. The formulation works effectively in all aerobic wastewater treatment processes such as SBR, MBR, MBBR, aerated lagoon, etc. The consortium of microbial strains handles shock load and process fluctuations of a typical wastewater plant. The formulation does not contain GMO, therefore, it is environment friendly. The formulation can be used in wastewater treatment plants without any activation stage. The formulation meets the standards and discharge norms of Central Pollution Control Board (CPCB), India.

The process of the present invention is capable of attaining high CFU count of the microbial strains in 48-96 h. of batch run of the fermenters, thereby reducing batch time by 20%, and reducing operational and maintenance cost of the fermenters.

CLAIMS:We claim:
1. A microbial formulation for biodegradation of wastewater, said formulation comprises microbial strains of Pseudomonas putida and Bacillus paramycoides in ratio of 3:1, and wherein viable cell count of Pseudomonas putida and Bacillus paramycoides is at about 1-5x1010 CFU per ml and 1-2x1010 CFU per ml, respectively.
2. The microbial formulation as claimed in claim 1, wherein said formulation has shelf life of more than 24 months.
3. The microbial formulation as claimed in claim 1, wherein said formulation is in the form of liquid, powder and granules.
4. The microbial formulation as claimed in claim 1, wherein the formulation exhibits minimum 77% improvement in non-performing sewage treatment plant (STP) without sludge and minimum 20% improvement in performing STP with sludge), minimum 10% increase in fat oil and grease degradation, and reduction in chemical oxygen demand (COD) from 1493 mg/L to 177.6 mg/L in aeration tank.
5. The microbial formulation as claimed in claim 1, wherein said formulation is effective to treat wastewater at a minimum concentration of 0.01 % of the aeration tank capacity of 1 MLD (megaliter per day).
6. The microbial formulation as claimed in claim 1 wherein the said formulation is effective to treat wastewater at a concentration ranging from 0.01% to 10%. of the aeration tank capacity of 1 MLD.
7. The microbial formulation as claimed in claim 1, wherein a single dose in the range of 0.01 % to 0.05 % is effective for aeration tank with capacity of 1 MLD.
8. The microbial formulation as claimed in claim 1, wherein a single dose of 0.025 % is effective for aeration tank with capacity of 1 MLD.
9. A method to obtain microbial formulation as claimed in claim 1, wherein said method comprises the steps of:
- screening and identification of desired microbial strains of Pseudomonas putida and Bacillus paramycoides from sludge,
- culturing microbial strains of Pseudomonas putida and Bacillus paramycoides separately in optimized culture medium to obtain pre-inoculum of each of the microbial strains,
- inoculating shake flasks containing optimized culture medium with pre-inoculum of each of the bacterial strains separately,
- maintaining shake flasks under pre-determined process parameters to attain shake flask cultures containing optimum growth of each of the microbial strains,
- inoculating fermenter media in fermenters with inoculum from shake flask cultures of Pseudomonas putida and Bacillus paramycoides in ratio of 3:1, incubating inoculated fermenter media under pre-determined process parameters to obtain fermented liquid having density of the cells of the microbial strains at about 1-5x1010 and 1-2x1010CFU per ml for Pseudomonas putida and Bacillus paramycoides, respectively,
10. The method to obtain microbial formulation as claimed in claim 9, wherein said pre-determined process parameters comprises agitation rate in a range of 210 RPM to 300 RPM (Rotation per minute), aeration in the range of 0.2 VVM to 1.0 VVM (Vessel Volume per minute), temperature in the range of 25° C to 30° C and process time in the range of 48 hr to 72 hr.
11. The method as claimed in claim 9, wherein said culture medium and fermenter medium comprises a carbon source, a nitrogen source, and micronutrients, wherein the carbon source is selected from jaggery or glycerol or a mixture thereof, the nitrogen source is selected from yeast extract or peptone or a mixture thereof and the micronutrients are selected from dipotassium hydrogen phosphate, ferrous sulphate, magnesium sulphate, triammonium citrate, manganese (II) sulphate, calcium carbonate, sodium chloride, ammonium chloride, mannitol, succinic acid and sodium nitrate.
12. The method as claimed in claim 11, wherein said culture medium and fermenter medium comprises jaggery 5% to 10 %, yeast extract 1% to 2 %, peptone 2% to 3%, dipotassium hydrogen phosphate 1% to 4 %, magnesium sulphate 1% to 2 %, sodium chloride 1% to 2 %, ammonium chloride 1% to 2 %, mannitol 5 %, succinic acid 2 %, sodium nitrate 2 %.