[001] The present invention generally relates to naturally deriving bio-available forms of nutrients such as nitrogen, phosphorous and potassium, more specifically it relates to naturally deriving bio-available forms of nitrogen, phosphorous and potassium using microbial fermentation

Background

[002] Fertilizer plays a major role in agricultural practices and forms an integral part among the farming community. With no alternate options available currently, rampant usage of chemical fertilizer is adopted to increase crop productivity creating deleterious impact on the soil causing long-term pH imbalances and fertility, besides paving way to related pest and diseases.

[003] As per current estimations, the fertilizer consumption globally is anticipated to reach to 208.0 million tons by 2020. However, despite the use of new and improved crop varieties and chemical fertilizers, the decline in crop yield is very evident. Various analysis proves that the application of chemical fertilizer especially the N input to N leached shows a declining trend ranging from 87% to 25%. As a consequence nutrient management through chemical farming soil fertility is destabilized. [004] Chemical fertilizer production by fossil fuel combustion (example: Urea through Haber process) leads to an increased concentration of greenhouse gases (GHG) which depletes the ozone layer in the atmosphere. These activities negatively affect land productivity, biomass accumulation, and biodiversity.

[005] The extensive use of chemical fertilizer resulted in food production but with insufficient concern for sustainability. Plants/ crops are able to use about 50% of the applied fertilizer N while 25% is lost from the soil -plant system through leaching, volatilization, denitrification. Also, many other factors causing not only an economical loss but also causes pollution to the environment.

[006] Further, most of the commercially available bio fertilizers are less stable. Due to stability issues, they need to be utilized in a short duration from manufacturing time. [007] The present invention addresses all the drawbacks associated with the prior arts and it provides eco-friendly and cost effective organic nutrients. Summary of the invention

[008] One embodiment of the present invention is to provide a bio fertilizer comprising a) a blend comprising about 15:15:15 NPK in powder form, b) about 10% of NPK in liquid form c) not less than 60% of organic carbon and d) about 3% of sulphur.

[009] Another embodiment of the present invention provides a process for preparation of bio available form of nitrogen comprising; a) growth of Pseudomonas strutzeri (AFN-1601) under the medium of specialized protein complex (beef extract-2%, humic acid -10%) and b) salts comprising magnesium and sodium salts or a combination thereof. [010] Another embodiment of the present invention provides a bio available form of phosphorous comprising; a) growth of Streptomyces albus strain (AFN001) in a media comprising 10% of sucrose, 2% o of Sodium chloride and 10% of humic acid and b) metal ion selected from 0.1% each of Magnesium, manganese and ferrous ions or a combination thereof [011] Another embodiment of the present invention provides a process for preparing bio available form of potassium comprising; a) growth of Methylophilismethylotrophus (AFN-1516) under a suitable buffer medium comprising high sugar source - glucose upto the range of 10%, b) protein source peptone in the range of 3-5% and c) 0.5% NaCl or 0.5% Zinc Oxide.

[012] Another embodiment of the present invention provides a drying process of broth culture comprising a) addition of solvent to the broth culture at solvent broth ratio of 27: 75, b) agitation, c) isolation of the precipitation obtained in step (b) and d) drying to obtain powder form of nitrogen, phosphorous and potassium.

Brief Description of Drawings [013] Fig. 1 illustrates the Ninhydrin test.

[014] Fig. 2 illustrates phenotypic characterizations of M-9

[015] Fig. 3 illustrates phenotypic characterizations of M-9

[016] Fig. 4 illustrates phenotypic characterizations of potash producing microbes

[017] Fig. 5 illustrates a flow chart for cell lysis to obtain potassium

Detailed Description of the Invention

Definitions

[018] The "bio-available form of nitrogen" refers to the nitrogen from the soil in the form of nitrate (N03-), ammonium (NH4+), free amino nitrogen, and free ammonium coupled with carbon chains.

[019] The "bio-available form of phosphorous" refer to the phosphate in the form of P205.

[020] The "bio-available form of potassium" refer to the form of ions- K+ and K20. [021] A "biologically pure culture" refer to a culture of the microorganism that does not include other materials (i) which are normally found in soil in which the microorganism grows, and/or (ii) from which the microorganism is isolated.

[022] "Culturing" refers to the propagation of organisms on or in nutrition media of various kinds. [023] The term "microbial fermentation" refers to method, wherein, micro-organisms used to grow on media with particular ingredients for particular time to obtain a product. [024] A "plant" refers to any living organism belonging to the kingdom Plantae (i.e., any genus/species in the Plant Kingdom). This includes familiar organisms such as but not limited to trees, herbs, bushes, grasses, vines, ferns, mosses and green algae. The term refers to both monocotyledonous plants also called monocots, and dicotyledonous plants also called dicots.

[025] The "Supernatant" refers to the liquid broth remaining when cells grown in broth are removed by centrifugation, filtration, sedimentation or other means well known in the art. The present invention relates to a method, wherein, microbial strains are cultured, isolated and extracted. The Microbial extract are further formulated and blended to obtain the product. The product of present invention on application helps in replenishing the microbial multiplication rate to maintain a balanced rate of microbial biomass and plant growth.

[026] In a preferred embodiment of the present invention, microbial fermentation methods are used for producing naturally derived bio-available forms of nutrients such as nitrogen, phosphorous and potassium. Use of the aforementioned methods would constitute an economical and/or ecofriendly method of extracting sufficient amounts of nutrients to meet crop production needs.

[027] One embodiment of the present invention is to provide a biologically pure culture of Pseudomonas stutzeri (AFN-1601 ).

[028] One embodiment of the present invention provides a biologically pure consortium of plant growth promoting microorganism selected from Pseudomonas stutzeri

(AFN-1601), Streptomyces albus ( AFN001 ) and Methylophilismethylotrophus ( AFN-

1516). [029] In an exemplary embodiment of the current invention, the three major elements; Nitrogen(N), Potassium(P) and Phosphorus(K) required for the plants are synthesized naturally through a consortium of microbial cultures. The microbial extracts are formulated and blended of about 15: 15: 15 in powder form and about 10% range of NPK in liquid form with more than 60% organic carbon and about 3% sulphur . The product on application helps in replenishing the microbial multiplication rate to maintain a balanced ratio of microbial biomass and plant growth.

[030] Another embodiment of the present invention provides a biologically pure culture of Pseudomonas stutzeri (AFN-1601 ) which is 99% identical to the gene sequence accession number KC660146.1, GU594474.1, KC166815.1 and KJ956671.1 as described in Bergey’s Manual of Systematic Bacteriology.

[031] Another embodiment of the present invention provides a bio fertilizer comprising a) a blend comprising about 15 : 15 : 15 of NPK in powder form, b) about 10% of NPK in liquid form c) not less than 60% of organic carbon and d) about 3% of sulphur.

[033] The invention comprehends on the low-cost production technology with high productivity and profitability of about 70-80%.With additional benefit of replenishing the plant system with sulphur. Sulfur is a vital part of all plant proteins, and certain plant hormones. It is also used in the formation of certain oils and volatile compounds. Sulfur is essential for nitrogen-fixing nodules on legumes, and necessary in the formation of chlorophyll. Plants use sulfur in the processes of producing proteins, amino acids, enzymes and vitamins. Sulfur also helps the plant's resistance to disease, aids in growth, and in seed formation.

[034] In an implementation of the invention, the following process is used to identify the microorganism that produces nitrogen.

[035] Various water samples, soil samples, waste water, plant samples as well as samples from different effluent treatment plant units are collected. Temperature and pH while collecting the sample were estimated to be 30°C and 6 respectively. The samples are taken and diluted serially up to 10-6 about 0.1ml of serially diluted sample is taken and the spread plate technique is performed by using yeast peptone dextrose agar plate with ampicillin of pH-7 (2% dextrose, 1% peptone, 0.5% yeast extract, ampicillin 0.01%). The inoculated plates are then incubated for 48hours at 30°C. 9 cultures were identified, each of which were grown in different media conditions a. Subculture technique and media optimization; [036] The 9 isolates of microbes obtained were sub-cultured on a suitable nutrient such as agar and yeast peptone dextrose broth media, and incubated at 30°C for 48hours.

The microbe isolated was grown in different media with the following composition:

The following media compositions were used: Media 1:

Glucose -3%

Casein -0.1%

Beef Extract - 1 % Peptone - 3%

Nacl- 1% pH-6

All the 9 isolates were grown aerobically under room temperature at 120 rpm for a period of 5 days. The colony count was optimized spectrophotometrically at OD greater than 9 at

600nm wavelength.

Media 2:

Beef extract-2%

Humic acid - 10%

Amino powder- 0.1%

Magnesium suphate-0.5%

Nacl- 0.1%

All the 9 isolates were grown aerobically at 120 rpm or 5 days and measured spectrophotometrically.

Media 3:

SD broth /Glucose

Ammonium sulfate - 5 g/1

Glucose - 20 g/1

Copper sulfate - 40 μg/l Potassium iodide - 100 μ g/l Ferric chloride - 10 μ g/1 Manganese sulfate·· 10 m g/1 All the 9 isolates were grown in the aforementioned media for period of 7 days at 120 RPM aerobically.Purified cultures were routinely maintained on nutrient agar kept at -4°C. b.Screening for amino acid production [037] The different isolates grown in different media compositions were tested for their ability to produce amino acids. Ninhydrin, which is originally yellow, reacts with amino acid and turns deep purple. It is this purple color that is detected in this method. Ninhydrin will react with a free alpha-amino group, NH2-C-COOH. This group is present in all amino acids, proteins or peptides. Whereas, the decarboxylation reaction will proceed for a free amino acid, it will not happen for peptides and proteins. When amino acids with a free alpha amino group are treated with an excess of ninhydrin, they yield a purple colored product. Under appropriate conditions, the color intensity produced is proportional to the amino acid concentration. [038] As illustrated in Fig.1, the colour intensity through ninhydrin test revealed maximum the production of amino acids. Isolate M-9 produced maximum colour intensity in the media- 1 at 25 degree Celsius in 24 hours incubation period. Theisolate M-9 was subjected to ribotyping. Phenotypic characterizations of M-9:

[039] The morphological and biochemical characterizations of the selected strain M-9 were carried out as described in Bergey’s Manual of Systematic Bacteriology. For cell and colony morphology (shape, size, color, motility) determination of the isolate, it was studied under microscope.The isolate was plated in sterile petri plates by spread plate method and plates incubated at 28 °C. As illustrated in Fig. 2 and Fig. 3, the 16S rRNA gene of the strain with sequences of the nearest type species retrieved from the ribosomal database project (RDP), this strain showed high homology with Pseudomonas stutzeri. Following the physiological and biochemical characteristics and comparison of its 16S rRNA gene sequence, the selected strain was identified as Pseudomonas stutzeri.

[040] Another embodiment of the present invention provides a process for preparation of bioavailable form of nitrogen comprising; a) growth of Pseudomonas strutzeri (AFN-1601) under the medium of specialized protein complex (beef extract-2%, humic acid -10%) and b) salts comprising magnesium and sodium salts or a combination thereof.

[041] The identified microbial strain -Pseudomonas strutzeri named as AFN-1601 is used for the production of nitrogen represented by the following mechanism under the medium of specialized protein complex (beef extract-2%, humic acid -10%) with minimal salts comprising combination of magnesium and sodium salts including trace elements which triggers the active transport mechanism under fermentation process during a time period of 2 days. [042] The microbe AFN-1601 during the growth phase, transports amino acid and small peptides into the cell via an active transport process that utilizes specialized membrane proteins. The difference in the pH gradient and the near neutral pH of cytoplasm of the microbial cells enhances the nitrogen efflux. The specialized transporter protein complex present in the plasma membrane of the microbial cell AFN-1601 brings amino acid residues and small peptides into the cell coupled with an hydrogen ion that later gets expelled by the cell which remains in the broth culture in the range of 2-3% and on drying reported to be in the range of 15% in fully water soluble form.

[043] Another embodiment of the present invention provides a drying process of broth culture comprising a) addition of solvent to the broth culture at solvent broth ratio of 27: 75, b) agitation, c) isolation of the precipitation obtained in step (b) and d) drying to obtain powder form of nitrogen..

[044] The drying process involved a series of precipitation process where solvent was added to the broth at solvent broth ratio of 27: 75. The mixture on agitation for 24 hrs helps precipitation of protein causing cell lysis to form combination of free amino nitrogen, ammonia and ammonium coupled with carbon chains. The precipitate was separated, The spent broth subjected to evaporation for solvent recovery and collection any additional precipitates. The precipitate was dried in a tray drier at 45 degree Celsius for 2 days and further pulverized to fine powder Another embodiment of the present invention provides a biologically pure culture of

Streptomyces albus strain (AFN001).

[045] Another embodiment of the present invention provides a process for preparation of biologically pure Streptomyces albus strain (AFN001) comprising; a) Collection of samples, b) dilution of the samples serially up to 10-6, c) inoculation of diluted sample onto yeast peptone dextrose agar plate with ampicillin of pH-7, d) incubation of inoculated plates for about 48 hours at about 30°C, e) isolation of the microbes and sub-culturing on a suitable media, f) screening of strains and g) morphological and biochemical characterization of the selected strain. [046] In an implementation of the invention, the following process is used to identify the microorganism that produces phosphorus. Various water samples, soil samples, waste water, plant samples as well as samples from different effluent treatment plant units are collected. Temperature and pH while collecting the sample were estimated to be 30°C and 6 respectively. The samples are taken and diluted serially up to 10-6 about 0.1ml of serially diluted sample is taken and the spread plate technique is performed by using yeast peptone dextrose agar plate with ampicillin of pH-7 (2% dextrose, 1% peptone, 0.5% yeast extract, ampicillin 0.01%). The inoculated plates are then incubated for 48hours at 30°C. 9 cultures were identified, each of which were grown in different media conditions. a. Subculture technique and media optimization:

[047] The 9 isolates of microbes obtained were sub-cultured on a suitable nutrient such as agar and yeast peptone dextrose broth media, and incubated at 30°C for 48hours.

The microbe isolated was grown in different media with the following composition:

The following media compositions were used:

Media 1:

Glucose -3% Beef Extract - 1%

Peptone - 3%

Nacl - 1% pH-6

All the 9 isolates were grown aerobically under room temperature at 120 rpm for a period of 5 days. The colony count was optimized spectrophotometrically at OD greater than 9 at

600nm wavelength.

Media 2:

Beef extract-2% Humic acid - 10%

Magnesium suphate-0.5%

Nad- 0.1% All the 10 isolates were grown aerobically at 120 rpm or 5 days and measured spectrophotometrically.

Media 3: SD broth /Glucose

Sucrose - 10%

Humic acid - 10%1 Nacl - 2%

Magnesium sulphate - 0.1 % Ferric chloride - 0.1 %

Manganese sulfate-0.1 %

All the 9 isolates were grown in the above said media for period of 5 days at 120 RPM aerobically. Purified cultures were routinely maintained on nutrient agar kept at -4°C.

[048] Another embodiment of the present invention provides a process for preparing bio available form of phosphorous comprising; a) growth of Streptomyces albus strain (AFN001) in a media comprising 10% of sucrose, 2% o of Sodium chloride and 10% of humic acid and b) metal ion selected from 0.1% each of Magnesium, manganese and ferrous ions or a combination thereof [049] Each of cultures were subjected to phosphorous estimation after completion of growth cycle. Of the 9 cultures isolated AFN001 was found to be the maximum producer of phosphorous. The microbe Streptomyces albus strain AFN001 was isolated and screened for various studies for the production of phosphate under different parameters. The AFN001 grown in a media with sucrose-10%, NaCl-2% and humic acid -10% in specific pH and temperature maintained with metal ions such as Magnesium, manganese and ferrous ions each @0.1%. The strain AFN001 was studied in different temperature, pH and different time period of incubations. It was found that a great efflux of phosphate was obtained under 28 degree Celsius incubation for 2 days in pH ranging from 6-7.4. Trace elements and PBS saline is a major part of media which provides a suitable environment for AFN001 for the release of phosphate in the form of P2O5.

[050] Strain AFN001 was grown aseptically under the parameters as mentioned earlier. Physiological background behind the process is that the PBS is a balanced salt solution commonly used in the bio-laboratory. The essential function of a balanced salt solution is to maintain pH and osmotic balance as well as provide cells with essential water and essential ions. The mitochondrial pH always remains at the range of 7.4-7.5. The same condition is provided by PBS outside the cell. This results in the extended release of energy molecules out of the cell. PBS is utilized to maintain cells for the short term in a viable condition while the cells are manipulated outside of their regular growth environment. The pH of most microbes falls under 5-7.4 and the pH of PBS is set to be in range of 7-7.4 which creates a better osmolarity (isotonic) which is non-toxic to the cells. Fermentation of AFN001 in the presence of sucrose results in the formation of sugar phosphates which are the energy molecules produced by glycolysis. PBS same time maintains osmolarity and also washes out all the sugar phosphates from the cell and dilutes all the biological substances. This results in the conversion of sugar phosphate in to P205 form. And the final concentration of phosphate after solvent extraction and heat drying and blending will be between 15% with a good percentage of carbon. Another embodiment of the present invention provides a drying process of broth culture comprising a) addition of solvent to the broth culture at solvent broth ratio of 27: 75, b) agitation, c) isolation of the precipitation obtained in step (b) and d) drying to obtain powder form of phosphorous.

[051] The drying process involved a series of precipitation process where solvent was added to the broth at solvent broth ratio of 25: 75. The mixture on agitation for 24 hrs helps precipitation of protein causing cell lysis to form combination of free amino nitrogen, ammonia and ammonium coupled with carbon chains. The precipitate was separated ,The upper solvent removed and further fresh solvent added with the same solvent broth ratio of 25:75.Thios was carried out three to four times to recover all the cell contents. The spent broth subjected to evaporation for solvent recovery and collection any additional precipitates. The precipitate was dried in a tray drier at 45 degree Celsius for 2 days and further pulverized to fine powder Another embodiment of the present invention provides a biologically pure culture of Methylophilismethylotrophus.

[052] In an implementation of the invention, the following process is used to identify the microorganism that produces potassium. The isolates collected were screened on agar plates for potash production containing 0.5% chitin, 0.03% peptone, 0.03% yeast extract,

0.07% K2HP04, 0.03% KH2P04, 0.05% MgS04. 7H20, 1.5% Agar, 0.2% NH4N03, 0.1% NaCl, (w/v) and 0.1% v/v trace elements (pH 7.8). Cultures were incubated for 2 days at 30°C. Culture media and growth conditions:

[053] For the production of potash, the best strain obtained from the primary screening, was cultured at first in pre-culture medium consisting of 0.8% nutrient broth, 1% malt extract, 1% peptone, 0.5% chitin, and 0.1% NaCl (w/v) for 24 hours at 30°C on a shaker incubator (200 rpm). A 2 ml sample of the pre-culture was added to 50 ml of the production medium (primary screening medium but without agar). The resultant inoculated medium was cultured at 28°C for 36hours on a rotary shaker (200 rpm). A 2 ml sample of the culture medium was removed every 24 hours and centrifuged (10000 g for 20 min at 4°C). The resulting supernatants were then collected and used for subsequent potash estimation using the method described below. The strain, which showed the highest potash was isolated, maintained on nutrient agar and used.

Phenotypic characterization: [054] The morphological and biochemical characterizations of the selected strain were carried out as described in Bergey’s Manual of Systematic Bacteriology. The isolates were plated in sterile petri plates by pour plate method and plates will be incubated at 30 °C. For cell and colony morphology (shape, size, color, motility) determination, the isolates was studied under microscopes.

As illustrated in Fig. 4, comparison of the isolates 16S rRNA gene sequence, the selected strain was identified as Methylophilismethylotrophus (AFN-1516).

Concentration of Potash through vacuum evaporation after cell lysis: [055] Solvent - Methanol in the ratio of 1:1 is added to the isolate and allowed to agitate for 24horrs at 30 degrees. The solvent added helps to lyse the cells. The extract is further vacuum evaporated to remove the solvent and obtain protein rich substrate.

Media optimization of Methylophilismethylotrophus (AFN-1516) [056] Different media composition used for optimum growth and production of

Methylophilismethylotrophus ( AFN- 1516).

Media - 1 Potato - 20%

Dextrose - 2% Distilled water - lOOmlk

Media -2

Peptone -3.5% Navl c 0.5%

Zinc oxide - 0.5%

Media -3 Yeast extract - 0.05%

Dextrose- 1 %

Calcium phosphate - 0.5%

Ammonium sulphate -0.05%

Potassium chloride -0.02%

Different pH and temperature studies showed that optimum growth of AFN-1516 was obtained in Media 2 at temperature 35 degree at 3days incubation and at pH 6.

Mutagenesis of AFN-1516 [057] Mutagenesis of AFN-1516 was performed using EMS method. Cells were washed twice in 0.1M phosphate buffer and re-suspended in the same solution. The suspension was treated with EMS at 20 ° C for 20 minutes at final concentration ranging from 2 - 20 μg/ml respectively. Cells were washed twice by suspension in 1ml of 0.1M- phosphate buffer (Ph-7) to remove the mutagen and inoculated into 3ml of PDB. Overnight cultures were plated on PDA and colonies were screened.

[058] The mutant of AFN-1516 exhibited the greatest increase of about 2 times at 27 degree in 48 hours than that of the parent strain. Another embodiment of the present invention provides a process for preparing bio available form of potassium comprising; a) growth of Methylophilismethylotrophus (AFN-1516) under a suitable buffer medium comprising high sugar source - glucose upto the range of 10%, b) protein source peptone in the range of 3-5% and c) 0.5% NaCl or 0.5% Zinc Oxide.

AFN-1516 as Methylophilismethylotrophus under a suitable buffer medium which includes high sugar source - glucose upto the range of 10% in combination with other protein source peptone in the range of 3-5%, NaCl 0.5% or Zinc Oxide 0.5% leading to high potash output. Mutation was carried out on the shortlisted microbial strain to enhance the productivity.

Out of 105 mutants, 1 mutant revealed highest yield of cells. K ⁺ is the major cation in any cytoplasm. K ⁺ -channel genes are found in nearly all genomes mostly in prokaryotic cells due to its crystal structure. The ions present within the crystal structures helps in permeation and selectivity of ions with in the cells which is controlled majorly by all the known stimuli including trace elements and under controlled mechanical force like heat treatment. A

K ⁺ channel can pass some 10 ⁷ K ⁺ per second and discriminate against the smaller Na ⁺ at the same time (PK+ : PNa ₊ > 1,000 : 1).

[058] So by checking the density of the cells in broth by photospectrometer every 8 hours, after 48hours the broth was harvested and subjected to mechanical force in order to lyse the cells as well as to control the channel leading to infinite release of K ⁺ ions. Under such circumstances the potash content in the broth is 5%. This broth was dried and tested for Potash by Flame photometer and line num around 15% of potash was found in the form of K20.

Another embodiment of the present invention provides a drying process of broth culture comprising a) addition of solvent to the broth culture at solvent broth ratio of 27: 75, b) agitation, c) isolation of the precipitation obtained in step (b) and d) drying to obtain powder form of potassium. [059] The drying process involved a series of precipitation process where solvent was added to the broth at solvent broth ratio of 27:75.The mixture on agitation for 24 hrs helps precipitation of protein causing cell lysis to form combination of free amino nitrogen, ammonia and ammonium coupled with carbon chains. The precipitate was separated ,The spent broth subjected to evaporation for solvent recovery and collection any additional precipitates. The precipitate was dried in a tray drier at 45 degree Celsius for 2 days and further pulverized to fine powder.