WO2019034992A1

WO2019034992A1 - Naturally deriving bio-available forms of nitrogen, phosphorous and potassium using microbial fermentation

Info

Publication number: WO2019034992A1
Application number: PCT/IB2018/056104
Authority: WO
Inventors: Nisha M M
Original assignee: Miklens Bio Private Limited
Priority date: 2017-08-17
Filing date: 2018-08-14
Publication date: 2019-02-21

Abstract

The present invention provides a microbial fermentation method for identification of microbial strains producing naturally derived bio-available form of nutrients such as nitrogen, phosphorus and potassium. The present invention also 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.

Description

NATURALLY DERIVING BIO-AVAILABLE FORMS OF NITROGEN, PHOSPHOROUS AND POTASSIUM USING MICROBIAL FERMENTATION

Field of Invention

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 of Invention

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.

As per current estimations, the fertilizer consumption globally is anticipated to reach to 208 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 input of nitrogenand leaching of nitrogen shows a declining trend ranging from 87% to 25%. As a consequence nutrient management through chemical farming soil fertility is destabilized.

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.

Thus, there was a need to develop an environmentally safe organic fertilizer, which should not have adverse effect on nature and its resources.

Object of Invention

Object of the invention is to develop an organic fertilizer obtained by microbial fermentation, wherein, the fertilizer contains bio-available forms of nitrogen, phosphorous and potassium.

Summary

The present invention provides a microbial fermentation method for identification of microbial strains producing naturally derived bio-available form of nutrients such as nitrogen, phosphorus and potassium. The method comprises following steps: a) Collection of sample

b) Dilution of 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 48 hours at 30°C

e) Isolation of microbes and sub-culturing on a suitable media

f) Screening for strains

g) morphological and biochemical characterization of the selected strain

Brief Description of Drawings

Fig. 1 illustrates the Ninhydrin test.

Fig. 2 illustrates phenotypic characterizations of M-9

Fig. 3 illustrates phenotypic characterizations of M-9

Fig. 4 illustrates phenotypic characterizations of potash producing microbes

Fig. S illustrates a flow chart for cell lysis to obtain potassium

Detailed Description of Invention

Definitions

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.

The "bio-available form of phosphorous" refer to the phosphate in the form of P2O5.

The "bio-available form of potassium" refer to the form of ions- K+ and K₂0.

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.

"Culturing" refers to the propagation of organisms on or in nutrition media of various kinds.

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.

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.

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.

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.

In an exemplary embodiment of the current invention, the three major elements; Nitrogen(N), Potassium(K) and Phosphorus(P) required for the plants are synthesized naturally through a consortium of microbial cultures. The microbial extracts are formulated and blended in the range of 10:10:10 in powder form and 3-4% range of NPK in liquid form.

The broth obtained will have Nitrogen , phosphorous and potash to the extent of 3-4% each, the remaining being the cell mass rich in proteinaceous substance with different forms of amino acids constituenting about 60% and the remaining about 35-40% organic carbon.

Formulation of liquid into powder form: To the individual NPK broth, 30% of methanol is added. Every single molecule of carbon and hydrogen binds with the nitrogen element, this results in precipitation of nitrogen in its purest form which settles down at the bottom, this is then separated through filtration process and dried and converted into pure powder form. Similarly butanol and ethanol are added to phosphorous and potash broth respectively and subjected to similar filtration and separation process. These individual powders are blended with carrier preferable dextrose in the required NPK ratios to form 100% completely water soluble powder formulation.

Following examples further describes the invention in stepwise manner. Example 1:

Following process is used to identify the microorganism that produces nitrogen.

Step 1: Sample Collection

Various water reservoir samples, soil samples, waste water samples, plant samples as well as samples from different effluent treatment plant units were collected. Temperature and pH while collecting the sample were estimated to be 30°C and pH 6 respectively. The samples were taken and diluted serially up to 10-6, wherein, about 0.1ml of serially diluted sample was taken and the spread plate technique was 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 were then incubated for 48 hours at 30°C. Total 9 cultures were identified.

Step 2: Subculture technique and media optimization:

All 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 48 hours.

The isolated microbes were grown in different media with the following composition(s):

Media 1:

All the 9 isolates were grown aerobically under room temperature at 120 rpm for a period of S days.

The colony count was optimized spectrophotometrically at OD greater than 9 at 600nm wavelength.

Glucose -3%

Beef Extract - 1%

Peptone - 3%

Nacl - 1%

pH-6

Media 2:

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

OD greater than 9 at 600nm wavelength.

Beef extract-2%

Humic acid - 10%

Magnesium suphate-0.5%

Nacl- 0.1 %

Media 3:

All the 9 isolates were grown in the aforementioned media for period of S days at 120 rpm aerobically and measured spectrophotometrically at OD greater than 9 at 600nm wavelength.

SD broth /Glucose

Ammonium sulfate - 5 g/1

Glucose - 20 g/1

Copper sulfate - 40 pg/l

Potassium iodide - 100 μg/l

Ferric chloride - 10 μg/l

Manganese sulfate- 10 pg/l

The purified cultures were routinely maintained on nutrient agar kept at -4°C. Step3: Screening for amino acid production

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 was detected in this method. Ninhydrin reacted 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 were 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.

As illustrated in Fig.l, 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 at S days incubation period. The isolate M-9 was further subjected to ribotyping.

Step 4: Phenotypic characterizations of M-9:

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 PseudomonasstutzerL

Step 4: Product testing

The identified microbial strain-Pseudomonas strutzeri named as MKN-1601 was 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.

The microbe MKN-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 MKN-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 10-12% in fully water soluble form. This when regulated under different levels of evaporation the range of nitrogen can be increased.

Further, through cell lysis under a solvent extraction process, the protein content can be precipitated in the form of combination of free amino nitrogen, ammonia and ammonium coupled with carbon chains.

Example 2:

The following process used to identify the microorganism that produces phosphorus. Step 1: Sample Collection

Various water reservoir samples, soil samples, waste water samples, plant samples as well as samples from different effluent treatment plant units were collected. Temperature and pH while collecting the sample were estimated to be 30°C and pH 6 respectively. The samples were taken and diluted serially up to 10-6, wherein, about 0.1ml of serially diluted sample was taken and the spread plate technique was 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 were then incubated for 48 hours at 30°C. Total 9 cultures were identified.

Step 2: Subculture technique and media optimization:

Media 1:

All the 9 isolates were grown aerobically under room temperature at 120 rpm for a period of 5 days.

Glucose -3%

Beef Extract - 1%

Peptone - 3%

Nacl - 1%

pH-6

Media 2:

All the 9 isolates were grown aerobically at 120 rpm or S days and measured spectrophotometrically at OD greater than 9 at 600nm wavelength.

Beef extract-2%

Humic acid - 10% Magnesium suphate-0.5%

Nacl- 0.1%

Media 3:

All the 9 isolates were grown in the above said media for period of S days at 120 rpm aerobically and measured spectrophotometrically at OD greater than 9 at 600nm wavelength.

SD broth /Glucose

Sucrose - 10%

Humic acid - 10%1

Nacl - 2%

Magnesium sulphate - 0.1%

Ferric chloride - 0.1 %

Manganese sulfate-0.1%

Purified cultures were routinely maintained on nutrient agar kept at -4°C. Step 3:Screening and testing

Each of cultures were subjected to phosphorous estimation after completion of growth cycle. Amongstthe 9 cultures isolated and cultured, MKN001 was found to be the maximum producer of phosphorous. The microbe Streptomyces albuswhich was identified as strain MKN001 was further isolated and screened for various studies for the production of phosphate under different parameters. The MKN001 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 at 0.1%. The strain MKN001 was further studied in different temperature, pH and different time period of incubations. It was found that a great efflux of phosphate was obtained under temperature 28°Celsius and 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 MKN001 for the release of phosphate in the form of P2O5.

Example 3:

The following process is used to identify the microorganism that produces potassium. Step 1: Sample collection and primary screening

Various water reservoir samples, soil samples, waste water samples, plant samples as well as samples from different effluent treatment plant units were collected. Temperature and pH while collecting the sample were estimated to be 30°C and pH 6 respectively. The samples collected were initially screened on agar plates of media with following composition at pH 7.8. Cultures were incubated for 2 days at 30°C.

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.

The best grown strain obtained from the primary screening was further tested as described further. Step 2: Culture media and growth conditions:

For the production of potash, the best strain obtained from the primary screening, was cultured at first in pre-culture media of following composition for 24 hours at 30°C on a shaker incubator (200 rpm).

0.8% nutrient broth,

1% malt extract,

1% peptone,

0.5% chitin, and

0.1% NaCl (w/v)

Step 3: Product screening

A 2ml of the pre-culturesample was added to 50ml of the production medium (primary screening medium but without agar). The resultant inoculated medium was cultured at 28°C for 36 hours on a rotary shaker (200 rpm). A 2ml 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 MKN1516, which showed the highest potash, was isolated and maintained on nutrient agar and used further.

Concentration of Potash through vacuum evaporation after cell lysis:

Solvent - Methanol in the ratio of 1:1 is added to the isolate and allowed to agitate for 24 hours at 30°C. The solvent added helps to lyse the cells. The extract is further vacuum evaporated to remove the solvent and obtain protein rich substrate. Step 4: Phenotypic characterization

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 petriplates by pour plate method and plates incubated at 30°C.

For cell and colony morphology (shape, size, color, motility) determination, the isolates were studied under microscopes.

As illustrated in Fig. 4, comparison of the isolates 16S rRNA gene sequence, the selected strain MKN1516 was identified as MethylophiUs methylotrophus.

Step 5: Media optimization of /MKN 1516

Different media composition used for optimum growth and production of MKN1516

Media -1

Potato - 20%

Dextrose - 2%

Distilled water - 100mlk

Media -2

Peptone -3.5%

Nacl - 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 MKN-1516 was obtained in Media 2 at temperature 35°Cat 3days incubation and at pH 6.

Step 6: Mutagenesis of MKN1516 for potash in the form of K₂0

Mutagenesis of MKN-1516 was performed using ETHYL METHANE SULPHONATE method. Ethyl methanes ulfonate (EMS) is a mutagenic organic compound with formula CH.SO,. It produces random mutations in genetic material by nucleotide substitution; particularly by guanine alkylation. This typically produces only point mutations. It can induce mutations at a rate of 5x10" to 5x10^~! per gene without substantial killing. The ethyl group of EMS reacts with guanine in DNA, forming the abnormal base O⁶- ethylguanine. During DNA replication, DNA polymerases that catalyze the process frequently place thymine, instead of cytosine, opposite O-ethylguanine. Following subsequent rounds of replication, the original G:C base pair can become an A:T pair (a transition mutation). This changes the genetic information,.

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 pg/ml respectively. Cells were washed twice by suspension in 1ml of O.lM-phosphate buffer at pH-7 to remove the mutagen and inoculated into 3ml of Potato Dextrose Broth. Overnight cultures were plated on Potato Dextrse Agar and colonies were screened.

The mutant of MKN-1516 exhibited the greatest increase of about 2times at 35 degree in 3days than that of the parent strain.

MKN-1516 as Methylophilis methylotrophus 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 10⁷ K⁺ per second and discriminate against the smaller Na⁺ at the same time (PK+ : PNa+> 1,000 : 1).

Upon checking the density of the cells in broth by photospectrometer every 8 hours, after 48 hours 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 2 to 3%. This broth was dried and tested for Potash by Flame photometer and around 10% of potash was found in the form of K₂0.

Claims

CLAIMS We Claim:

1. A biologically pure culture of Pseudomonas strutter strainMKN-1601 for the production of bio-available form of nitrogen (N).

2. A biologically pure culture of the Pseudomonas strutter strain of claim 1 isolated and identified by method comprising of following steps:

a) collection of a sample

b) dilution of the samples serially up to 10-6

c) inoculation of the diluted sample onto yeast peptone dextrose agar plate with ampicillin of pH-7

d) incubation of inoculated plates for 48 hours at 30°C

e) isolation of microbes and sub-culturing on a suitable media

f) screening for amino acid producing strain

g) morphological and biochemical characterization of the selected strain.

3. The bio-available form of nitrogen production obtained from said strain of claim lis selected from the group of nitrates (N03-), ammonium (NH4+), free amino nitrogen, and free ammonium coupled with carbon chains.

4. A biologically pure culture of the Streptomyces albus strain MKN001 for the production of bio-available form of phosphorus (P).

5. A biologically pure culture of Streptomyces albusstrain of claim 4 isolated and identified by method comprising of following steps:

a) collection of a sample

b) dilution of the samples serially up to 10-6

d) incubation of inoculated plates for 48 hours at 30°C

e) isolation of the microbes and sub-culturing on a suitable mediaof sucrose and humic acid

f) screening for the phosphorus producing strain

g) morphological and biochemical characterization of the selected strain

6. The bio-available form of phosphorous production obtained from said strain of claim 4is in the form of P₂O₅.

7. A biologically pure culture of Methylophilis methylotrophus strain MKN-1516 and mutants thereof for the production of bio-available form of potassium (K).

8. A biologically pure culture of Methylophilis methylotrophusstram of claim 7 isolated and identified by method comprising of following steps: a) collection of a sample

b) dilution of the sample serially up to 10-6

d) incubation of inoculated plates for 48 hours at 30°C

e) isolation of the microbes and sub-culturing on a suitable mediaof peptone and zinc oxide

f) screening for the potassium producing strain

g) morphological and biochemical characterization of the selected strain

9. The bio-available form of potassium production obtained from said strain of claim 7is selected from the group of K+ and K₂O.

10. A biological culture of mutant strain of Methylophilis methylotrophusof claim 7 isolated and identified by method comprising of following steps:

a) washing and resuspending of cells in 0.1 M-phosphate buffer

b) EMS treatment of suspension of step (a) at 20°C for 20 minutes at final concentration ranging from 2 - 20 μg/ml

c) washing of the cells in O.lM-phosphate buffer at pH 7 to remove the mutagen and inoculation into 3ml of PDB and incubated overnight.

d) sub-culturing on PDA and screening of mutant of MKN-1516 which exhibited the greatest increase of about 2 times at 3S°Cin 3days than that of the parent strain.

11. Formulation of microbial extracts of the preceding claims, comprising of NPK in the ratio of 10:10:10 in powder form wherein the application of formulation helps in replenishing the microbial multiplication rate to maintain a balanced ratio of microbial biomass and plant growth.

12. Formulation of microbial extractsof the preceding claims, comprising of NPK in the 3-4% range of NPK in liquid form wherein the application of formulation helps in replenishing the microbial multiplication rate to maintain a balanced ratio of microbial biomass and plant growth.