Description:FIELD OF INVENTION:
The present invention generally relates to field of agricultural technology and microbiology. The present invention also relates to process to prepare enriched organic manure.
BACKGROUND OF INVENTION:
Anaerobic digestion (AD) is a widely used technology for the production of biogas from organic wastes and biomass residue. It involves a controlled biological process whereby organic waste and biomass residue is transformed by the activity of certain bacteria under anaerobic conditions into a desired methane rich biogas, yielding a plant nutrient rich residue (digestate) as by-product. The digestate is known to have soil fertilizing properties leading to improved soil heath. The digestate which comes from AD process is semisolid, fibrous solid and liquid mass containing both organic and inorganic matter. If digestate is subjected for solid liquid separation and low types of organic fertilizers are produced: Solid known a Fermented Organic Manure (FOM) and liquid, known as Liquid Fermented Organic Manure (LFOM). The FOM and LFOM need to be stabilized before use. Both the FOM and LFOM obtained after anaerobic digestion has nutrient value for plant growth, but it needs to be stabilized before its field application. Further, lot of nutrients are not in the crop available form in the digestate which needs to be solubilized to harness complete benefit of the available nutrients in the digestate. In current practice, the separated solid waste and liquid effluent subject to stabilization in an open area by mixing in intervals. Existing method of stabilization requires high processing time and couldn’t increase any crop available nutrient content.
Disadvantages of existing method:
• Highly time consuming, i.e., from 7-8 weeks for solid manure and 4-6 weeks for liquid manure.
• Composition of final manure contains low inorganic carbon content due to incomplete conversion.
• The nitrogen, phosphorus and potash (N, P and K) contents are not enriched.
• It provides nutrient to the soil but will not be able to increase the soil fertility for crop rotation.
• Needs to be dosed at higher rates with every crop.
Few inventions have been disclosed related to the enriching the solid/liquid manure from biogas plant and are described here under.
RU2628411C2 relates to a method for inoculating the soil with specific microorganisms to improve soil fertility. This invention does not talk about the stabilization of digestate from biogas plants, reduction in composting time, improving the manure quality, production of plant growth promoting substances.
JP2003009848A relates to a method for degrading wastes at faster rate using specific microbes and under controlled temperature (40-65 °C). This invention does not talk about the stabilization of digestate from biogas plants, reduction in composting time, improving the manure quality, production of plant growth promoting substances. However, in the present disclosure, there is no need of controlling temperature.
JP2010088309A relates to a process of decomposing organic waste using specific microbes at faster rate. This invention does not teach about the stabilization of digestate from biogas plants, reduction in composting time, improving the manure quality, production of plant growth promoting substances.
All the existing inventions are based on preparing microbial blends and process development for degradation/decomposition of organic waste directly but not for stabilization of the digestate from biogas plant. Further, they are focused only on stabilization of manure and improving quality but not on enriching the manure with desired microbes and increasing soil fertility through addition of these specific microbes into manure.
SUMMARY OF THE INVENTION:
The present invention relates to a process for stabilization of a nutrient rich residue and preparation of an enriched fermented solid or liquid organic manure, wherein the process comprises the steps of: obtaining a solid or a liquid fermented organic manure from an anaerobic digester; mixing amicrobial blend 1to the fermented organic manure obtained from step a); adding a microbial stimulant 1 to the fermented organic manure obtained from step b); adding a microbial stimulant 2 to the solid fermented organic manure obtained from step c); adding a microbial blend 2 to the fermented organic manure obtained from step d) to obtain the enriched fermented organic manure.
The present invention also relates to a combination to prepare enriched organic manure comprising: a microbial blend 1; a microbial blend 2; a microbial stimulator 1; a microbial stimulator 2; a carrier, wherein the microbial blend 1 comprises microbes of Seq. ID 1, Seq. ID 2, Seq. ID 3, Seq. ID 4, Seq. ID 5 and Seq. ID 6; and microbial blend 2 comprises microbes of Seq. ID 7, Seq. ID 8, Seq. ID 9, Seq. ID 10, Seq. ID 11, Seq. ID 12; Seq. ID 13, Seq. ID 14, Seq. ID 15, Seq. ID 16 and Seq. ID 17; wherein the concentration of microbial blend 1 is in a range of 45-55 L/Ton in fermented organic manure and in a range of 45-55 L/kL in liquid fermented organic manure; wherein the microbial blend 2 along with carrier is added in the concentration in a range of 45 -55 L/Ton in fermented organic manure and in the range of 45-55 L/kL without any carrier in liquid fermented organic manure.
The present invention includes combination of different microbes which harmoniously work together and synergistically promote the composting.
DESCRIPTION OF THE ACCOMPANYING DRAWINGS:
These and other features, aspects, and advantages of the present disclosure will become better understood when the following detailed description is read with reference to the accompanying drawings in which like characters represent like parts throughout the drawings, wherein:
Figure 1 illustrates enzyme activity (cellulase, laccase and peroxidase) of isolates.
Figure 2 illustrates N2 fixation activity of isolates.
Figure 3 illustrates solubilization ability of isolates for phosphorus and potassium.
Figure 4 illustrates production of plant growth promoting substances by isolates.
Figure 5 illustrates siderophores production ability of isolates.
Figure 6 illustrates schematic representation of current invention.
Figure 7 illustrates change in cellulase activity of with time in case of conventional stabilization and present invention (solid line: conventional and dashed line: present invention; PI: present invention).
Figure 8 illustrates change in laccase activity of with time in case of conventional stabilization and present invention (solid line: conventional and dashed line: present invention; PI: present invention).
Figure 9 illustrates change in peroxidase activity of with time in case of conventional stabilization and present invention (solid line: conventional and dashed line: present invention; PI: present invention).
Figure 10 illustrates seed germination rate from soil dosed with conventional FOM and enriched FOM.
Figure 11 illustrates gibberelic acid from soil dosed with conventional FOM and enriched FOM.
Figure 12 illustrates IAA from soil dosed with conventional FOM and enriched FOM.
Figure 13 illustrates siderophores from soil dosed with conventional FOM and enriched FOM.
DETAILED DESCRIPTION OF THE INVENTION:
The present disclosure addresses the drawbacks of the art and provides for a process for stabilization of a nutrient rich residue and preparation of an enriched solid fermented organic manure and enriched liquid fermented manure. In some embodiments, the present disclosure also provides a process for preparation of enriched fermented organic manure, by employing microbial blends and microbial stimulants.
For the purpose of promoting an understanding of the principles of the disclosure, reference will now be made to the embodiment illustrated in the drawings and specific language will be used to describe the same. It will nevertheless be understood that no limitation of the scope of the disclosure is thereby intended, such alterations and further modifications in the illustrated system, and such further applications of the principles of the disclosure as illustrated therein being contemplated as would normally occur to one skilled in the art to which the disclosure relates. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skilled in the art to which this disclosure belongs. The system, methods, and examples provided herein are illustrative only and not intended to be limiting.
Although any methods and materials similar or equivalent to those described herein can also be used in the practice or testing of the present disclosure, the preferred methods and materials are now described. All publications mentioned herein are incorporated herein by reference to disclose and describe the methods and/or materials in connection with which the publications are cited.
It must be noted that as used herein and in the appended claims, the singular forms "a", "and", and "the" include plural referents unless the context dictates otherwise. Thus, for example, reference to "a compound" includes a plurality of such compounds, and reference to "the step" includes reference to one or more steps and equivalents thereof known to those skilled in the art, and so forth.
The term “some” as used herein is defined as “none, or one, or more than one, or all.” Accordingly, the terms “none,” “one,” “more than one,” “more than one, but not all” or “all” would all fall under the definition of “some.” The term “some embodiments” may refer to no embodiments or to one embodiment or to several embodiments or to all embodiments. Accordingly, the term “some embodiments” is defined as meaning “no embodiment, or one embodiment, or more than one embodiment, or all embodiments.”
The terminology and structure employed herein is for describing, teaching and illuminating some embodiments and their specific features and elements and does not limit, restrict or reduce the spirit and scope of the claims or their equivalents.
More specifically, any terms used herein such as but not limited to “includes”, “comprises”, “has”, “consists” and grammatical variants thereof is inclusive or open-ended and does not exclude additional, unrecited elements or method steps. The specification will be understood to also include embodiments which have the transitional phrase “consisting of” or “consisting essentially of” in place of the transitional phrase “comprising.” The transitional phrase “consisting of” excludes any element, step, or ingredient not specified in the claim, except for impurities associated therewith. The transitional phrase “consisting essentially of” limits the scope of a claim to the specified materials or steps “and those that do not materially affect the basic and novel characteristic(s)” of the claimed disclosure.
Whether or not a certain feature or element was limited to being used only once, either way it may still be referred to as “one or more features” or “one or more elements” or “at least one feature” or “at least one element.” Furthermore, the use of the terms “one or more” or “at least one” feature or element do NOT preclude there being none of that feature or element, unless otherwise specified by limiting language such as “there NEEDS to be one or more. ” or “one or more element is REQUIRED.”
As used herein, the term “about” is used to indicate a degree of variation or tolerance in a numerical or quantitative value. It indicates that the disclosed value is not intended to be strictly limiting, and may vary by plus or minus 5%, without departing from the scope of the invention.
Unless otherwise defined, all terms, and especially any technical and/or scientific terms, used herein may be taken to have the same meaning as commonly understood by one having an ordinary skill in the art.
Reference is made herein to some “embodiments”. It should be understood that an embodiment is an example of a possible implementation of any features and/or elements presented in the attached claims. Some embodiments have been described for the purpose of illuminating one or more of the potential ways in which the specific features and/or elements of the attached claims fulfil the requirements of uniqueness, utility and non-obviousness.
Use of the phrases and/or terms such as but not limited to “a first embodiment,” “a further embodiment,” “an alternate embodiment,” “one embodiment,” “an embodiment,” “multiple embodiments,” “some embodiments,” “other embodiments,” “further embodiment”, “furthermore embodiment”, “additional embodiment” or variants thereof do NOT necessarily refer to the same embodiments. Unless otherwise specified, one or more particular features and/or elements described in connection with one or more embodiments may be found in one embodiment, or may be found in more than one embodiment, or may be found in all embodiments, or may be found in no embodiments. Although one or more features and/or elements may be described herein in the context of only a single embodiment, or alternatively in the context of more than one embodiment, or further alternatively in the context of all embodiments, the features and/or elements may instead be provided separately or in any appropriate combination or not at all. Conversely, any features and/or elements described in the context of separate embodiments may alternatively be realized as existing together in the context of a single embodiment.
As used herein, the term "enriched manure" refers to a value-added product produced by co-compositing various organic wastes with microbial biofertilizers inoculum.
As used herein, the term “waste-water” refers to used water, which includes substances such as dairy wastewater, food industry waste, food scraps, juice industry wastewater, molasses and oils/ oil processing waste-water.
As used herein the terms “method” and “process” have been used interchangeably.
The present invention relates to a process for stabilization of a nutrient rich residue and preparation of an enriched fermented solid or liquid organic manure.
In some embodiments, the process comprises the steps of:
a. obtaining a solid or a liquid fermented organic manure from an anaerobic digester;
b. mixing amicrobial blend 1to the fermented organic manure obtained from step a);
c. adding a microbial stimulant 1 to the fermented organic manure obtained from step b);
d. adding a microbial stimulant 2 to the solid fermented organic manure obtained from step c);
e. adding a microbial blend 2 to the fermented organic manure obtained from step d) to obtain the enriched fermented organic manure.
The present invention provides for a quick and efficient method for stabilization of FOM and LFOM as well as enriching them with desired microbes for enhancing soil fertility. The microbial communities digest most of the organic matter FOM and LFOM converting them into inorganic compounds.
In an embodiment, there is provided the process, wherein the process comprises the steps of:
a. obtaining the solid fermented organic manure from the anaerobic digester;
b. reducing a moisture content of the solid fermented organic manure obtained from step a) to 40-60%;
c. mixing the microbial blend 1to the fermented organic manure with reduced moisture content obtained from step b);
d. adding the microbial stimulant 1 to the fermented organic manure obtained from step c);
e. adding the microbial stimulant 2 to the fermented organic manure obtained from step d) and forming a heap;
f. adding the microbial blend 2 to the fermented organic manure obtained from step e);
g. leaving the manure from step f) for 8 to 10 days to obtain enriched solid fermented organic manure.
In an embodiment, there is provided the process, wherein the process comprises the steps of:
a. obtaining the liquid fermented organic manure from the anaerobic digester;
b. stabilizing the liquid fermented organic manure obtained from step a) for 1-2 days;
c. adding the microbial blend 1 and the microbial stimulant 1 to the fermented liquid organic manure obtained from step b);
d. adding of the microbial blend 2 to the fermented liquid organic manure obtained from step c); and
e. leaving the fermented liquid organic manure from step d) for at least 1 day to obtain enriched liquid fermented organic manure.
In an embodiment, there is provided the process, wherein the fermented organic manure is a nutrient rich residue obtained from the anaerobic digester.
In an embodiment, there is provided the process, wherein the microbial blend 1 is a consortia of Seq. ID 1, Seq. ID 2, Seq. ID 3, Seq. ID 4, Seq. ID 5 and Seq. ID 6 and microbial blend 2 is a consortia of Seq. ID 7, Seq. ID 8, Seq. ID 9, Seq. ID 10, Seq. ID 11, Seq. ID 12; Seq. ID 13, Seq. ID 14, Seq. ID 15, Seq. ID 16 and Seq. ID 17.
In an embodiment, there is provided the process, wherein the Seq. ID 1 is Streptomyces viridosporus MTCC 25541, Seq. ID 2 is Thermoanaerobacterium thermosaccharolyticum MTCC 25529, Seq. ID 3 is Glomus fasciculatum MTCC 25543, Seq. ID 4 is Aspergillus fumigates MTCC 25544, Seq. ID 5 is Tricoderma sp. MTCC 25547, Seq. ID 6 is Aspergillus niger MTCC 25548, Seq. ID 7 is Serratia marcescens MTCC 25532, Seq. ID 8 is Aneurinibacillus aneurinilyticus MTCC 25531, Seq. ID 9 is Pseudomonas fluorescens MTCC 25530, Seq. ID 10 is Rhizobium leguminosarum MTCC 25538, Seq. ID 11 is Azotobacter vinelandii MTCC 25537, Seq. ID 12 is Azospirillum lipoferum MTCC 25535, Seq. ID 13 is Bradyrhizobium japonicum MTCC 25536, Seq. ID 14 is Bacillus megaterium MTCC 25540, Seq. ID 15 is Frateuria aurantia MTCC 25539, Seq. ID 16 is Brevibacillus brevis MTCC 25533, Seq. ID 17 is Bacillus pumilus MTCC 25534.
In an embodiment, there is provided the process, wherein the Seq. ID 1, Seq. ID 2, Seq. ID 7, Seq. ID 8, Seq. ID 9, Seq. ID 10, Seq. ID 11, Seq. ID 12, Seq. ID 13, Seq. ID 14, Seq. ID 15, Seq. ID 16 and Seq. ID 17 are 16S rRNA and Seq. ID 3, Seq. ID 4; Seq. ID 5 and Seq. ID 6 are 18S rRNA.
In an embodiment, there is provided the process, wherein the microbial stimulant 1 is added 3-4 days after adding the microbial blend 1; the microbial stimulant 2 is added 4-5 days after adding the microbial stimulant 1; the microbial blend 2 is added 7-8 days after adding the microbial stimulant 2 and left for 4-5 days to obtain the enriched fermented organic manure.
In an embodiment, there is provided the process, wherein the microbial blend 1 and the microbial stimulant 1 are added simultaneously; the microbial blend 2 is added 2-3 after adding the microbial blend 1 and the microbial stimulant 1 and left for 2-3 days to obtain the enriched liquid fermented organic manure.
In an embodiment, there is provided the process, wherein the microbial blend 1 is the consortia comprising of a cellulolytic, a laccase-producing and a peroxidase producing micro-organisms and the microbial blend 2 is the consortia comprising of a nitrogen fixer, a phosphate solubilizers, a potash solubilizers, a siderophores producers and a plant growth promoters.
In an embodiment, there is provided the process, wherein the microbial blend 1 is added in a concentration in a range of 45-55 L/ Ton in fermented organic manure and in a range of 45-55 L/ kL in liquid fermented organic manure.
In an embodiment, there is provided the process, wherein the microbial blend 2 is added in a concentration in a range of 45-55 kg/Ton in fermented organic manure and in a range of 45-55 L/kL in liquid fermented organic manure mixed with a carrier.
In an embodiment, there is provided the process, wherein the carrier is selected from cake obtained after oil extraction from nuts selected from ground nut, palm, sunflower, mustard, rice bran, combination thereof; cocopeat; saw dust or rice bran; activated carbon powder or carbon powder or graphite powder or combination thereof and edible oil.
In an embodiment, there is provided the process, wherein the microbial stimulator 1 comprises FeCl2 (0.08-0.1% w/v), MgSO4 (0.1-0.15% w/v), MnSO4 (0.1-0.15% w/v), NaHCO3 (0.15-0.2% w/v), KNO3 (0.05-0.15% w/v), KH2PO4 (0.2-0.3 % w/v) and K2HPO4 (0.2-0.3% w/v) in water, waste-water or combination thereof.
In an embodiment, there is provided the process, wherein the microbial stimulant 1 is added in the concentration in a range of 45 to 55 L/Tonin fermented organic manure and in a range of 45-55 L/kL in liquid fermented organic manure.
In an embodiment, there is provided the process, wherein the microbial stimulant 2 comprises yeast extract (0.2-0.25% w/v), ZnCl2 (0.2-0.3% w/v), CaCO3 (0.3-0.35% w/v), (NH4)2SO4 (0.1-0.15% w/v) in water, waste-water or combination thereof.
In an embodiment, there is provided the process, wherein the microbial stimulant 2 is added in the concentration in a range of 45-55 L/Ton.
In an embodiment, there is provided the process, wherein the tilling or the mixing is carried out at a time interval in a range of 5 -7 hours.
In an embodiment, there is provided the process, wherein the mixing is carried out using aerotiller, rotavator or tiller.
In an embodiment, there is provided the process, wherein the time required for producing the enriched manure is reduced to 4-5 weeks.
The present invention also discloses a combination to prepare enriched organic manure comprising: a microbial blend 1; a microbial blend 2; a microbial stimulator 1; a microbial stimulator 2; a carrier, wherein the microbial blend 1 comprises microbes of Seq. ID 1, Seq. ID 2, Seq. ID 3, Seq. ID 4, Seq. ID 5 and Seq. ID 6; and microbial blend 2 comprises microbes of Seq. ID 7, Seq. ID 8, Seq. ID 9, Seq. ID 10, Seq. ID 11, Seq. ID 12; Seq. ID 13, Seq. ID 14, Seq. ID 15, Seq. ID 16 and Seq. ID 17;wherein the concentration of microbial blend 1 is in a range of 45-55 L/Ton in fermented organic manure and in a range of 45-55 L/kL in liquid fermented organic manure; wherein the microbial blend 2 along with carrier is added in the concentration in a range of 45 -55 L/Ton in fermented organic manure and in the range of 45-55 L/kL without any carrier in liquid fermented organic manure.
In an exemplary and non-limiting embodiment, the concentration of essential elements such as Carbon, Nitrogen, Phosphorus and Potassium is higher in the organic manure of the present invention for healthy growth of crops/plants.
The present disclosure is further illustrated by reference to the following examples which is for illustrative purpose only and does not limit the scope of the disclosure in any way. These examples are not intended to be inclusive of all aspects of the subject matter disclosed herein, but rather to illustrate representative features, methods, compositions, and results. These examples are not intended to exclude equivalents and variations of the present disclosure, which are apparent to one skilled in the art.
EXAMPLES:
Material and methods
Biological Materials:
The biological materials (Streptomyces viridosporus MTCC 25541, Thermoanaerobacterium thermosaccharolyticum MTCC 25529, Glomus fasciculatum MTCC 25543, Aspergillus fumigates MTCC 25544, Tricodermasp. MTCC 25547, Aspergillus niger MTCC 25548, Serratia marcescens MTCC 25532, Aneurinibacillus aneurinilyticus MTCC 25531, Pseudomonas fluorescens MTCC 25530, Rhizobium leguminosarum MTCC 25538, Azotobacter vinelandii MTCC 25537, Azospirillum lipoferum MTCC 25535, Bradyrhizobium japonicum MTCC 25536, Bacillus megaterium MTCC 25540, Frateuria aurantia MTCC 25539, Brevibacillus brevis MTCC 25533 and Bacillus pumilus MTCC 25534) were procured from Indian Oil Research and Development Center, Sector-13, Faridabad 121007.
Example 1: Screening and isolation of specific microbes
Isolation of the microbes was carried out in a step wise protocol. Sample from different composting sites were collected and mixed in equal proportion to prepare a composite sample. 1% of composite sample was added in a flask containing MMM (mineral minimum medium, pH 7.2) (NaNO3 2.5?g; KH2PO4 2?g; MgSO4 0.2?g; NaCl 0.2?g; CaCl2·6H2O 0.1?g in a litre) along with 2% digestate from biogas plant and was mixed thoroughly. The flasks were incubated for 5 days in a shaker incubator at 37°C at 100?rpm. After 5days, 5 ml of the culture broth was taken and transferred to flask having same composition. Similar stepswere repeated for 3-5 cycles. After 3-5 cycles, fungi were isolated on Potato DextroseAgar (PDA) and bacteria were isolated using Nutrient Agar (NA) as per standard microbiology practices.
Microbes (bacteria/fungi) capable of producing cellulase, laccase and peroxidase at higher rate were considered for preparing MB-1 which were selectively enriched and isolated using standard microbiological practices. The enzyme activities, viz., cellulase, laccase and peroxidase, were carried out for each isolate separately and as a mixture. For this, all the cultures were grown on media and once growth was obtained, the supernatant of the centrifuged culture was used for enzyme activity assay. Procedure adapted for each enzyme assay is as follows.
Cellulase activity
Cellulases are group of enzymes, viz., endo-1,4-glucanase, exo-1,4-glucanase, and ß-D-glucosidase that catalyze the degradation of cellulose into reducing sugars. Cellulase activity was examined with carboxymethyl cellulose (CMC) as substrate. The assay mixture consisted of 0.25% substrate and 0.2 mL of sample in 50 mM phosphate buffer with a pH of 6.8. The reducing sugars produced from CMC were measured continuously to estimate the activity.
Laccase activity
Laccase activity was determined through the oxidation of ABTS (2,2-azino-bis-3-ethylbenzothiazoline-6-sulphonic acid) and color change is an indication of activity. The ABTS is a nonphenolic dye and its oxidation by laccase to stable cation radical produce intense blue-green color which was read at 420?nm. The assay mixture contained 0.5?mM ABTS, 0.1?M sodium acetate (pH 4.5), and a suitable amount of enzyme. One unit was defined as the amount of the laccase that oxidized 1?µmol of ABTS substrate per min.
Peroxidase activity
The peroxidase was measured through oxidation of veratryl alcohol. 25 mM sodium tartrate buffer (pH 3.0) with 2 mM veratryl alcohol and 0.4 mM H2O2was incubated for 10 min in presence of enzyme. The activity was measured through change in absorbance at 310 nm due to the H2O2 dependent oxidation of veratryl alcohol to veratraldehyde.
For preparing MB-2, different microbes were screened as follows:
Microbial species that belongs to Rhizobium colonizes plant roots and helps in nitrogen fixation. Rhizobium is also known to produce gibberellin and Indole 3-Acetic Acid (IAA), stimulating plant growth. Rhizobium was selectively isolated on media containing Mannitol 10 g/L, Dipotassium phosphate 0.5 g/L, Magnesium sulphate 0.2 g/L, Yeast extract 1 g/L, Sodium chloride 0.1 g/L and grown at 37ºC. Nitrogen fixation ability of all isolates was measured using Jenson’s media known in prior art.
Microbes capable of hydrolyzing organic and inorganic insoluble phosphorus compounds to soluble phosphorus form, that can easily be assimilated by plants, are known as phosphate solubilizing microbes. Screening and isolation of these microbes was carried out on selective media containing glucose 10 g/L, Ca3(PO4) 25 g/L, CaCO3 5g/L, (NH4)2SO4 0.5 g/L, NaCl 0.2 g/L, MgSO4.7H2O, 0.1 g/L, KCl 0.1 g/L, MnSO4 0.002 g/L, FeSO4.7H2O 0.002 g/L, pH 7.0. The soluble phosphorus content of growth media was determined using molybdenum antimony scandium colorimetric method.
Siderophore production by microbes was confirmed by growing different isolates on selective chrome azurol sulfonate (CAS) agar medium at 37ºC for 24 hours which is known in prior art. The quantitative analysis of siderophores was carried out using known prior art where CAS reagent turns to orange red color by production of sideropores and was quantified at 480 nm.
The plant growth promoting (PGP) substances like gibberelic acid (GA) and indole-3-acetic acid (IAA) were also produced by some bacteria/fungi which help in plant growth. The ability of producing PGP substances was identified by using method known in prior art. For this, the pH of filtrate of growth media was adjusted to 2.5 followed by liquid-liquid extraction using ethylacetate and NaHCO3. GA in the ethylacetate phase was quantified at 254 nm. Similarly, IAA production was measured by mixing the cell free supernatant with Salkowski’s reagent at 1:2 ratio and incubated for 30 min followed by measuring OD at 600 nm.
All the isolates were firstly screened through morphological examination depending on the colors of colony formed at the top and reverse of the cultures. The microscopic examination of the shape of the spore-producing structures was used for further identification. The molecular identification was carried out by DNA barcoding using the ITS region sequencing. The ITS rDNA sequences were compared to those in the databases using NCBI-BLAST.
Example 2: Development of consortia
All the isolates were checked for different biochemical and metabolic functions (Figures 1, 2, 3, 4 and5) based on which two different consortia were prepared (MB-1 and MB-2).
Among the isolates, six isolates were found to have higher enzyme activities, viz., cellulase, laccase and peroxidase and these six isolates were mixed in equal proportion and prepared MB-1 consortia.
Apart from the six isolates, other 11 isolates have different biochemical functions like nitrogen fixation, phosphate solubilization, potash solubilization, siderophores production, plant growth promoters production. They all were mixed in equal proportion and MB-2 was prepared.
Example 3: Preparation of enriched solid/ liquid organic manure (FOM/LFOM)
Further, a step-wise methodology was developed for faster stabilization of FOM and LFOM, and improving their quality to premium grade. The steps involved in the preparation of enriched FOM/LFOM are as follows:
1. The biomass/organic waste is subjected to biomethanation and the outlet is obtained as slurry which is further separated as solid and liquid portion. The solid portion is called as fermented organic manure (FOM) and liquid portion as liquid FOM (LFOM).
2. The FOM contains about 50-70% moisture content which need to be reduced and stabilized prior to its use as manure. Similarly, the LFOM contains >95% moisture which also needs to be stabilized prior to its use as manure.
3. The FOM was initially added with a defined microbial blend (MB-1) in a concentration of 50 L/Ton of Streptomyces viridosporus MTCC 25541, Thermoanaerobacterium thermosaccharolyticum MTCC 25529, Glomus fasciculatum MTCC 25543; Aspergillus fumigates MTCC 25544; Tricoderma sp. MTCC 25547; Aspergillus niger MTCC 25548 that effectively fasten the stabilization process. These microbes specifically facilitate hydrolytic and depolymerizing functions.
4. After adding MB-1, the FOM was mixed thoroughly through tilling at 6h/day rate to allow aeration into the material and this was continued for 3-4 days. This facilitated a combination of microaeropohilic and aerobic (18 hours / 6 hours) conditions in a single day which enabled faster and efficient degradation of residual components. This process also eliminated the pathogenic microbes in the manure.
5. After 3-4 days, the FOM was added with microbial stimulant (MS-1) at 50L/Ton and tiling was continued for 4-5 days. The composition of MS-1 included FeCl2 (0.08-0.1% w/v), MgSO4 (0.1- 0.15% w/v), MnSO4 (0.1-0.15% w/v), NaHCO3 (0.15-0.2% w/v), KNO3 (0.05-0.15% w/v), KH2PO4 (0.2-0.3 % w/v) and K2HPO4 (0.2-0.3% w/v). The contents were mixed in raw water and sprayed on the material followed by tilling for proper mixing. Further, the MS-1 can also be prepared in waste-water of any food industry origin, if available.
6. After 4-5 days, the FOM is added with MS-2 at 50 L/Ton followed by tilling for 2-4 hours for proper mixing and then leave FOM as heap to attain 55-65 °C temperature inside the heaps. The composition of MS-2 includes yeast extract (0.2-0.25% w/v), ZnCl2 (0.2-0.3% w/v), CaCO3 (0.3- 0.35% w/v), (NH4)2SO4 (0.1-0.15% w/v).
7. The heap was left for 7-8 days without tilling to enable complete degradation of leftover organics and obtaining compost texture.
8. After 7-8 days, the FOM was added with another defined microbial blend (MB-2) that enriched the manure quality. The MB-2 included Serratia marcescens MTCC 25532; Aneurinibacillus aneurinilyticus MTCC 25531; Pseudomonas fluorescens MTCC 25530; Rhizobium leguminosarum MTCC 25538; Azotobacter vinelandii MTCC 25537; Azospirillum lipoferum MTCC 25535; Bradyrhizobium japonicum MTCC 25536; Bacillus megaterium MTCC 25540; Frateuria aurantia MTCC 25539; Brevibacillus brevis MTCC 25533; and Bacillus pumilus MTCC 25534. These microbes exhibited specific biochemical functions like nitrogen fixation, solubilization of potassium, phosphorus, zinc, etc., and produce plant growth promoting substances like siderophores and GA.
9. The MB-2 was added along with carrier (50 L/Ton) to safely carry the microbes to the soil where they can proliferate and increase soil fertility. The carrier consists of cake obtained after oil extraction from any nut, preferably ground nut, palm, sunflower, mustard and rice bran (0.3-0.4% w/w). Further it also consists of coco peat (0.2-0.3% w/w), saw dust or rice bran (0.2-0.3% w/w), activated carbon or carbon powder or graphite powder (0.2-0.3% w/w), used cooking oil (0.1- 0.2% w/w).
10. After adding MB-2 and carrier, the FOM was mixed thoroughly at a rate of once in a day for 8-10 days.
11. After 8-10 days, the compost was ready for dispatch. Prior to dispatch, the material was processed through pulverizer, if required to remove any lumps. The final moisture of enriched FOM was maintained at 25-30%.
12. In case of LFOM, the liquid was initially added with MB-1 and MS-1 at a concentration of 50 L/kL each simultaneously and mixed thoroughly for 2-3 days.
13. After, 2-3 days, the LFOM was added with MB-2 along with carrier (50 L/kL). The carrier in case of LFOM includes activated carbon powder or carbon powder or graphite powder (0.5-0.6% w/w) and any edible oil, preferably used cooking oil (0.4-0.5% w/w).
14. The carrier and MB-2 were mixed thoroughly with LFOM and left for 2-3 days with mixing once in a day. The enriched LFOM was ready for dosing after 2-3 days.
Example 4: Role of each microbial blend (MB-1 and MB-2) and stimulators (MS-1 and MS-2)
The role of each microbial blend and stimulator in the current process of composting was established using digestate from different feedstock, viz., kitchen waste, press mud and paddy straw. For this, experiments were conducted using different combinations of MB-1, MB-2, MS-1 and MS-2. All other conditions and methodology were kept same as mentioned in Figure 6.
Table 1: Consolidated data obtained from different combinations of microbial blend (MB-1 and MB-2) and stimulators (MS-1 and MS-2) using digestate of kitchen waste

Table 2: Consolidated data obtained from different combinations of microbial blend (MB-1 and MB-2) and stimulators (MS-1 and MS-2) using digestate of press mud

Table 3: Consolidated data obtained from different combinations of microbial blend (MB-1 and MB-2) and stimulators (MS-1 and MS-2) using digestate of paddy straw

Example 5: Faster and efficient composting process
The efficiency of proposed stabilization and enrichment process of FOM/LFOM over conventional stabilization process was evaluated using digestate obtained from biomethanation of different feedstock, viz., Kitchen waste (KW), Organic fraction of municipal solid waste (OF-MSW), Press mud (PM), Cattle dung (CD) and Paddy straw (PS) (Table 4 and 5). Different experiments were performed using the protocol detailed in figure 6. The stabilization of digestate in conventional manner is considered as control. Briefly, the digestate spread over for drying for 8-10 days and mixed thoroughly through tilling. Then, FOM is kept as heap for 15-20 days followed by again spreading for 10-15 days. Finally, the FOM is pulverized and used as it is.
Table 4: Characteristics of FOM and enriched FOM obtained from digestate of different feedstock

Table 5: Characteristics of LFOM and enriched LFOM obtained from digestate of different feedstock

Results of the experiment depicted substantial improvement in the quality of FOM/LFOM in terms of N, P, K, organic carbon and C/N ratio, which are crucial parameters for a premium quality manure. Substantial improvement in reducing the particle size also observed even up to >98% in case of FOM indicating the improved process.
Example 6: Monitoring of different enzyme activities
During all the experiments, samples were collected at regular time intervals from both conventional stabilization and present invention, for analyzing activities of cellulase, peroxidase and laccase which are crucial enzymes involved in decomposition of the organic matter. The data obtained from digestate of different feedstock, viz., KW, OF-MSW, PM, CD and PS were compared against time (Figures 7, 8 and 9).
Results indicated substantial improvement in all the desired enzyme activities during enrichment of FOM compared to conventional stabilization indicating efficient and faster stabilization process. Improved characteristics of FOM/LFOM is also supporting this increment of enzyme activity.
Example 7: Effect of enriched FOM on soil fertility and crop production
The enriched FOM/LFOM and conventional FOM/LFOM were mixed into soil at 2 Ton/acre rate and 10 kg mixture placed in flat trays for experiment. These trays were sowed with fenugreek seeds 5g/tray rate (approx. 250 seeds). Proper moisture maintained in trays and left it for 15 days. Then samples were collected and mixed thoroughly in water (10% w/v) and supernatant was estimated for the siderophores, gibberelic acid and IAA using standard methods from literature. Further, seed germination was calculated from both trays. This experiment was conducted for FOM from all selected digestates (Figures 10, 11, 12 and 13). In addition to that, the soil fertility characteristics were also assessed to establish the efficiency of enriched FOM/LFOM in improving the native soil nature.
The results indicated significant improvement in all the parameters of plant growth promotion. Presence of specific microbes that produce plant growth promoters and also solubilizes the nutrients required for plant growth in soil showed a clear demarcation between the conventional FOM and enriched FOM. The seed germination was about 60% in case of conventional FOM which was increased to more than 93% in case of dosing enriched FOM. Further, the soil characteristics were also analyzed after the crop harvesting to assess the change in soil fertility (Table 6).
Table 6: Change in soil fertility factors after dosing FOM and enriched FOM obtained from different digestates

Significant improvement in soil fertility characteristic observed in the soil dosed with enriched FOM irrespective of the feedstock used for generating digestate. The organic carbon content of soil enhanced to a large extent along with N, P, K content in the soil dosed with enriched FOM. The results indicate the efficiency of enriched FOM to regain the soil fertility over the period of time and reducing the load of inorganic fertilizers.
Advantages of present invention:
The present invention provides a quick and efficient method for stabilization of FOM and LFOM as well as enriching them with desired microbes for enhancing soil fertility. The microbial communities digest most of the organic matter FOM and LFOM converting them into inorganic compounds. These added selective microbes mineralize the nutrients during composting process as well as make them crop available during use of such enriched manure in field. The inorganic nutrients in the FOM and LFOM are present in plant-utilizable forms at a markedly higher level compared to the without adding microbes, due to the mineralization of organic matter by the added microbes.
Addition of the designed microbial blend to digestate (FOM/LFOM) from any biogas plant has following advantages:
• Fasten the composting/stabilization process (4-5 weeks in comparison to 7-8 weeks) by secreting both extra/intra cellular enzymes
• Chemical free and highly effective in enhancing soil fertility
• Microbes help in enhancing the bioavailable micro and macro nutrients in soil
• The enriched compost enhances the soil fertility by enhancing organic/inorganic constituents and minerals of the soil that helps in plant growth.
• The inorganic carbon content is increases through mineralization of the organic constituents.
• The fixed carbon in soil that is unavailable to plant will be converted to free carbon enabling easy access to plants.
• Nitrogen content increased by converting the ammoniacal nitrogen to inorganic nitrogen (mostly nitrates). Further, nitrogen fixation is continued once these microbes colonize on the plant root.
• Phosphorus, potassium, zinc, sulfur, etc., embedded in the organic molecules are converted to soluble form so that plant can easily absorb the same.
• Microbes present in enriched premium compost deliver siderophores that balance the phytohormones enabling plant growth.
• Microbes of enriched compost delivers plant growth promoting substances like gibberelic acid. ,
Claims:1. A process for stabilization of a nutrient rich residue and preparation of an enriched fermented solid or liquid organic manure, wherein the process comprises the steps of:
a. obtaining a solid or a liquid fermented organic manure from an anaerobic digester;
b. mixing amicrobial blend 1to the fermented organic manure obtained from step a);
c. adding a microbial stimulant 1 to the fermented organic manure obtained from step b);
d. adding a microbial stimulant 2 to the solid fermented organic manure obtained from step c);
e. adding a microbial blend 2 to the fermented organic manure obtained from step d) to obtain the enriched fermented organic manure.
2. The process as claimed in claim 1, wherein the process comprises the steps of:
a. obtaining the solid fermented organic manure from the anaerobic digester;
b. reducing a moisture content of the solid fermented organic manure obtained from step a) to 40-60%;
c. mixing the microbial blend 1to the fermented organic manure with reduced moisture content obtained from step b);
d. adding the microbial stimulant 1 to the fermented organic manure obtained from step c);
e. adding the microbial stimulant 2 to the fermented organic manure obtained from step d) and forming a heap;
f. adding the microbial blend 2 to the fermented organic manure obtained from step e);
g. leaving the manure from step f) for 8 to 10 days to obtain enriched solid fermented organic manure.

3. The process as claimed in claim 1, wherein the process comprises the steps of:
a. obtaining the liquid fermented organic manure from the anaerobic digester;
b. stabilizing the liquid fermented organic manure obtained from step a) for 1-2 days;
c. adding the microbial blend 1 and the microbial stimulant 1 to the fermented liquid organic manure obtained from step b);
d. adding of the microbial blend 2 to the fermented liquid organic manure obtained from step c); and
e. leaving the fermented liquid organic manure from step d) for at least 1 day to obtain enriched liquid fermented organic manure.

4. The process as claimed in claim 1, wherein the fermented organic manure is a nutrient rich residue obtained from the anaerobic digester.
5. The process as claimed in claim 1, wherein the microbial blend 1 is a consortia of Seq. ID 1, Seq. ID 2, Seq. ID 3, Seq. ID 4, Seq. ID 5 and Seq. ID 6 and microbial blend 2 is a consortia of Seq. ID 7, Seq. ID 8, Seq. ID 9, Seq. ID 10, Seq. ID 11, Seq. ID 12; Seq. ID 13, Seq. ID 14, Seq. ID 15, Seq. ID 16 and Seq. ID 17.
6. The process as claimed in claim 1, wherein the Seq. ID 1 is Streptomyces viridosporus MTCC 25541, Seq. ID 2 is Thermoanaerobacterium thermosaccharolyticum MTCC 25529, Seq. ID 3 is Glomus fasciculatum MTCC 25543, Seq. ID 4 is Aspergillus fumigates MTCC 25544, Seq. ID 5 is Tricodermasp. MTCC 25547, Seq. ID 6 is Aspergillus niger MTCC 25548, Seq. ID 7 is Serratia marcescens MTCC 25532, Seq. ID 8 is Aneurinibacillus aneurinilyticus MTCC 25531, Seq. ID 9 is Pseudomonas fluorescens MTCC 25530, Seq. ID 10 is Rhizobium leguminosarum MTCC 25538, Seq. ID 11 is Azotobacter vinelandii MTCC 25537, Seq. ID 12 is Azospirillum lipoferum MTCC 25535, Seq. ID 13 is Bradyrhizobium japonicum MTCC 25536, Seq. ID 14 is Bacillus megaterium MTCC 25540, Seq. ID 15 is Frateuria aurantia MTCC 25539, Seq. ID 16 is Brevibacillus brevis MTCC 25533, Seq. ID 17 is Bacillus pumilus MTCC 25534.
7. The process as claimed in claims 1 and 6, wherein the Seq. ID 1, Seq. ID 2, Seq. ID 7, Seq. ID 8, Seq. ID 9, Seq. ID 10, Seq. ID 11, Seq. ID 12, Seq. ID 13, Seq. ID 14, Seq. ID 15, Seq. ID 16 and Seq. ID 17 are 16S rRNA and Seq. ID 3, Seq. ID 4; Seq. ID 5 and Seq. ID 6 are 18S rRNA.
8. The process as claimed in claims 1 and 2, wherein the microbial stimulant 1 is added 3-4 days after adding the microbial blend 1; the microbial stimulant 2 is added 4-5 days after adding the microbial stimulant 1; the microbial blend 2 is added 7-8 days after adding the microbial stimulant 2 and left for 4-5 days to obtain the enriched fermented organic manure.
9. The process as claimed in claims 1 and 3, wherein the microbial blend 1 and the microbial stimulant 1 are added simultaneously; the microbial blend 2 is added 2-3 after adding the microbial blend 1 and the microbial stimulant 1 and left for 2-3 days to obtain the enriched liquid fermented organic manure.
10. The process as claimed in claim 1, wherein the microbial blend 1 is the consortia comprising of a cellulolytic, a laccase-producing and a peroxidase producing micro-organisms and the microbial blend 2 is the consortia comprising of a nitrogen fixers, a phosphate solubilizers, a potash solubilizers, a siderophores producers and a plant growth promoters.
11. The process as claimed in claim 1, wherein the microbial blend 1 is added in a concentration in a range of 45-55 L/ Ton in fermented organic manure and in a range of 45-55 L/ kL in liquid fermented organic manure.
12. The process as claimed in claim 1, wherein the microbial blend 2 is added in a concentration in a range of 45-55 kg/Ton in fermented organic manure and in a range of 45-55 L/kL in liquid fermented organic manure mixed with a carrier.
13. The process as claimed in claims 1 and 12, wherein the carrier is selected from cake obtained after oil extraction from nuts selected from ground nut, palm, sunflower, mustard, rice bran, combination thereof; cocopeat; saw dust or rice bran; activated carbon powder or carbon powder or graphite powder or combination thereof and edible oil.
14. The process as claimed in claim 1, wherein the microbial stimulator 1 comprises FeCl2 (0.08-0.1% w/v), MgSO4 (0.1-0.15% w/v), MnSO4 (0.1-0.15% w/v), NaHCO3 (0.15-0.2% w/v), KNO3 (0.05-0.15% w/v), KH2PO4 (0.2-0.3 % w/v) and K2HPO4 (0.2-0.3% w/v) in water, waste-water or combination thereof.
15. The process as claimed in claim 1, wherein the microbial stimulant 1 is added in the concentration in a range of 45 to 55 L/Ton in fermented organic manure and in a range of 45-55 L/kL in liquid fermented organic manure.
16. The process as claimed in claims 1 and 2, wherein the microbial stimulant 2 comprises yeast extract (0.2-0.25% w/v), ZnCl2 (0.2-0.3% w/v), CaCO3 (0.3-0.35% w/v), (NH4)2SO4 (0.1-0.15% w/v) in water, waste-water or combination thereof.
17. The process as claimed in claims 1 and 2, wherein the microbial stimulant 2 is added in the concentration in a range of 45-55 L/Ton.
18. The process as claimed in claim 1, wherein the tilling or the mixing is carried out at a time interval in a range of 5 -7 hours.
19. The process as claimed in claim 1, wherein the mixing is carried out using aerotiller, rotavator or tiller.
20. The process as claimed in claim 1, wherein the time required for producing the enriched manure is reduced to 4-5 weeks.
21. A combination to prepare enriched organic manure comprising:
a. a microbial blend 1;
b. a microbial blend 2;
c. a microbial stimulator 1;
d. a microbial stimulator 2;
e. a carrier
wherein the microbial blend 1 comprises microbes of Seq. ID 1, Seq. ID 2, Seq. ID 3, Seq. ID 4, Seq. ID 5 and Seq. ID 6; and
microbial blend 2 comprises microbes of Seq. ID 7, Seq. ID 8, Seq. ID 9, Seq. ID 10, Seq. ID 11, Seq. ID 12; Seq. ID 13, Seq. ID 14, Seq. ID 15, Seq. ID 16 and Seq. ID 17;
wherein the concentration of microbial blend 1 is in a range of 45-55 L/Ton in fermented organic manure and in a range of 45-55 L/kL in liquid fermented organic manure;
wherein the microbial blend 2 along with carrier is added in the concentration in a range of 45 -55 L/Ton in fermented organic manure and in the range of 45-55 L/kL without any carrier in liquid fermented organic manure.