Description:FIELD OF INVENTION:
The present disclosure generally relates to the field of agricultural science. More specifically, the present invention relates to a method of preparing a crop residue decomposer bioinoculant and its application thereof. The crop residue decomposer bioinoculant described in the present invention is cost effective, simple, efficient, rapid and requires less time to manage residual wastes as well as facilitate the in-situ sustainable and eco-friendly farming.

BACKGROUND OF INVENTION:
Agriculture waste management is one of the major challenges in front of human race to balance the escalating demand for food and its associated climate change impacts. About 998 million tons of agricultural residue waste is produced every year. Besides this, cattle houses and agriculture-based industry produces about 20 MMTPA waste on an average, which is a rich source of nutrients and manures. Agricultural residual wastes create major environmental problems without effective means of management methods. Specifically, paddy straw and wheat straw management is major challenge as they occupy the major fraction of agriculture residue. Burning of crop residue is one of the major causes in North India every year that creates major environmental (air pollution) and health issues. Further, burning of crop residue not only creates pollution but it also burns the top layer off the soil and deprives the agriculture fields of much needed nutrients. So, there is a need for an effective and sustainable waste disposal technology for converting this waste into some valuable form. As the farmers are unaware of the sustainable and ease of use methods for crop rotation by removing the residues is the basic cause for crop burning.

Decomposition of the crop residues is one of the sustainable methods that is adapted at ground level to overcome the climate mitigation challenges arriving due to crop burning. The effective decomposition methods also provide a quick means of crop rotation and help farmers to retain the nutrients from crop residue in their fields. Further, the decomposition also helps in retaining the major portion of the nutrients left over in stalk and the roots through microbial action that helps in increasing the soil fertility. Biological decomposition is an efficient decomposition method in which specific microbes (bacteria and fungi) facilitate the degradation of the crop residue at faster rate. The decomposition process depends upon the ability of different microbes used and the exocellular enzymes released by them. The successive catabolic reactions carried by microbial metabolism will mineralize the organic constituents into soil essential constituents, which will be the most effective sources of macro- and micronutrients for the soil fertility. The efficiency of these microbes depends upon different parameters like moisture, temperature, pH, etc., and the efficiency can be enhanced by optimization of microbial composition/blend. This will reduce the time required for decomposition and also enhance the nutrient composition of soil.

CN101638622A discloses a microbial inoculum for degrading straw and the active ingredients of the microbial inoculum comprise Trichoderma viride, Trichoderma aureoviride and forage Paenibacillus pabuli. The colony formation unit ratio of Trichoderma viride to Trichoderma aureoviride and forage Paenibacillus pabuli in the microbial inoculum is (0.8-1.2):(0.8-1.2):(0.8-1.2). The microbial inoculum for degrading straw of the invention can mature straw fast to prepare organic fertilizer, and the prepared organic fertilizer can be uses as a base fertilizer and can obviously improve the quality of vegetables.

CN102174398A discloses a composite microbiological bacterial agent used for returning maize straws to a field and a preparation method and applications thereof. The composite microbiological bacterial agent comprises Arthrobactersp, Bacillus subtilis, Phanerochaetechrysosporium, Trichoderma koningii, Aspergillus niger, Saccharomyces cerevisiae, Streptomyces alboniger, and Streptomyces hygroscopicus. The preparation method comprises the following steps: carrying out slant culture, fermenting one-grade seeds and second-grade seeds and mixing, culturing, and fermenting the one-grade seeds and the second-grade seeds, realizing the optimization of compound bacterial biomass and the matching of different strains, and the compound bacterium effective viable count can reach 1010cfu per gram. By utilizing the compound microbiological bacterial agent, the decomposition velocity of organic matters can be accelerated, the straw can be decomposed and blackened within 10 days at the low temperature of 10-15 DEG C; the straw is basically well-rotted; and the composite microbiological bacterial agent has certain disease-resisting effect and provides necessary condition for preventing the diseases of wheat roots.

CN103641522A discloses a crop straw decomposition agent. The crop straw decomposition agent is composed of the following components by mass percent: 20%-30% of bacillus subtilis, 20%-30% of aspergillus niger, 10%-15% of candida utilis, 15%-20% of trichoderma viride, 10%-15% of aspergillus oryzae and 1%-2% of cellulase. In order to solve the problems such that current straws are directly returned to fields, and the temperature rise is slow, the decomposition is slow and the decomposition effect is poor when the straws are used for composting an organic fertilizer, crop straw decomposition is tested for a plurality of years, microorganism strains and biological enzyme preparations are continuously screened, and finally, the bacillus subtilis, the Aspergillus niger, the candida utilis, the trichoderma viride, the Aspergillus oryzae and the cellulase are prepared into the crop straw decomposition agent according to the scheme. The four fungi are not antagonistic and combined with the cellulase to have a synergistic interaction effect and the straws can be rapidly decomposed.

CN101885624A discloses a decomposition maturing agent for degrading straw. The agent comprises active ingredients, such as candida tropicalis, aspergillus oryzae, trichoderma viride and bacillus subtilis. The decomposition maturing agent can rapidly decompose wheat straw, rice straw, grain straw, sweet potato stems, broad bean stems, rape stalks, weeds, leaves and household garbage of which the fibrous matter content is high so as to prepare an organic fertilizer. The decomposition maturing agent has the advantages of short degradation time and capability of effectively killing diseases and pests and providing nutrients for crops.

Juikar et al., 2017, Industrial crops & products 109 (2017) 420-425; depicts about nano lignin extraction from coconut fibers by a controlled microbial hydrolysis process and compared with the nano lignins prepared by high shear homogenization and ultrasonication processes. Further, the produced nano lignin was characterized using different analytical methods. However, this work doesn’t represent any nano lignin formation on field from the crop residue.

Hellequin et al., 2018, PLOS One 13(12) 2018; depicted the development of a bio stimulant to increase the mineralization of organic carbon and to increase the microbial biomass content of soil. However, this paper does not present any development of microbial blend for efficient and faster crop residue decomposition.

Kaushik etal., 2015, International biodeterioration & biodegradation 100 (2015) 70-78; describes an attempt to produce myco-granules targeted for multiple industrial applications including dye and heavy metal sequestration and xylanase production. Authors tried to produce myco granules for application in dye removal, metal removal and xylanase enzyme production. The prior art does not disclose any microbial blend preparation for crop residue decomposition.

Gola et al., 2019 Biotechnology research and innovation (2019) 3, 242-251; describes about production of three different formulations, viz., myco-granules, myco-tablets and myco-capsules of specific microbe, Beauveria bassiana, targeted for metal removal. Prior art reveals the formation of granules, tablets and capsules followed by their shelf life and stability over the period of one year. However, this study has not discussed any microbial blend for crop residue decomposition, use of bio stimulant, etc.

All the above-mentioned literature/prior arts do not talk about the isolation of microorganisms based on the activity of most desired enzymes for decomposition of cellulose, hemicelluloses and lignin components of biomass. None of this inventions/literature mentioned about the spore fermentation for safe handling of the product, no additional growth time required before dosing the culture and direct field application of the product. Further, no literature has shown the use of microbe adjuvant for increasing the rate of decomposition.

All the existing decomposition consortia are developed based on their degradation. The microbial blend must be regenerated at site in large volumes before dosing in the field. The time required for growth of the culture is in addition to the decomposition time. Health hazard to the farmers during its growth and dosing is another limitation for these consortia.

Thus, there is a need to develop a method of preparing a crop residue decomposer bioinoculant which has increased rate of decomposition, cost effective, safe, and environment friendly to provide commercial advantages.

SUMMARY OF THE INVENTION:
This summary is provided to introduce a selection of concepts, in a simplified format, that are further described in the detailed description of the invention. This summary is neither intended to identify key or essential inventive concepts of the invention and nor is it intended to determine the scope of the invention.

The present application describes a method of preparing a crop residue decomposer bioinoculant and its application. The crop residue decomposer bioinoculant of the present invention promotes the quick composting of crop residues in the field. The crop residue decomposer bioinoculant includes the synergistic combination of the facultative micro-aerophilic microorganisms with decomposition function for cellulose, hemicellulose, lignin, pectin, proteins and lipids present in biomass. Different microbes secrete complementary enzyme that degrade or decompose straw in the field facilitating the sustainable, eco-friendly and environmentally benign farming.

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 activities of individual isolates and StubVorous.

Figure 2 illustrates degradation pattern of major components of paddy straw.

Figure 3 illustrates SEM images of paddy straw degradation a) control b) present invention.

DETAILED DESCRIPTION OF THE INVENTION:
The present disclosure addresses the drawbacks of the art and provides for a method of preparing a crop residue decomposer bioinoculant and its application. Further, the crop residue decomposer bioinoculant is cost effective, simple, efficient, rapid and requires less time to manage residual wastes as well as facilitate the in-situ sustainable and eco-friendly farming.

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 “polymer composite” refers to a multi-phase material in which reinforcing fillers or fibres are integrated with a polymer matrix, resulting in synergistic mechanical properties that is not achieved from either component alone.

As used herein, the term “ligand” is an ion or molecule which donates a pair of electrons to the central metal atom or ion to form a coordination complex.

As used herein the terms “method” and “process” have been used interchangeably.

The present application describes a method of preparing a crop residue decomposer bioinoculant and its application. The crop residue decomposer bioinoculant described in the present invention is cost effective, simple, efficient, rapid and requires less time to manage agricultural and other residual wastes as compared to other existing methods.

The crop residue decomposer bioinoculant of the present invention promotes the quick composting of crop residues in the field. Which includes the synergistic combination of the facultative micro-aerophilic microorganisms with decomposition function for cellulose, hemicellulose, lignin, pectin, proteins, and lipids present in biomass, each member of the consortium harmoniously coexists, mutually promote, reach mutual supplement with each other’s advantages. Different microbes secrete complementary enzyme that degrade or decompose straw in the field facilitating the sustainable, eco-friendly, and environmentally benign farming.

The present invention relates to a method of preparing a crop residue decomposer bioinoculant, wherein the method comprises the steps of:
- isolating and identifying microorganisms capable of producing cellulase, xylanase, laccase, lignin peroxidase, manganese peroxidase, biphenyl 2,3-dioxygenase (BPDO), versatile peroxidase, ß-etherize, pectinase, protease, urease and lipase;
- culturing the isolated and identified microaerophilic microorganisms;
- mixing the cultured microorganisms in an equal proportion which is 1 ml of each culture, to develop a consortia;
- converting the consortia into safe to use granules, capsules or tablets by employing carriers;
- adding bio-stimulant composition for obtaining a crop residue decomposer bioinoculant,
wherein the bio-stimulant composition comprises:
- dextrose in a concentration range of 400-500ppm;
- sodium bicarbonatein a concentration range of100-140ppm;
- diammonium phosphate in a concentration range of 100-150ppm;
- phosphorus in a concentration range of 15-30ppm;
- sulfur in a concentration range of 10-20ppm;
- calcium in a concentration range of 15-20ppm;
- magnesium in a concentration range of 40-60ppm;
- potassium in a concentration range of 15-25ppm;
- nickel in a concentration range of 10-20ppm;
- zincin a concentration range of 15-20 ppm;
- molybdenum in a concentration range of 5-10 ppm; and
- amino acids in a concentration range of 5-10 ppm.
- carrier material in a concentration range of 8 to 10 % (w/w) to balance the total composition.

In an aspect, there is provided a method, wherein crop residue decomposer bioinoculant of microbial consortium comprises microaerophilic microorganism.

In an another aspect, there is provided a method, wherein crop residue decomposer bioinoculant of microbial consortium comprises microorganisms and the microorganisms are lignocellulolytic fungi and lignocellulolytic bacteria.

In yet another aspect, there is provided a method, wherein the crop residue decomposer bioinoculant of microbial consortium contains the fungi are Aspergillus fumigates (MTCC 25544), Glomus fasciculatum (MTCC 25543, Tricoderma Sp. (MTCC 25547), Humicola lanuginose (MTCC 25546) and Aspergillus niger (MTCC 25548).

In still another aspect, there is provided a method, wherein the crop residue decomposer bioinoculant of microbial consortium contains bacteria, wherein the bacteria are Actinomycetes Sp. (MTCC 25542) and Streptomyces viridosporus (MTCC 25541).

In yet another aspect, there is provided a method, wherein the carrier is selected from starch powder, potato starch powder, corn powder, soy meal powder.

In another aspect, there is provided a method, wherein converting the cultures comprises the steps of:
- collecting spores from cultures by using stirring and filtration;
- adding the spore rich media as obtained above with carriers and incubating the same for 24 hours; and
- drying the spore rich media with carriers above till the moisture comes to less than 2% for obtaining safe to use form of the consortia.

In still another aspect, there is provided a method, wherein the crop residue decomposer bioinoculant maintains the moisture content of the soil and comprises the following steps:
- proper mixing of stubble, biomass, residue in soil by using a device selected from mulcher machine, rotavator, polydisc harrow or combination thereof; and
- adding the crop residue decomposer bioinoculant and bio-stimulant to the field.

In another embodiment, the crop residue decomposer bioinoculant of microbial consortium contains at least one microbe producing cellulase, xylanase, laccase, lignin peroxidase, manganese peroxidase, pectinase, protease, urease and lipase.

In another embodiment, there is provided the method, wherein the crop residue decomposer bioinoculant of consortium is capable of producing nano-lignin from crop residue by microbial hydrolysis process.

In another embodiment, there is provided the method, wherein the crop residue decomposer bioinoculant of consortium degrades residue from cereals, pulses, vegetables, oilseed, perennial grass crop.

In another embodiment, the crop residue decomposer bioinoculant of consortium is designed for in situ degradation of wheat/paddy stubbles remaining after mechanical harvesting of wheat and paddy.

In another embodiment, there is provided the method, wherein the crop residue decomposer bioinoculant of consortium takes 7-21 days to degrade the crop residue. the crop residue decomposer bioinoculant of microbial consortium degrades crop residue in soil.

In another embodiment, there is provided the method, wherein the crop residue decomposer bioinoculant of microbial consortium degrades crop residue to compost.

In another embodiment, the microbial consortium is used for composting of diverse agricultural wastes such as paddy straw, soybean trash, pearl millet, maize residues and mustard stover effectively.

In another embodiment, there is provided the method, wherein the crop residue decomposer bioinoculant of consortium of efficient lignocellulolytic fungi cause in situ degradation of paddy/ wheat stubbles in the soil/field.

In another embodiment, there is provided the method, wherein the crop residue decomposer bioinoculant of consortium is used for composting of crop residues.

In another embodiment, there is provided the method, wherein the crop residue decomposer bioinoculant of consortium is grown in large scale using synthetic media.

In another embodiment, there is provided the method, wherein the crop residue decomposer bioinoculant of consortium is subjected for solid-state fermentation for fungal spore production.

In another embodiment, there is provided the method, wherein the consortium is used in liquid form or in the form of the granules, table, capsule using carrier (8-10% w/w).

In another embodiment, there is provided the method, wherein the crop residue decomposer bioinoculant of consortium is required to be spread on the crop residue at the rate of 103 cfu/ton of crop residue.

In another embodiment, there is provided the method, wherein an optimized micro-adjuvant formulation needs to be added at the beginning of the decomposition process as well on 5th day at the rate 45-55 mg/kg soil.

In another embodiment, there is provided the method, wherein the micro-adjuvant formulation help the microbes present in crop residue decomposer bioinoculant of microbial consortium to grow faster as well cause bio-stimulation.

In another non-limiting embodiment, there is provided the method, wherein the crop residue decomposer bioinoculant of microbial consortium has no harmful effect on yield of next crop.

The present invention addresses several issues of handling crop residue and facilitates farmers with sustainable agriculture by resolving the following major issues:
• Elimination of stubble burning issue and thus reducing the pollution load
• Decomposition of crop residue in lesser time facilitating crop rotation
• Safe handling of the microbial product by farmers, thus reducing health hazard
• Purely organic product without any hazardous chemical agents
• Faster and efficient decomposing process (21-24 days)
• Synergistic blend of potential microbes for efficient degradation
• Addition of specific bio stimulant enhance the degradation rate
• On-site germination of spores and growth of consortia reducing the time requirement
• Easy to use form of product for field application.

EXAMPLES:
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.

Materials and method:
Biological material:
The biological materials ( Aspergillus fumigates (MTCC 25544), Glomus fasciculatum (MTCC 25543), Tricoderma Sp. (MTCC 25547), Humicola lanuginose (MTCC 25546), Aspergillus niger (MTCC 25548), Actinomycetes Sp. (MTCC 25542) and Streptomyces viridosporus (MTCC 25541)) were procured from Institute of Microbial Technology, Shanti Path, 39A, Sector 39, Chandigarh, 160036.

Example 1: Isolation of crop residue decomposer microbes:
Isolation of the microbes carried out in a step wise protocol. Soil sample from waste biomass composting site was collected. Microbes capable of producing cellulase, xylanase, laccase, lignin peroxidase, manganese peroxidase, Biphenyl 2,3-dioxygenase (BPDO), versatile peroxidase, ß-etherize, pectinase, protease, urease and lipase were selectively enriched and isolated using standard microbiological practices.

Example 2: preparing of crop residue decomposer (CRD) bioinoculant:
In a flask mineral minimum medium (MMM), of 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 liter) was taken and this medium is supplemented with 2% ( w/v) fraction of plant biomass i.e., cellulose or hemicellulose or lignin or pectin . To this solution 0.5% soil from waste biomass composting site is added. The flasks were incubated for 7 days in a shaker incubator at 37°C at 100?rpm. Shaking was done 8 hour/day to enrich the microbes with ability to grow at micro-aerophilic conditions. After 7 days, from each flask 5 ml of the culture broth was taken and transferred to flask having same fraction of plant biomass (2% (w/v) cellulose or hemicellulose or lignin or pectin) along with MMM. The flasks were incubated for 7 days in a shaker incubator at 37°C at 100?rpm. Shaking was done 8 hour/day to enrich the microbes with ability to grow at micro-aerophilic conditions. Similar steps followed for 3-5 more cycles after every 7 days. After 7 days of 3-5 cycles, from each flask 5 ml of the culture broth was taken and transferred to flask having paddy straw (cut to 0.5-2.0 cm in length) along with MMM. The flasks were incubated for 7 days in a shaker incubator at 37°C at 100?rpm. Shaking was done 8 hour/day to enrich the microbes with ability to grow at micro-aerophilic conditions. After 7 days, fungi are isolated on Potato Dextrose Agar (PDA) and bacteria were isolated using Nutrient Agar (NA) as per standard microbiology practices. The hemicellulose, cellulose, pectin, lignin that are required for isolation, can be extracted from paddy straw using any art known in prior art for the purpose. Alternately, commercially available hemicellulose, cellulose, pectin, lignin can be used. The isolates were firstly identified to a genus level using a morphological examination depending on the colors of colony formed at both sides, the top and reverse of the cultures. The microscopic examination of the shape of the spore-producing structures was used for further identification. The morphological examination and identification of isolates are useful for the identification of isolates up to the family or genus level. 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. The microbes were identified as Streptomyces viridosporus IOC 618(MTCC 25541), Aspergillus fumigates IOC 705(MTCC 25544), Glomus fasciculatum IOC 704(MTCC 25543), Trichoderma Sp. IOC 712(MTCC 25547), Humicoli lanuginose IOC 709(MTCC 25546), Actinomycetes sp. IOC 702(MTCC 25542), Aspergillus niger IOC 845(MTCC 25548).

S No. Sequence ID No. Biological material(The 16s and 18s rRNA sequences of isolated strains was determined)

1 1 Aspergillus fumigates (MTCC 25544)
2 2 Glomus fasciculatum (MTCC 25543)
3 3 Tricoderma Sp. (MTCC 25547)
4 4 Humicola lanuginose (MTCC 25546)
5 5 Aspergillus niger (MTCC 25548)
6 6 Actinomycetes Sp. (MTCC 25542)
7 7 Streptomyces viridosporus (MTCC 25541)

Example 3: Development of consortia
All the isolates were checked for different enzyme activities, viz., cellulase, xylanase, laccase, lignin peroxidase, manganese peroxidase, Biphenyl 2,3-dioxygenase (BPDO), peroxidase, pectinase, protease, urease, and lipase. Further, the isolated cultures were mixed in equal proportion, prepared consortia and evaluated for its ability in terms of enzyme activities. The enzyme activities, viz., cellulase, xylanase, laccase, lignin peroxidase, manganese peroxidase, Biphenyl 2,3-dioxygenase (BPDO), versatile peroxidase, ß-etherize, pectinase, protease, urease and lipase, were carried out for each isolate separately and as a mixture. For this, all the cultures were grown on media and once growth obtained, the supernatant of the centrifuged culture used for enzyme activity assay. Procedure adapted for each enzyme assay is as follows.

A. 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.

B. Xylanase activity: The procedure for xylanase estimation is similar to cellulase with the substrate being 0.2% Birchwood xylan. The activity measured in terms of xylose released against. Measurements were made in 0.1 M sodium acetate buffer during 30 min of incubation at pH 4.8 and 50 0C. Enzyme activity has been expressed in International Units (U) viz., the amount of the enzyme that liberated 1 µmol of substrate equivalent per minute under the assay conditions.

C. Pectinase: The pectinase (polygalacturonase) activity was determined against standard curve of galacturonic acid. Measurements of galacturonic acid were made in 0.1 M sodium acetate buffer during 30 min of incubation using 0.1% pectin at 50 0C and at pH 4.2. Enzyme activity has been expressed in International Units (U) viz., the amount of the enzyme that liberated 1 µmol of substrate equivalent per minute under the assay conditions.

D. Laccase activity: Laccase activity was determined through the oxidation of ABTS 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 is 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.

E. Lipase activity: Lipase activity was determined through hydrolysis of p-nitrophenylpalmitate (p-NPP). The reaction was carried out in sodium acetate buffer (pH 8; 50?mM) added with 2,1% Triton X-100 p-NPP solution and crude enzyme extract. The lipase activity was determined by the rate of p-nitrophenol production (p-NP) at 412 nm. One unit of activity was defined as the amount of lipase required to release one µmol of the p-NP per minute.

F. Lignin peroxidase activity: Lignin is one of the complex aromatic structure of agriculture residue and its degradation is essential for complete decomposition of the crop residue. The lignin peroxidase is one of the lignin degrading enzymes and its activity can be measured through oxidation of veratryl alcohol. 25 mM sodium tartrate buffer (pH 3.0) with 2 mM veratryl alcohol and 0.4 mM H202 incubated for 10 min in presence of enzyme. The activity was measured through change in absorbance at 310 nm due to the H202 dependent oxidation of veratryl alcohol to veratraldehyde.

G. Manganese peroxidase activity: The activity of MnP was measured using 1mM MnSO4 in sodium malonate buffer (50mM) in the presence of 0.1mM H2O2 and crude extract of enzyme.. Mn+3 form a complex with malonate when incubated at 25 0C and the change in absorbance was measured at 270 nm after 10 min interval. One unit of Manganese peroxidase activity corresponds to the change of 1 µmol Mn3+ per minute.

H. Biphenyl 2,3-dioxygenase (BPDO) enzyme activity: BPDO enzymatic activity was measured in 50 mM phosphate buffer using 2,3-dihydroxybiphenyl as substrate at pH 7.5 and 25 0C. The activity was measured through change in absorbance at 434 nm.

I. Versatile peroxidase activity: Versatile peroxidase is a typical ligin degrading enzyme that mostly found in bacteria. Its activity was assessed through oxidation of RB5 dye. Oxidation of 10 µM RB5 in 100 mM sodium tartrate buffer (pH 3) in presence of 0.1 mM H2O2 and was estimated at 598 nm.

J. Protease activity: Protease activity was measured using casein as substrate, where the free tyrosine liberated from casein digestion reacts with Folin & Ciocalteus reagent to produce a blue colored chromophore. The intensity of color can be measure at 280 nm and the enzyme activity is represented as international units, which is the amount in micromoles of tyrosine equivalents released from casein per minute.

K. ß-Etherase: The activity of ß-Etherase enzyme activity was measured in 10 mM Tris-HCl buffer (pH 7.5) having 1 mM DTT, 1°70 detergent (MEGA-8), 0.1 mM fluorogenic lignin model compound and enzyme crude extract. The mixture was incubated at 28°C for 30 min and reaction was terminated by addition of 500 mM glycine-NaOH buffer (pH 10). The reaction of ether bond cleavage of the fluorogenic lignin model compound a-O-(ß-methylumbelliferyl)-acetovanillone results in the release of fluorescent 4-methylumbelliferone (with excitation at 360 nm and emission at 450 nm). The fluorescence measured to quantify the ß-etherase activity.

L. Urease activity: Urea is the product of decarboxylation of amino acids. Hydrolysis of urea produces ammonia and CO2. The formation of ammonia alkalinizes the medium, and the pH shift is detected by the color change of phenol red from light orange at pH 6.8 to magenta (pink) at pH 8.1. Rapid urease-positive organisms turn the entire medium pink within 24 hours. The enzyme activity of each isolate and their mixture is given in Figure 1. Enzyme activities varied significantly among the individual cultures but as consortia, all the enzyme activities were significantly increased due to the synergistic association of the consortia.

M. pH and temperature growth pattern:
The consortia were evaluated for their growth under wide range pH (ranging from 4-10) and temperature (10–45 0C) and ability to degrade the major components of biomass. For this, the CRD was inoculated onto minimal media containing 5% biomass at different pH and temperature and incubated for 21 days. Samples were collected and analyzed for microbial growth and degradation of cellulose, hemicelluloses and lignin. The results obtained were given in Tables 2 and 3.

Table 2: Effect of pH on growth and metabolism of CRD

pH Growth (CFU/ml) Cellulose degradation (%) Hemicellulose degradation (%) Lignin degradation (%)
4 5.3X106 39.64 38.63 16.22
5 7.6X106 44.36 43.22 19.64
6 8.3X106 49.33 50.02 23.61
7 6.1X107 53.19 55.21 28.73
8 5.9X107 51.26 51.38 27.13
9 7.3X106 47.28 47.63 24.76
10 2.9X105 32.34 33.29 17.55

Table 3: Effect of temperature on growth and metabolism of CRD

Temperature Growth (CFU/ml) Cellulose degradation (%) Hemicellulose degradation (%) Lignin degradation (%)
10 5.3X104 18.63 17.69 10.63
15 8.3X104 22.34 21.38 12.44
20 6.6X105 29.21 27.32 15.87
25 3.6X106 37.46 36.58 21.34
30 9.1X106 49.18 47.23 26.89
35 6.1X107 53.19 55.21 28.73
40 5.8X107 53.88 54.26 28.34
45 1.3X107 50.26 51.18 25.21

The CRD has shown good growth along with degradation of cellulose, hemicelluloses and lignin under wide range of pH and temperature indicating its ability.

N. Bio-stimulation and micro-adjuvant support
A unique combination of activating materials has been prepared that facilitate faster growth and metabolic activity of the prepared microbial blend on-site, that results in faster decomposition of biomass. The composition of bio-stimulant as given in table 4.:
Table 4: Typical composition of bio-stimulant

Component Concentration (ppm)
Dextrose 400-500
Sodium bicarbonate 100-140
Diammonium phosphate 100-150
Phosphorus 15-30
Sulfur 10-20
Calcium 15-20
Magnesium 40-60
Potassium 15-25
Iron 4-6
Manganese 20-30
Copper 10-15
Nickel 10-20
Zinc 15-20
Molybdenum 5-10
Amino acids
Isoleucine 5-8
Leucine 5-8
Lycine 5-7
Cystine 5-8
Methionine 5-8
Threonine 5-7
Tryptophan 6-8
Valine 5-7
Alanine 6-8
Arginine 4-7
Aspargine 4-6
Glycine 4-7
Histidine 4-6
Serine 3-5
Glutamic acid 8-10
Aspartic acid 6-10

Example4: Production and shelf-life evaluation of field formulations
The growth and multiplication of designated consortia in the liquid media by end user, may cause severe health hazards due to the ignorance and illiteracy. Hence, in order to make the application easy and safe, the developed consortia are converted to granules, capsules or tablets. For this, each of the fungal strain cultured on media containing dextrose (10 g/l), potato starch (4 g/l), molasses (10 g/l), peptone (5 g/l), yeast extract (5 g/l) and sodium chloride (2 g/l) and after obtaining growth, spores were collected. For spore collection, the culture grown media stirred continuously for about 5-10 min to detach the spores from fungal mat to the maximum possible extent. Further, the content of flask is passed through thin layer of absorbent cotton and the filtrate is checked for spore count. Once, the spore rich media obtained, it is added with carriers (8-10% w/w) and incubated for 24 hours and then dried till the moisture comes to less than 2%. The spore carriers include but not limited to starch powder, potato starch powder, corn powder, soy meal powder. Once the spore coated carrier is ready, its viability and shelf life was evaluated up to 3 months at regular time intervals of 30 days. The shelf life of each isolate spores studied by adding 1g of dried spore powder (on carrier) on 100 ml media containing dextrose (10 g/l), potato starch (4 g/l), molasses (10 g/l), peptone (5 g/l), yeast extract (5 g/l) and sodium chloride (2 g/l). After incubation, spore count was measured for 72h at every 24h interval as given in table 5.

Table 5: Data of shelf- life of the CRD with respect to storage time

Organism 30 days (spores/ml) 60 days (spores/ml) 90 days (spores/ml)
Initial 24h 48h 72h Initial 24h 48h 72h Initial 24h 48h 72h
Streptomyces viridosporus 48 280 11X102 56X103 46 290 10X102 59X103 44 290 12X102 57X103
Aspergillus fumigates 42 310 9X102 84X103 44 310 11X102 83X103 41 300 10X102 82X103
Glomus fasciculatum 44 290 12X102 62X103 43 280 9X102 63X103 43 290 9X102 63X103
Trichoderma Sp. 39 300 11X102 57X103 40 300 10X102 56X103 40 310 10X102 58X103
Humicoli lanuginose 41 320 10X102 73X103 42 310 12X102 74X103 42 320 12X102 72X103
Actinomycetes sp. 46 310 10X102 77X103 45 320 11X102 76X103 45 290 11X102 76X103
Aspergillus niger 47 290 12X102 81X103 43 290 11X102 82X103 46 310 11X102 79X103

It was observed from the experiment that the growth of spores was sustained at same rate after 90 days also indicating the shelf life of the spore powder more than 90days (3 months). The spore powder is made into pellets, tablets, granules and capsules for ease of storage and application in the field.

Example 5: Application in soil for decomposition of crop residue:
Experiment was carried out in rectangular plastic trays in 5 kg soil added with 3% paddy straw. The spore powder of each organism mixed in equal proportion and prepared a crop residue decomposer (CRD) bioinoculant for soil application. CRD was added to these trays at a rate of 1g/kg soil and mixed thoroughly. Bio-Stimulant for boosting the performance of CRD was dosed on 5th day at a rate of 50 mg/kg soil. Control experiment has also been set up along with test where no additional microbes or bio-stimulant were added. Moisture content of the soil was maintained at least 20% by spraying the water whenever required. After setting up experiment, samples collected at regular time intervals for analyzing various parameters as shown in table 6.
Table 6: Consolidate data of the field application of CRD as product in soil
Parameter Control (Without bio-stimulant) Test (With bio-stimulant)
0 7 14 21 0 7 14 21
Enzyme activities (Units/ml)
Cellulase 0.73 1.87 3.18 1.63 0.74 2.38 5.33 1.39
Xylanase 0.82 1.24 2.04 1.33 0.83 2.01 4.12 2.87
Lacasse 3.96 4.22 4.68 3.91 3.98 5.87 6.36 4.11
Lipase 2.21 2.44 2.76 2.31 2.26 4.18 5.23 4.31
Lignin Peroxidase 3.36 4.97 5.12 4.28 3.48 6.08 8.16 4.36
Biphenyl 2,3-dioxygenase 0.21 0.86 1.09 1.23 0.34 1.88 3.09 2.67
% degradation of major constituents of biomass
Cellulose 11.68 35.22 53.19 18.21 46.73 76.6
Hemicellulose 10.23 31.99 55.21 21.63 42.11 87.3
Lignin 6.27 19.46 28.73 10.68 29.32 56.7
Colony count in soil (CFU/g) 3.1X10 2.6X103 8.6X105 6.1X107 3.2X10 4.2X106 9.1X109 6.6X1011
Total C in soil (% w/w) 0.17 0.21 0.38 0.54 0.17 0.31 0.48 0.89
N in soil (% w/w) 0.07 0.09 0.11 0.14 0.07 0.14 0.17 0.22
P in soil (% w/w) 0.02 0.03 0.06 0.07 0.03 0.07 0.13 0.18
K in soil (% w/w) 0.02 0.04 0.05 0.09 0.02 0.08 0.19 0.32
C/N ratio in soil 2.42 2.33 3.45 3.85 2.42 2.21 2.82 4.04

The data has shown substantial improvement in the degradation of major constituents of paddy straw. The respective enzyme activities were also increased significantly. The degradation of major components of paddy straw was also significantly increased with the addition of CRD (Figure 2). SEM images (Figure 3) showed clear demarcation in the degradation of paddy straw between control and present invention.

Example 6: Effect of carrier addition on the CRD performance:
The performance of CRD in presence and absence of carrier was also studied to establish its role in enhancing the decomposition process. For this, experiment was carried out in rectangular plastic trays in 5 kg soil added with 3% paddy straw. The CRD was added to these trays at a rate of 1 g/kg soil and mixed thoroughly. In addition, bio-Stimulant for boosting the performance of CRD was dosed on 5th day at a rate of 50 mg/kg soil. Control experiment was set up where the CRD was added as it is in liquid form keeping all other conditions same. Moisture content of the soil was maintained at least 20% by spraying the water whenever required. After setting up experiment, samples collected at regular time intervals for analyzing various parameters as shown in table 7.

Table 7: Effect of carrier addition on CRD performance in soil
Parameter Control (CRD without carrier) Test (CRD with carrier)
0 7 14 21 0 7 14 21
Enzyme activities (Units/ml)
Cellulase 0.72 1.56 2.67 1.32 0.75 2.34 5.44 1.27
Xylanase 0.81 1.03 1.83 1.18 0.81 2.12 4.23 2.63
Lacasse 3.92 3.87 4.05 2.86 3.94 5.76 6.41 4.14
Lipase 2.26 2.11 2.29 1.93 2.22 4.22 5.37 4.23
Lignin Peroxidase 3.31 4.19 4.62 3.54 3.29 6.13 8.29 4.19
Biphenyl 2,3-dioxygenase 0.24 0.54 0.83 0.62 0.26 1.91 3.17 2.38
% degradation of major constituents of biomass
Cellulose 9.87 31.27 47.03 18.73 46.87 76.13
Hemicellulose 9.26 27.08 45.18 22.06 42.45 87.22
Lignin 4.21 14.52 21.44 10.36 29.78 56.36
Colony count in soil (CFU/g) 2.3X10 2.1X103 7.4X105 5.3X107 1.9X10 4.8X106 9.6X109 7.8X1011
Total C in soil (% w/w) 0.15 0.18 0.43 0.71 0.17 0.32 0.49 0.88
N in soil (% w/w) 0.07 0.11 0.15 0.18 0.07 0.13 0.18 0.21
P in soil (% w/w) 0.02 0.04 0.08 0.12 0.03 0.06 0.12 0.16
K in soil (% w/w) 0.03 0.06 0.11 0.23 0.02 0.09 0.17 0.29
C/N ratio in soil 2.14 1.64 2.87 3.94 2.43 2.46 2.72 4.19

Example 7: Effect of bio-stimulant addition on the CRD performance:
The performance of CRD in presence and absence of bio-stimulant was also studied to establish its role in enhancing the decomposition process. For this, experiment was carried out in rectangular plastic trays in 5 kg soil added with 3% paddy straw. The CRD was added to these trays at a rate of 1 g/kg soil and mixed thoroughly. In addition, bio-Stimulant for boosting the performance of CRD was dosed on 5th day at a rate of 50 mg/kg soil. Control experiment was set up where the bio-stimulant was not added but the CRD was added. Moisture content of the soil was maintained at least 20% by spraying the water whenever required. After setting up experiment, samples collected at regular time intervals for analyzing various parameters as shown in table 8.
Table 8: Effect of bio-stimulant addition on CRD performance in soil
Parameter Control (Without bio-stimulant) Test (With bio-stimulant)
0 7 14 21 0 7 14 21
Enzyme activities (Units/ml)
Cellulase 0.73 1.87 3.18 1.63 0.74 2.38 5.33 1.39
Xylanase 0.82 1.24 2.04 1.33 0.83 2.01 4.12 2.87
Lacasse 3.96 4.22 4.68 3.91 3.98 5.87 6.36 4.11
Lipase 2.21 2.44 2.76 2.31 2.26 4.18 5.23 4.31
Lignin Peroxidase 3.36 4.97 5.12 4.28 3.48 6.08 8.16 4.36
Biphenyl 2,3-dioxygenase 0.21 0.86 1.09 1.23 0.34 1.88 3.09 2.67
% degradation of major constituents of biomass
Cellulose 11.68 35.22 53.19 18.21 46.73 76.6
Hemicellulose 10.23 31.99 55.21 21.63 42.11 87.3
Lignin 6.27 19.46 28.73 10.68 29.32 56.7
Colony count in soil (CFU/g) 3.1X10 2.6X103 8.6X105 6.1X107 3.2X10 4.2X106 9.1X109 6.6X1011
Total C in soil (% w/w) 0.17 0.21 0.38 0.54 0.17 0.31 0.48 0.89
N in soil (% w/w) 0.07 0.09 0.11 0.14 0.07 0.14 0.17 0.22
P in soil (% w/w) 0.02 0.03 0.06 0.07 0.03 0.07 0.13 0.18
K in soil (% w/w) 0.02 0.04 0.05 0.09 0.02 0.08 0.19 0.32
C/N ratio in soil 2.42 2.33 3.45 3.85 2.42 2.21 2.82 4.04 , Claims:1. A method of preparing a crop residue decomposer bioinoculant, wherein the method comprises the steps of:
- isolating and identifying microorganisms capable of producing cellulase, xylanase, laccase, lignin peroxidase, manganese peroxidase, biphenyl 2,3-dioxygenase (BPDO), versatile peroxidase, ß-etherize, pectinase, protease, urease and lipase;
- culturing the isolated and identified microaerophilic microorganisms;
- mixing the cultured microorganisms in an equal proportion which is 1 ml of each culture, to develop a consortia;
- converting the consortia into safe to use granules, capsules or tablets by employing carriers;
- adding bio-stimulant composition for obtaining a crop residue decomposer bioinoculant,
wherein the bio-stimulant composition comprises:
- dextrose in a concentration range of 400-500ppm;
- sodium bicarbonatein a concentration range of100-140ppm;
- diammonium phosphate in a concentration range of 100-150ppm;
- phosphorus in a concentration range of 15-30ppm;
- sulfur in a concentration range of 10-20ppm;
- calcium in a concentration range of 15-20ppm;
- magnesium in a concentration range of 40-60ppm;
- potassium in a concentration range of 15-25ppm;
- nickel in a concentration range of 10-20ppm;
- zincin a concentration range of 15-20 ppm;
- molybdenum in a concentration range of 5-10 ppm; and
- amino acids in a concentration range of 5-10 ppm.
- carrier material in a concentration range of 8 to 10 % (w/w) to balance the total composition.

2. The method as claimed in claim 1, wherein the microorganism is a microaerophilic microorganism.

3. The method as claimed in claim 1, wherein the microorganisms are lignocellulolytic fungi and lignocellulolytic bacteria.

4. The method as claimed in claim 3, wherein the fungi are Aspergillus fumigates (MTCC 25544), Glomus fasciculatum (MTCC 25543, Tricoderma Sp. (MTCC 25547), Humicola lanuginose (MTCC 25546) and Aspergillus niger (MTCC 25548).

5. The method as claimed in claim 3, wherein the bacteria are Actinomycetes Sp. (MTCC 25542) and Streptomyces viridosporus (MTCC 25541).

6. The method as claimed in 1, wherein the carrier is selected from starch powder, potato starch powder, corn powder, soy meal powder.

7. The method as claimed in claim 1, wherein converting the cultures comprises the steps of:
- collecting spores from cultures by using stirring and filtration;
- adding the spore rich media as obtained above with carriers and incubating the same for 24 hours; and
- drying the spore rich media with carriers above till the moisture comes to less than 2% for obtaining safe to use form of the consortia.

8. The method as claimed in claim 1, wherein the crop residue decomposer bioinoculant maintains the moisture content of the soil and comprises the following steps:
- proper mixing of stubble, biomass, residue in soil by using a device selected from mulcher machine, rotavator, polydisc harrow or combination thereof; and
- adding the crop residue decomposer bioinoculant and bio-stimulant to the field.