FORM 2
THE PATENTS ACT 1970
(39 of 1970)
&
The Patents Rules, 2003
COMPLETE SPECIFICATION
(See Section 10; Rule 13)
1. TITLE OF THE INVENTION:
LIGNOCELLULOSE DEGRADING CONSORTIUM
2. APPLICANT(S)
(a) NAME: Excel Crop Care Limited
(b) NATIONALITY: An Indian Company
(c) ADDRESS:
184-87, Swami Vivekanand Road, Jogesh wari (West), Mumbai400 102, Maharashtra, India.
3. PREAMBLE TO THE DESCRIPTION:
The following COMPLETE specification particularly describes the nature of this invention and the manner in which it is to be performed.

LIGNOCELLULOSE DEGRADING MICROBIAL CONSORTIUM
FIELD OF INVENTION
The present invention relates to a consortium of microorganisms which efficiently degrades lignocelluloses and use of said consortium for degrading substrates containing lignocelluloses.
BACKGROUND AND PRIOR ART
Lignocellulose is the major structural unit of all plants. It consists of three components, viz., cellulose, hemicelluloses and lignin. Large quantities of lignocellulosic wastes are produced as a result of agricultural activities and it is also produced by many related industries.
Lignin is a heterogeneous phenylpropanoid polymer. It is resistant to degradation because of high molecular weight with biologically stable carbon-to-carbon and ether linkages. Fungi and actinomycetes are reported to degrade of lignin by oxidative process. Bacterial strains degrade low molecular weight part of lignin. However, degradation of high molecular weight backbone of the lignin polymer by bacterial strains is difficult.
Invention disclosed in US2006/0121592 Al provides a consortium of ligninolytic bacteria for degradation of lignin.
JP2011193848 discloses a lignin degrading bacterium. A fungus belonging to genus Marasmiellus has been used for degradation of lignin as reported in JP2010200707.

As disclosed in JP2000064185, lignin of scrap wood, pulp, etc., is decomposed by using a lignin decomposing agent obtained by mixing a laccase-containing substance composed of a cultured product of at least one laccase producing fungus selected from the group consisting of Schizophyllum commune, Coriolus versicolor, Pycnoporus coccineus, Pleurotus octreatus and Fomitella fraxinea or its treated substance with a composition containing terremutin and/or terreic acid composed of a cultured product of Aspergillus sp. capable of producing terremutin and terreic acid or its treated substance.
In JPH11253917, microorganisms in the digestive tracts of xylophagy insects having lignin decomposing activity have been used for lignin decomposition.
Solid phase bioremediation method using lignin degrading fungi has been disclosed in US5476788.
Microorganisms as pure cultures are not found to be efficient in lignin degradation. Consortium comprising combination of bacteria and fungi can be more effective. However, if such consortium is used for degradation of lignocelluloses, it needs to be thermo-stable in the sense that it should withstand elevated temperatures during thermophilic phase of degradation. Moreover, the microorganisms should be compatible among themselves. Further, it should be practicable to revive the selected organisms easily.
The inventors of present invention have overcome disadvantages of prior art and developed a culture of microorganisms with abovementioned desired properties which can be used for effective degradation of lignocelluloses.

OBJECTS OF INVENTION
The main object of invention is to provide a novel consortium of microorganisms which can be used for effective bio-degradation of lignocelluloses.
Another object of invention is to provide a consortium for bio-degradation of lignocelluloses wherein the microorganisms are compatible with each other.
Another object of invention is to provide a consortium comprising microorganisms for bio-degradation of lignocelluloses which can be revived easily.
Another object of invention is to provide a method of production of compost by biodegradation of lignocelluloses using invented consortium.
DESCRIPTION OF DRAWINGS
Fig. 1 indicates variation in temperature over a period of time (temperature Vs days) for test heap of compost.
Fig.2 indicates variation in temperature over a period of time (temperature Vs days) for control heap of compost.
DETAILED DESCRIPTION OF INVENTION
Various environmental samples were collected as a source of lignocellulose degrading microorganisms. The selection of samples was based on their likelihood for containing lignocellulose degrading microorganisms. Accordingly, samples such as soil from forest, humus, dead and decayed wood, compost heaps, paper mill effluent

etc. were collected. Samples were stored at 4°C till used, and were analyzed within 6 hrs. of collection.
ISOLATION OF FUNGI
5 gm of soil/humus/compost heap sample was added to 45 ml of sterile distilled water. It was kept on a rotary shaker at 150 rpm for 15 min. It was then allowed to settle and then 1 ml aliquot of the supernatant was serially diluted to lO-10.
Samples of dead and decaying wood were cut into pieces and transferred to tube containing sterile distilled water and vortex. 1 ml aliquot of supernatant was serially diluted to 10-10.
1 ml of effluent sample was serially diluted to 10-10.
From each dilution as mentioned above, 1 ml was transferred to sterile petri dish. Sterile potato dextrose agar (potatoes infusion from 200 gm, dextrose 20 gm, agar 15 gm, distilled water 1 L, pH 5.6) was poured to each petri dish. Streptomycin (110 jig/ml) was added to inhibit bacterial growth.
Plates were incubated at 30°C for 8-10 days and the individual colonies grown thereon were transferred to fresh media and were studied for their morphological characteristics.
ISOLATION OF BACTERIA
5 gm of soil/humus/compost heap sample was added to 45 ml of sterile distilled water. It was kept on a rotary shaker at 150 rpm for 15 min. It was then allowed to settle and then 1 ml aliquot of the supernatant was serially diluted to 10-10.

Samples of dead and decaying wood were cut into pieces and transferred to tube containing sterile distilled water and vortex. 1 ml aliquot of supernatant was serially diluted to 10-10.
1 ml of effluent sample was serially diluted to 10-10.
From each dilution as mentioned above, 0.1 ml was spread on following media:
Nutrient agar comprising 5 gm peptic digest of animal tissue, 1.5 gm beef extract, 1.5 gm yeast extract, 5 gm NaCl, 15 gm agar, 1 L distilled water;
Actinomycetes isolation agar comprising 2 gm Na-caseinate, 0.1 gm L-asparagine, 4 gm Na-propionate, 0.5 gm dipotassium phosphate, 0.1 gm magnesium sulfate, 0.001 gm ferrous sulfate, 15 gm agar, 1 L distilled water;
Yeast mannitol agar comprising 1 gm yeast extract, 10 gm mannitol, 0.5 gm dipotassium phosphate, 0.2 gm manessium sulfate, 0.1 gm sodium chloride, 0.025 gm Congo Red, 20 gm agar and 1 L distilled water.
90 (ig/ml of cycloheximide was added to each medium mentioned above to inhibit fungal growth.
Plates were incubated at 30°C for 8-10 days and the individual colonies grown thereon were transferred to fresh media and were studied for their morphological characteristics.

Several isolates (fungi as well as bacteria) were thus isolated and were screened for their cellulolytic and lignolytic activities by plate-assays.
DETERMINATION OF CELLULOLYTIC ACTIVITY
Cellulolytic activity of fungal and bacterial isolates was determined using carboxymethyl cellulose agar comprising Carboxymethyl cellulose sodium salt lOgm/L, sodium nitrate 6.5 gm/L, dipotassium hydrogen orthophosphate 6.5 gm/L, yeast extract 0.3 gm/L, potassium chloride 6.5 gm/L, magnesium sulfate heptahydrate 3 gm/L, D-glucose 0.65 gm/L and agar agar 20 gm/L.
For fungal isolates, 3 mm discs of actively growing culture were cut and placed on the CMC agar plate. Plates were incubated at 30°C for 8-10 days. After completion of incubation period, plates were flooded with 0.5% Congo Red for 15 min. and were subsequently destained with 1 Molar NaCl for 20 min. Isolates with positive cellulolytic activity showed yellow to colourless zones around their colonies. No colour change indicated no activity.
For determination of cellulolytic activity of bacterial isolates, 24 hr. old culture was streaked on CMC agar plates. Remaining procedure followed was same as that followed for fungal isolates as mentioned above.
DETERMINATION OF LIGNOLYTIC ACTIVITY
Lignolytic activity of fungal and bacterial isolates was determined using two media as follows:

Malt-extract glucose medium comprising 5 gm malt-extract, 10 gm glucose, 15 gm agar, pH 4.5;
Yeast-extract peptone glucose agar comprising 10 gm glucose, 5 gm peptone, 2 gm yeast extract, 1 gm KH2PO4, 0.5 gm MgS04.7H20, 15 gmagar, pH 4.5.
A dye such as Ramazol Brilliant Blue R (RBBR), poly R-478, poly s-119, Malachite Green was added to media at concentration of 200 ppm.
For determination of lignolytic activity of fungal isolates, 3 mm discs of actively growing culture were cut and placed on the abovementioned media and were incubated at 30°C for 8 days. After completion of incubation period, decolonization zone around the colony indicated positive activity, whereas no colour change indicated no activity.
For determination of lignolytic activity of bacterial isolates, 24 hr. old culture was streaked on abovementioned media. Remaining procedure followed was same as that followed for fungal isolates as mentioned above.
Out of a number of isolates, best performing isolates were identified.
Some isolates showed significant cellulolytic activity, some isolates
showed lignolytic activity and some isolates showed both, cellulolytic
as well as lignolytic activity. These isolates showing
cellulolytic/lignolytic activity based on their plate assays were identified. Best performing isolates were selected based on activity and ability to grow on nutrient poor media. It should be noted that a

set of isolates performs in nutrient rich media and therefore, they are also useful when the substrate such as bio-waste is rich in required nutrients.
From the set of best performing isolates, a subset of microorganisms was selected [see Table-1] and used for preparation of a consortium for degradation of lignocelluloses. The consortium exhibited all desirable properties viz., compatibility of microorganisms among themselves, effectiveness in lignocelluloses degradation, stability during thermophilic phase and ease of revival.
Table-1
[microorganisms selected for consortium]

Sr. No. Species MTCC accession No.
1 Bacillus subtilis MTCC 5843
2 Streptomyces sp. MTCC 5844
3 Aspergillus brasiliensis MTCC 5845
EXAMPLE-1
(Preparation of consortium)
In one embodiment of the present invention, a consortium of 3 micro-organisms including 2 bacteria viz., Bacillus subtilis [MTCC 5843] & Streptomyces sp. [MTCC 5844], and 1 fungi Aspergillus brasiliensis [MTCC 5845] was prepared. The consortium was prepared by taking young culture of fungi in potato dextrose broth comprising potatoes infusion from 200 gm, dextrose 20 gm, agar 15 gm, distilled water 1

L, pH 5.6; and bacteria in nutrient broth comprising 5 gm peptic digest of animal tissue, 1.5 gm beef extract, 1.5 gm yeast extract, 5 gm NaCl, and 1 L distilled water. Bacteria and fungi in abovementioned broths were loaded on a sterilized solid carrier selected from the group lignite, charcoal, cow dung, fly ash, pre-treated saw-dust or a combination thereof. The inoculum thus prepared contained 108 fungal spores/gm and 109 colony forming units of bacteria (cfu)/gm.
EXAMPLE-2
(use of the consortium for degradation of lignocelluloses containing waste):
626 gm of the consortium as prepared in Example-1 was added to a compost heap comprising 310 Kg shredded cotton waste, 310 Kg farm yard waste, 15 L cow urine and 440 L water. Turning of material was done every 15 days up to 90 days and water was added to maintain approximately 40% moisture. Before each turning, representative samples were drawn and analyzed for pH, % organic carbon, % Nitrogen, total bacterial count and total fungal count.
For effective composting, it is necessary that high temperature is sustained, which was achieved in the test heap in which the consortium of present invention was added. It was observed that compared to control, there was a sustained increase in temperature for the test heap as shown in Fig.l. On the other hand, in control, there was initial increase in temperature, however, it could not be sustained as that in the test heap Fig.2. Samples from test and control were drawn on 90th day for testing quality of compost.

Quality of compost for both, test and control was evaluated based on stability and maturity which were determined by enzyme assays (Table-2) and phytotoxicity tests (seed germination & root elongation) (Table-3). From the results, it is clear that use of consortium of present invention for composting of lignocellulose waste provides advantages in terms of phytotoxicity, stability and maturity compared to control.
ENZYME ASSAYS
Cellulases (Endoglucanase, Exoglucanase, p-Glucosidase, Cellulase) and lignin modifying enzymes i.e., lignolytic enzymes (Lignin Peroxidase, Manganese Peroxidase, Laccase) tend to level-off upon maturation indicating gradual disappearance of degradable substrates which in-turn indicates stability and maturity.
Upon testing the sample and control, these enzyme activities were found lower in test heap compared to control heap suggesting better maturation.
Table-2 (Enzyme assays)

Enzyme Test Heap Control
CELLULASES
Endo -glucanase 57.3 EU/ml 72.35 EU/ml
Exo -glucanase NIL 0.25 EU/ml
P - glucosidase NIL NIL
Total cellulase 5.5 EU/ml 10.2 EU/ml
LIGNIN MODIFYING ENZYMES
Lignin Peroxidase(LiP) 6.0 EU/ml 14 EU/ml
Manganese 0.005 EU/ml 0.015

Peroxidase (MnP) EU/ml
Laccase(Lac) 0.020 EU/ml 0.068 EU/ml
Dehydrogenase 915 μg
INTF/g
compost/h 765 μg
INTF/g
compost/h
Alkaline Phosphatase 320 EU/ml 400 EU/ml
Phytase 3.6 EU/ml 1.8 EU/ml
Protease NIL NIL
Saccharification 1.09% 0.75%
PHYTOTOXICITY TEST:
Phytotoxicity is determined in terms of germination index (GI).

Table-3
Results of Phytotoxicity Test (seed germination & root elongation)
Test Parameters:
Test type : static
Pretreatment : Rinsed with sterile distilled water and 0.1% mercuric chloride
Temperature : 25°C

Light: none
Test Vessel: 1x9 cm petridish with Whatman Filter Paper (sterilized)
Extract: 1 gm compost extracted with 5 ml sterile distilled water
Test volume : 2 ml/plate
No. of seeds : 10
Type of seeds : Phaseolus aureus Roxb.
Triticum aestivum L.
Lycopersicum esculentum
Lepidium sativum L.
Control : sterile distilled water
Test Duration : 6 days for Phaseolus aureus Roxb., Triticum aestivum L., Lycopersicum esculentum; 2 days for Lepidium sativum
L.
Germination Index (GI) of samples

Sr. No. Sample Seeds —»
Extract
Concentration(%) J, Phaseolus aureus Roxb. (Mung) Triticum aestivum
L. (Wheat) Lycopersicum
esculentum
(Tomato) Lepidium sativum L. (gardencress)

G« ;rmination I ndex GI (% of co ntrol)
1 Control 100 41 77 70 42

50 62 73 65 59

25 31 91 94 47

5 32 92 65 101
2 Test 100 105 128 126 403

50 43 88 100 279

25 64 54 74 200

5 60 36 44 152
3 Soil 100 52 86 34 252

50 106 61 89 215

25 26 58 75 148

5 74 112 104 272

INDICATORS:
GI MATURITY OF COMPOST
<60 immature
>60 average
>80 mature
> 100 completely mature
The use of consortium of present invention helps eliminating/reducing toxicity of soil, which is an important advantage.
Phytotoxicity assay for compost are universally accepted for Lepidium sativum as other seeds such as cereals and legumes generally depend more on their reserves for germination rather than the environmental conditions applied. In this context, GI value which takes into consideration root growth along with germinated seeds is rather more comprehensive in assessing the phytotoxicity.
It can be observed from Table-2 that only when the extract of test compost (in which the consortium of present invention was used) gave >100 GI at 100% test concentration in all four type of the seeds tested. Similar values were observed for soil only with Lepidium sativum L. The control did not give such values at its highest test concentration with any of the seeds.
It should be noted that when the GI values increase with dilution, then the extract is considered to be phytotoxic. On the other hand, when GI value decreases upon dilution it indicates not only non-

phytotoxic nature, but also indicates its positive effect on seed germination and root elongation which also decreases upon dilution.
Higher Gl values of test heap are also in line with results of enzyme assays to ascertain the stability and maturity by measuring its microbial activity.

WE CLAIM:
1. A microbial consortium for degradation of lignocelluloses comprising a bacterial strain selected from the group Bacillus subtilis corresponding to MTCC accession number MTCC5843, a streptomyces sp. corresponding to MTCC accession number MTCC5844, and a fungi selected from the group Aspergillus brasiliensis corresponding to MTCC accession number MTCC5845.
2. A method of preparing compost from waste containing lignocelluloses by any conventional method, wherein consortium as claimed in claim 1 has been added for degradation of lignocelluloses contained in the waste.