FORM 2
THE PATENTS ACT, 1970
(39 of 1970)
&
THE PATENTS RULES, 2003
PROVISIONAL SPECIFICATION
(See section 10, rule 13)
1. Title of the invention
MICROBIAL CONSORTIUM FOR COMPOSTING ORGANIC MATTER
2. Applicants)
Name Nationality Address
TATA CHEMICALS LIMITED INDIA BOMBAY HOUSE, 24 HOMI MODI STREET, MUMBAI-400001


3. Preamble to the description
PROVISIONAL SPECIFICATION
The following specification particularly describes the invention.

The invention relates to isolated bacterial strains capable of decomposing organic matter and their use for decomposing organic matter. More particularly the invention relates to a microbial consortium consisting of two or more of the isolated bacterial strains capable of decomposing organic matter and its use for decomposing organic matter.
RELATED STATE OF ART
Huge amounts of organic matter is generated as waste from cities each day in form of municipal solid waste, residual water sludge, from industrial units and agricultural activities. At the present a large portion of this organic waste is dumped off in open land, which occupies large land area and results in offensive odour and ground water contamination due to leaching.
A simple and effective manner of disposing this organic matter is by composting. Composting involves microbial decomposition of organic matter under aerobic conditions. The process not only helps to dispose organic matter in an ecologically safe manner but also adds economic value to the waste product by converting it into compost. Compost is a natural fertilizer with benefits far greater than the chemical fertilizers. Besides being environmental-friendly compost increase soil water-holding capacity in porous soils and prevents evaporation of moisture.
One of the factors that control the composting process is the presence of microorganisms capable of decomposing the biopolymers present in the organic waste and their successful adaptation to environmental conditions present at the time of composting. Inoculation of the waste to be composted with bacteria capable of decomposing it is a strategy of vital importance to accelerate these processes. This strategy consists of adding microorganism to the organic waste. The microorganisms which are added should be such


that they are able to survive symbiotically and work in a synergistic manner to expedite the composting process. The present invention relates to the isolation of bacteria and a bacterial consortium for the decomposition of organic matter.
BRIEF DESCRIPTION OF ACCOMPANYING DRAWINGS:
The accompanying drawings illustrate the preferred embodiments of the invention and together with the following details description serve to explain the principles of the invention.
Fig. 1: Microscopic observation (100X) of culture IC62
Fig. 2: Microscopic observation (100X) of culture IC67
Fig. 3: Microscopic observation (100X) of culture IC68
Fig. 4: Microscopic observation (100X) of culture IC73
Fig. 5: Microscopic observation (100X) of culture IC82
Fig. 6: Growth curve of culture IC62
Fig. 7: Growth curve of culture IC67
Fig. 8: Growth curve of culture IC68
Fig. 9: Growth curve of culture IC73
Fig. 10: Growth curve of culture IC82
Table 1: Carbohydrate utilization studies
Table 2: Total Viable Count of microbial consortium
Table 3: Optical density of microbial cultures at various NaCl concentrations


DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS
To promote an understanding of the principles of the invention, reference will be made to the embodiment illustrated in the drawing and specific language will be used to describe the same. It will nevertheless be understood that no limitation of scope of the invention is thereby intended, such alterations and further modifications in the described method and such further applications of the principles of the inventions as illustrated therein being contemplated as would normally occur to one skilled in art to which the invention relates.
It will be understood by those skilled in the art that the foregoing general description and the following detailed description are exemplary and explanatory of the invention and are not intended to be restrictive thereof.
The present invention relates to the isolated bacterial strains that are capable of decomposing organic matter. More particularly the invention relates to a consortium comprising of two or more of these isolated bacteria that are capable of decomposing organic matter.
The bacterial strains in the present invention have been isolated from a soil sample from Mithapur, Gujarat, India.
To isolate the bacteria, soil samples from Mithapur, Gujarat, India were collected, soil suspensions were made, the suspension thus made were spread on a plate containing medium and incubated at the required temperature. After the required incubation period colonies were selected based on their morphological characters. These colonies were purified by repeated sub-culturing. The cultures were observed under the microscope to determine purity and pure cultures were maintained by known methods.


In accordance with an aspect of the invention the medium used for the isolation of bacteria is Zobell Marine Agar (HiMedia, Mumbai) medium. The preferred temperature for incubation during isolation process is 37°C.
In accordance with an aspect of the invention sub-culturing of selected colonies of bacteria was carried out 5-7 times.
Moreover the purified cultures were maintained as 20 % glycerol stocks and used in further studies.
In accordance with an aspect of the invention, for characterization of the bacterial isolates carbohydrate utilization studies were carried out using Hi Carbohydrate Kit (HiMedia) comprising of growing the bacterial isolate cultures to OD 600 > 0.5, surface inoculating it on HiCarbohydrate containing plates and incubating it at 37 C for 24 hours. The change in color was noted and results interpreted as per the chart provided by the manufacturer. The results are provided in table no. 1.
In accordance with an aspect of the invention the bacterial isolates were characterized genetically using 16S rRNA gene sequencing. By way of a specific example, genomic DNA from individual bacterial strains was extracted using the DNA Extraction Kit (Zymo Research, USA). The 16S rDNA was selectively amplified from genomic DNA by using PCR with universal primers 27F and 1492R (DeLong et al., 1993). PCR amplification was undertaken with a Thermo Cycler model iCycler (BioRad) under the following conditions: 100-200 ng template DNA, IX reaction buffer (100 mM Tris-HCl, pH 8.3, 500 mM KC1, 15 mM MgC12 and 0.01% gelatin), 1 U Taq DNA polymerase (Sigma Aldrich), 1 uM upstream primer, 1 M downstream primer, 10 M dNTP mix (MBI Fermentas) and sterile water combined in a total volume of 50 l. The tubes were incubated at 95°C for 4 min and then


subjected to the thermal cycling programme: denaturation at 95 C for 45 s, primer annealing at 50°C for 45 s, and chain extension at 72°C for 90 s with an additional extension time of 7 min on the final cycle, for a total of 30 cycles. The amplified DNA was purified using Qiaquick PCR Purification Kit (Qiagen) and T/A cloned into T-tailed vector pTZ57R/T (MBI Fermentas) and sequenced. Nucleotide sequences of 16S rDNA (approximately 600 bp) were compared to the most similar sequences in the nucleotide sequence databases using NCBI BLAST programme. The species identification was based on the highest percentage similarities.
After the results obtained with the sequencing of 16S rRNA the bacterial isolates IC62, IC67, IC68, IC73, IC82 were identified as Bacillus subtilis strain, Bacillus licheniformis strain, Bacillus subtilis strain, Bacillus sp. strain, and Bacillus subtilis strain respectively.
These strains have been deposited for patent purposes in the The Microbial Type Culture Collection & Gene Bank at IMTECH, Chandigarh, India and have been given the access numbers MTCC 5415, MTCC 5416, MTCC 5417, MTCC 5418 and MTCC 5419 respectively.
The isolated strains of bacteria produce the enzymes cellulases, amylases, and proteases.
Preliminary results indicate that the isolated strains of the bacteria show a better capability to decompose various organic matter. The combination of these isolated strains of bacteria, also indicate a better capability to decompose various organic matter.
In accordance with an aspect of the invention the isolated strains of bacteria are salt
tolerant, as the soil from which they have been isolated is saline. These isolated bacteria are


therefore easily adapted for composting of organic waste in a region with high salinity or soil waste that contain high concentration of salt in them.
The salinity tolerance of the five microbial cultures was tested by growing them in SPN medium with varying salt concentrations: 0.85%, 1.5%, 2.5%, 4.0%, 6.0% and 8.0% NaCl. The cultures were incubated at 37°C for 24 hours and the growth was observed by taking optical density at 600 nm for 1.5 ml of culture (Table 3).
In accordance with an aspect of the invention bacterial consortium was prepared by culturing the strains of the bacteria in medium for required incubation period to get an inoculum, adding the inoculums to a fresh medium, incubating it for the required time and pooling these bacterial cultures to get a consortium.
In accordance with an aspect the medium for the growth of the bacterial consortium is any bacterial growth supporting medium including but not limited to SPN and ZoBell Marine Broth 2216. Medium may be supplemented with molasses.
In accordance with an aspect the consortium may be grown within a temperature range of 28 to 45°C with the preferred temperature being 30°C. The agitation of the consortium was carried out within the range of 50 to 200 m with the preferred rate being 180rmp.
In accordance with an aspect the consortium was incubated for 24-48 hour. The consortium contains 107to 1010 colony forming units/ml of each culture.
In accordance with an aspect of the invention bacterial growth over time was determined. The cultures were grown up to 48 h at 30°C at 180 rpm. Growth was monitored


in terms of cell density (Optical Density at 600nm) with respect to time and the data was plotted (Figures 6 -10).
Growth of the cultures was checked at different temperatures ranging from 28 to 45°C. All the five cultures grew well in the temperature range of 28 to 45°C after 24 - 48 h incubation, with an optimum temperature of 30°C.
In accordance with an aspect of the invention, the cultures were tested for growth in SPN, Zobell Marine Broth and molasses at 30°C. Results showed that SPN media gave better growth after 24 - 48 h incubation. SPN media contains 20 g of sucrose, 10 g of peptone and 8.5 g of NaCl in 1 liter of deionized water. ZoBell Marine Broth contains: Peptone 5.0 g, Yeast Extract 1.0 g, Ferric Citrate 0.1 g, Sodium Chloride 19.45 g, Magnesium Chloride 5.9 g, Magnesium Sulfate 3.24 g, Calcium Chloride 1.8 g, Potassium Chloride 0.55 g, Sodium Bicarbonate 0.16 g, Potassium Bromide 0.08 g, Strontium Chloride 34.0 mg, Boric Acid 22.0 mg, Sodium Silicate 4.0 mg, Sodium Fluoride 2.4 mg, Ammonium Nitrate 1.6 mg, Disodium Phosphate 8.0 mg per liter of water.
Total Viable Cell counts (TVC) were determined for the five microbial cultures. An aliquot (50 ul) of the respective microbial culture was spread over the nutrient agar plates and incubated at 37°C for 24 h and colony counts were taken (Table 2).
The following example is provided to explain and illustrate certain preferred embodiments of the process of the invention.
Example 1:
Five bacterial cultures were inoculated overnight in tubes containing SNP media and the grown cultures were used as inoculum for culturing in larger volumes after taking the OD


(600 run) at 'O'hours. After 18 hours of culturing at 30 C, OD of each culture was taken. It was observed that all the cultures were in late log phase. The five cultures were pooled together to constitute microbial consortia.
Dated this day of 2nd April 2008