FIELD OF THE INVENTION

This invention particularly relates to novel process for synthesis of a new class of

NiO-Nanocellulose (NiO-NC) Hybrid Nanocomposites of formula 5 having Antibacterial and Antifungal activities and possess overall yield 90% after purification. This invention also relates to the Antimicrobial Activities against (03) three gram positive; (02) two gram negative bacterial strains and fungal strain Candida albicans MTCC 3017. Particularly, Gram positive bacteria are Staphylococcus aureus MTCC 6908, Bacillus toyonensis BCT 7112 and Bacillus cereus MTCC 1272; Gram negative bacteria are Pseudomonas putida ATCC 17642 and Escherichia coli MTCC 739 and Fungal strain are Candida albicans MTCC 3017 and their Antifungal activity against plant pathogenic fungus Colletotrichum gleosporioides.

Also the present invention relates to the synthesis of a new class of NiO-

Nanocellulose Hybrid Nanocomposites. Furthermore, this invention relates to the synthesis of Nanocellulose from renewable agrowaste i.e. rice straw.

The process of the present invention does not involve the use of any costly and environmentally toxic reagents and catalysts and has been developed using a non-toxic reagents under mild reaction conditions.

Accordingly, the present invention provides for the production of nanocellulose in a very cost-effective and convenient method from a renewable agriculture resource, rice straw.

BACK GROUND AND PRIOR ART OF THE INVENTION

It is pertinent to note that the process for NiO-Nanocellulose (NiO-NC) Hybrid

Nanocomposites is recently gaining importance owing to their potent biological activity. So far, no literature reported on NiO-NC, which has potent antibacterial and antifungal activity against bacterial strains and fungal strain such as Candida albicans MTCC3007, Gram positive bacteria Staphylococcus aureus MTCC 6908, Bacillus toyonensis BCT 7112 and Bacillus cereus MTCC 1272; and Gram negative bacteria such as Pseudomonas putida ATCC 17642 and Escherichia coli MTCC 739; Fungal strain Candida albicans MTCC 3017 and plant pathogenic fungus Colletotrichum gleosporioides.

Furthermore, it is pertinent to mention that in the present invention synthesis of Nanocellulose was carried out from renewable agrowaste i.e. rice straw. The hitherto known prior art generally related is referred to herein below:

Reference may be made to patent number: WO2014087053A1, entitled "Method of

Manufacturing A Nanocellulose Composite" which describes the method of nanocellulose composites and a method of manufacturing nanocellulose composites which comprise nanocellulose material mixed with a polymer.

Patent number CN102505546A, entitled "Method for preparing nanocellulose by using homogeneous method" discloses a method for preparing nanocellulose by using a homogeneous method, relating to a method for preparing modified cellulose, i.e., the nanocellulose. The method comprises the following steps of adding ionic liquid into crushed wood fibers to ensure that the mass percentage of the cellulose is 1-15%.

The article "Chemically and mechanically isolated nanocellulose and their self- assembled structures. Feng Jiang, You-Lo Hsieh, Carbohydr Polym. 2013 Jun 5;95(1):32- 40" describes the method for isolation of cellulose nanocrystals (CNCs) and nanofibrils (CNFs) from pure rice straw cellulose via sulfuric acid hydrolysis, mechanical blending and TEMPO-mediated oxidation to 16.9%, 12% and 19.7% yields, respectively.

The study "Cellulose Nanocrystals/ZnO as a Bifunctional Reinforcing Nanocomposite for Poly(vinyl alcohol)/Chitosan Blend Films: Fabrication, Characterization and Properties. Susan Azizi, Mansor B. Ahmad, Nor Azowa Ibrahim, Mohd Zobir Hussein, Farideh Namvar, Int J Mol Sci. 2014 Jun; 15(6): 11040-11053." elaborates the synthesis of cellulose nanocrystals/zinc oxide (CNCs/ZnO) nanocomposites, which were dispersed as bifunctional nano-sized fillers into poly(vinyl alcohol) (PVA) and chitosan (Cs) blend by a solvent casting method to prepare PVA/Cs/CNCs/ZnO bio-nanocomposites films.

The article "Nano and nanocomposite antimicrobial materials for food packaging applications; Paulraj Kanmani & Jong -Whan Rhim, Progress in Nanomaterials for Food Packaging, 2014, 34-48" describes the antimicrobial function of some nano and nanocomposite materials.

The paper "Synthesis of methylcellulose-silver nanocomposite and investigation of mechanical and antimicrobial properties; Dipanwita Maitya, Md. Masud Rahaman Mollicka, Dibyendu Mondala, Biplab Bhowmicka, Mrinal Baina, Kalipada Bankuraa, Joy Sarkarb, Krishnendu Acharyab, Dipankar Chattopadhyaya, Carbohydrate Polymers, 90 (4), 1818-1825" discloses the preparation of methylcellulose-silver nanocomposite films by mixing aqueous solution of methylcellulose with silver nitrate followed by casting. The silver nanoparticles were generated in methylcellulose matrix through reduction and stabilization by methylcellulose.

The article "Novel Approaches to Metallization of Cellulose by Reduction of Cellulose-Incorporated Copper and Nickel Ions. N. Kotelnikoval,*, U. Vainio2, K. Pirkkalainen2 and R. Serimaa, Macromolecular Symposia, Volume 254, Issue 1, pages 74- 79, August 2007" describes the synthesis of copper and nickel nanoparticles in the insoluble microcrystalline cellulose support by reduction of metal ions with several reducers in various media resulting in cellulose-metal nanocomposites.

In view of the above reports the process for NiO-Nanocellulose (NiO-NC) Hybrid Nanocomposites of the formula 5 described in the present invention is completely new and has not been reported so far by any other workers.

OBJECTIVES OF THE INVENTION

The main objective of the present invention is to provide a novel process to synthesize a new class of NiO-Nanocellulose (NiO-NC) Hybrid Nanocomposites of formula 5 starting from nickel chloride hexahydrate (N1CI ₂ .6H ₂ O) and nanocellulose (NC). The size of this new class of NiO-Nanocellulose (NiO-NC) Hybrid Nanocomposites is in the range of 0.4 nm shown in Figure 4.

Yet another objective of the present invention is to provide antimicrobial activities against (03) three gram positive; (02) two gram negative bacterial strains and fungal strain Candida albicans MTCC 3017. Gram positive bacteria are Staphylococcus aureus MTCC 6908, Bacillus toyonensis BCT 7112 and Bacillus cereus MTCC 1272; Gram negative bacteria are Pseudomonas putida ATCC 17642 and Escherichia coli MTCC 739; and Fungal strain Candida albicans MTCC 3017 are shown in Figure 1.

Yet another objective of the present invention is to provide a general method of preparation of a new class of NiO-Nanocellulose (NiO-NC) Hybrid Nanocomposites of the formula 5 starting from Nickel oxide (NiO) and nano cellulose (NC) with one step reaction which could be useful as potent antifungal inhibitors against plant pathogenic fungus Colletotrichum gleosporioides shown in Figure 2.

Still another objective of the present invention is to provide a one pot process for synthesis or construct a new class of NiO-Nanocellulose (NiO-NC) Hybrid Nanocomposites of formula 5.

Yet another objective of the present invention is to provide a method for the production of nanocellulose in a very cost-effective and convenient way from a renewable agriculture waste, rice straw. Still another objective of the present invention is to provide a process for the synthesis of NiO-Nanocellulose (NiO-NC) Hybrid Nanocomposites.

SUMMARY OF THE INVENTION

Accordingly, the present invention provides a NiO-Nanocellulose hybrid nanocomposites having antibacterial and antifungal activity, wherein the said hybrid nanocomposite exhibit the particle size ranging from 04-10 nm.

In an embodiment of the present invention, the hybrid nanocomposites exhibit good antimicrobial activity viz. antibacterial activity against Gram+ve ,Gram-ve bacterial strains and Fungal strain Candida albicans MTCC 3017, wherein Gram positive bacteria are Staphylococcus aureus MTCC 6908, Bacillus toyonensis BCT 7112 and Bacillus cereus MTCC 1272; Gram negative bacteria are Pseudomonas putida ATCC 17642 and Escherichia coli MTCC 739.

In yet another embodiment of the present invention, wherein the compound possesses good antifungal activity against plant pathogenic fungus Colletotrichum gleosporioides.

In yet another embodiment of the present invention, wherein a process for preparation of NiO-Nanocellulose Hybrid Nanocomposites of the formula 5 comprising the steps of:

i) . mixing nickel chloride hexahydrate (N1CI ₂ .6H ₂ O) and nanocellulose in alcoholic solvent and further stirring the reaction for a period of 1 to 3 hrs at a temperature range 15 to 45 °C to get transparent solution, weight ratio of N1CI ₂ .6H ₂ O : nanocellulose was 1:2;

ii) . added base solution dropwise to the above reaction mixture with gentle stirring for a period of 11 to 15 hrs at a temperature range 65 to 90 °C and allowed to settle down;

iii) . centrifuged the reaction mixture of step (ii) to separate nickel oxide nanocellulose nanocomposites (NiO-NC), washing with distilled water for several times to remove the byproducts;

iv) . dried the samples for a period of 1 to 3 hrs at a temperature range 65 to 98 °C to get the compound of formula 5.

In one more embodiment, base solution used for the reaction is selected from the group consisting of potassium hydroxide, sodium hydroxide, calcium hydroxide and sodium carbonate and in the range of 100 ml of 2 mol/L - 100 ml of 10 mol/L per 1 gm of nanocellulose used.

In one more embodiment of the present invention, wherein for the preparation of nanocellulose the renewable agrowaste, rice straw was used. In yet another preferred embodiment of the present invention, the new class of NiO- Nanocellulose (NiO-NC) Hybrid Nanocomposites of formula 5 possess overall yield 90% after purification and have been found to show good antibacterial activity in general determined by disc diffusion method as shown in Figure 1.

In yet another embodiment of the present invention, the new class of NiO-

Nanocellulose (NiO-NC) Hybrid Nanocomposites of formula 5 also has been found to show potent antifungal activity against plant pathogenic fungus Colletotrichum gleosporioides determined by using the poisoned food technique as shown in Figure 2. Colletotrichum gleosporioides with the inhibition percentage 98% as depicted in the Figure 2 which was determined by the standard antifungal activity test, viz., Poison Food Technique.

In yet another embodiment of the present invention, Antibacterial test was performed on four different types of the pathogens and all were found to be sensitive to compound 5 with inhibition zones varying from 2.67-15 mm.

In yet another embodiment of the present invention, the product of formula 5 derived from nanocellulose with nickel chloride hexahydrate (N1CI ₂ .6H ₂ O) of the formula 1 and 2 were show highest inhibition against Candida albicans of 15+00 mm at concentration 50μ§/ιη1 followed by Bacillus toyonensis of 7+1 mm. In Pseudomonas putida and Staphylococcus aureus inhibition zones were 4.67+0.57 mm and 5.33+1.15 mm respectively at concentration of 50μ§/ιη1. A little inhibition zones were detected against Escherichia coli and Bacillus cereus. From the analyzed result, these NiO-Nanocellulose (NiO-NC) Hybrid Nanocomposites of the formula 5 has been found to be the most promising bactericide against both Gram positive and Gram negative bacteria with MIC values 0.05-2 μg respectively.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1: Zone of inhibition of compound against 1) Pseudomonas putida 2) Escherichia coli 3) Bacillus toyonensis 4) Staphylococcus aureus 5) Bacillus cereus and 6) Candida albicans

Figure 2: Antifungal activity against plant pathogenic fungus Colletotrichum gleosporioides.

Figure 3: TEM image of nanocellulose.

Figure 4: TEM image of the product 5.

Figure 5: SAED photograph of product 5. DETAILED DESCRIPTION OF THE INVENTION

This invention relates to the process for synthesis of a new and novel compound of formula 5 NiO-Nanocellulose Hybrid Nanocomposites prepared from nickel chloride hexahydrate (NiCl ₂ .6H ₂ 0) and nanocellulose

1 + 2 ^a ' ^b 5

Nano cellulose (NC) Nickel chloride hexahydrate NiO-Nanocellulose Hybrid Nanocomposites

(NiCl ₂ .6H20)

Reagents and conditions: (a) ROH (R= Et/Me/Pr/Bu), stirring for 1-3 hrs, 15 to 45 °C, NiCl ₂ .6H ₂ 0 : nanocellulose :: 1:2 (b) ROH (R =Na/K/Ca)/ Na ₂ C0 ₃ , stirring for 11 - 15 hrs at 65 to 90 °C and allowed to settle down, centrifuge and then washed with distilled water for several times, dryed for 1 to 3 hrs at 65 to 98 °C to get the compound 5.

The representative compounds used in invention comprising of :

1: Nano cellulose (NC) 2 : Nickel chloride hexahydrate (NiCl ₂ .6H ₂ 0) 3 : Ethanol (EtOH) 4 : Sodium hydroxide (NaOH)

5 : NiO-Nanocellulose Hybrid Nanocomposites

This invention also relates to the process for synthesis of new class of NiO- Nanocellulose Hybrid Nanocomposites and their Antifungal Activity against plant pathogenic fungus Colletotrichum gleosporioid.es.

This invention particularly relates to the process for synthesis of new class of NiO- Nanocellulose Hybrid Nanocomposites and their Antimicrobial Activities against (03) three gram positive and (02) two gram negative bacterial strains and fungal strain Candida albicans MTCC 3017. Gram positive bacteria are Staphylococcus aureus MTCC 6908, Bacillus toyonensis BCT7112 and Bacillus cereus MTCC 1272; Gram negative bacteria are Pseudomonas putida ATCC 17642 and Escherichia coli MTCC 739. Fungal strain is Candida albicans MTCC 3017.

The process of invention is found to have an overall yield 90% after purification. The process of the present invention does not involve the use of any costly and environmentally toxic reagents and catalysts and has been developed using a non-toxic reagents under mild reaction conditions.

Accordingly, the present invention provides for the production of nanocellulose in a very cost-effective and convenient method from a renewable agriculture resource, rice straw of North East India. The present investigation is to provide an one pot process for synthesizing or constructing a new class of NiO-Nanocellulose Hybrid Nanocomposites of the formula 5 with yield of 90% from nickel chloride hexahydrate (NiCl ₂ .6H ₂ 0) of the formula 1 and nanocellulose of the formula 2 accompanying this specification which comprises the following steps :-

Step I: Solvent extraction of rice straw plant material is done with the help of Soxhlet apparatus using non polar solvent (petroleum ether/hexane) and polar solvent (methanol/ethanol) .

Step II : After solvent extraction rice straw was dried in an oven in a period of 3-6 hrs at the temperature range 40-60 °C. Alkaline hydrolysis was done using base in the range of 100-250 ml of NaOH and Na ₂ C0 ₃ and stirred the mixture for a period of 6-12 hours. The reaction mixture was then cooled and neutralized with mineral acid selected from the group consisting of hydrochloric acid, sulphuric acid or perchloric acid etc, particularly hydrochloric acid and in the range of 10-20 % per 10 gm of pretreated rice straw used. Filtered the biomass and treated with H ₂ O ₂ solution in the range of 10-20 ml per 10 gm of pretreated rice straw and stirred in a period of 4-8 hrs at the temperature range 40-50 °C. The reaction mixture was then cooled and filtered and then washed with distilled water for several times to get the desired product cellulose.

Step III : Cellulose was acid hydrolysed with mineral acid selected from the group consisting of sulphuric acid, hydrochloric acid or perchloric acid etc, particularly sulphuric acid in the range of 4-6% per 10 gm of cellulose. Refluxed the reaction mixture in a period of 4-6 hrs at the temperature range 60-80 °C. Then the reaction mixture was filtered and washed with distilled water for several times to get the desired product nanocellulose .

Step IV : Nickel oxide nanocellulose hybrid nanocomposites were synthesized by using nanocellulose with nickel chloride hexahydrate (NiCl ₂ -6H ₂ 0) mixing in alcoholic solvent with weight ratio of N1CI ₂ .6H ₂ O : nanocellulose was 1:2. Stirred the reaction mixture for a period of 1 to 3 hrs at a temperature ranging 15 to 45°C to get transparent solution, adding sodium hydroxide solution dropwise to the above reaction mixture with gentle stirring for a period of 11 to 15 hrs at a temperature ranging 65 - 90 °C and allowing to settle down, centrifuging the reaction mixture to separate nickel oxide nanocellulose nanocomposites (NiO-NC), washing with distilled water for several times to remove the byproducts, drying the samples for a period of 1 to 3 hrs at a temperature ranging 65 - 98 °C to get the compound of the formula 5. This is the first ever method where this new class of NiO-Nanocellulose (NiO-NC) Hybrid Nanocomposites of the formula 5 has been found to show good antibacterial activity in general determined by disc diffusion method as shown in Figure 1.

Antimicrobial test was performed on four different types of the pathogens and all were found to be sensitive to compound 5 with inhibition zones varying from 2.67-15 mm. The highest inhibition was calculated against Candida albicans of 15+00 mm at concentration 50μ§/ιη1 followed by Bacillus toyonensis of 7+1 mm. In Pseudomonas putida and Staphylococcus aureus inhibition zones were 4.67+0.57 mm and 5.33+1.15 mm respectively at concentration of 50μ§/ιη1. A little inhibition zones were detected against Escherichia coli and Bacillus cereus. From the analyzed result, these NiO-Nanocellulose (NiO-NC) Hybrid Nanocomposites of the formula 5 has been found to be the most promising bactericide against both Gram positive and Gram negative bacteria with MIC values 0.05-2 μg respectively.

The new and novel class of NiO-Nanocellulose (NiO-NC) Hybrid Nanocomposites of formula 5 also has been found to show potent antifungal activity against plant pathogenic fungus Colletotrichum gleosporioides determined by using the poisoned food technique as shown in Figure 2. Colletotrichum gleosporioides with the inhibition percentage 98% as depicted in the Figure 2 which was determined by the standard antifungal activity test, viz., Poison Food Technique.

EXAMPLES

The following examples are given by way of illustration of the working of the invention in actual practice and should not be construed to limit the scope of the present invention in any way.

Example 1

Step A: Preparation of nanocellulose:

Step I: Dry powdered RS sample was extracted with non polar solvent -petroleum ether and polar solvent -methanol with the help of Soxhlet apparatus for 15 hrs at 45 °C to remove oil/wax etc.

Step II : After solvent extraction rice straw was dried in an oven for 4 hrs at 40 °C. Alkaline hydrolysis was done using mixture of 5% NaOH of 150 ml and 5% Na ₂ C0 ₃ of 150 ml and stirred the mixture for 7 hours. The reaction mixture was then cooled and neutralized with 10% mineral acid - 10 ml HC1 per 10 gm of pretreated rice straw. Filtered the biomass and treated with 20% H ₂ 0 ₂ solution of 10 ml per 10 gm of pretreated rice straw and stirred for 6 hrs at 45 °C. The reaction mixture was then cooled and filtered and then washed with distilled water for several times to get the desired product cellulose with the yield of 75%. Step III : Cellulose was acid hydrolysed with 5% mineral acid- 5 ml H ₂ S0 ₄ per 10 gm of cellulose. Reaction mixture was refluxed for 5 hrs at 70 °C. After that reaction mixture was filtered and washed with distilled water for several times to get the desired product nanocellulose with the yield 90%.

Step B: Synthesis of NiO-Nanocellulose (NiO-NC) Hybrid Nanocomposites :

Nickel oxide nanocellulose hybrid nanocomposites were synthesized by mixing nanocellulose with nickel chloride hexahydrate (NiCl ₂ .6H ₂ 0) in alcoholic solvent -EtOH and further stirring the reaction for a period of 3 hrs at 45 °C to get transparent solution with weight ratio of NiCl ₂ .6H ₂ 0 : nanocellulose as 1:2. After that sodium hydroxide solution was added dropwise to the above reaction mixture with gentle stirring for 12 hrs at 75 °C and allowing to settle down, centrifuging the reaction mixture to separate nickel oxide nanocellulose nanocomposites (NiO-NC) and washing with distilled water for several times to remove the byproducts. Sample was dried for 2 hrs at 70 °C to get the compound 5 with yield 90%.

Example 2

Step A: Preparation of nanocellulose:

Step I: Dry powdered RS sample was extracted with non polar solvent - hexane and polar solvent -ethanol with the help of Soxhlet apparatus for 18 hrs at 50 °C to remove oil/wax etc.

Step II : After solvent extraction rice straw was dried in an oven for 6 hrs at 45°C. Alkaline hydrolysis was done using mixture of 5% KOH of 150 ml and 5% Na ₂ C0 ₃ of 150 ml and stirred the mixture for 9 hours. The reaction mixture was then cooled and neutralized with 10% mineral acid -10 ml H ₂ S0 ₄ per 10 gm of pretreated rice straw. Filtered the biomass and treated with 20% H ₂ 0 ₂ solution of 10 ml per 10 gm of pretreated rice straw and stirred for 8 hrs at 55 °C. The reaction mixture was then cooled and filtered and then washed with distilled water for several times to get the desired product cellulose with the yield of 60%. Step III : Cellulose was acid hydrolysed with 10% mineral acid -10 ml HC1 per 10 gm of cellulose. Reaction mixture was refluxed for 7 hrs at 90 °C. After that reaction mixture was filtered and washed with distilled water for several times to get the desired product nanocellulose with the yield 80%.

Step B : Synthesis of NiO-Nanocellulose (NiO-NC) Hybrid Nanocomposites: Nickel oxide nanocellulose hybrid nanocomposites were synthesized by mixing nanocellulose with nickel chloride hexahydrate (NiCl ₂ .6H ₂ 0) in alcoholic solvent -MeOH and stirred the reaction for a period of 2 hrs at 40 °C to get transparent solution with weight ratio of NiCl ₂ .6H ₂ 0 : nanocellulose as 1:2. After that potassium hydroxide solution was added dropwise to the above reaction mixture with gentle stirring for 15 hrs at 80 °C and allowing to settle down, centrifuging the reaction mixture to separate nickel oxide nanocellulose nanocomposites (NiO-NC) and washing with distilled water for several times to remove the byproducts. Sample was dried for 3 hrs at 75°C to get the compound 5 with yield 70%.

Example 3

Step A: Preparation of nanocellulose:

Step I: Dry powdered RS sample was extracted with non polar solvent - hexane and polar solvent - propanol with the help of Soxhlet apparatus for 10 hrs at 45 °C to remove oil/wax etc.

Step II : After solvent extraction rice straw was dried in an oven for 8 hrs at 55 °C. Alkaline hydrolysis was done using mixture of 3% NaOH of 150 ml and 3% Na ₂ C0 ₃ of 200 ml and stirred the mixture for 10 hours. The reaction mixture was then cooled and neutralized with 10% mineral acid- 10 ml HC10 ₄ per 10 gm of pretreated rice straw. Filtered the biomass and treated with 15% H ₂ 0 ₂ solution of 20 ml per 10 gm of pretreated rice straw and stirred for 10 hrs at 60 °C. The reaction mixture was then cooled and filtered and then washed with distilled water for several times to get the desired product cellulose with yield of 50%.

Step III : Cellulose was acid hydrolysed with 10% mineral acid -10 ml HC10 ₄ 10 ml per 10 gm of cellulose used. Reaction mixture was refluxed for 9 hrs at 90 °C. After that reaction mixture was filtered and washed with distilled water for several times to get the desired product nanocellulose with yield 65%.

Step B: Synthesis of NiO-Nanocellulose (NiO-NC) Hybrid Nanocomposites :

Nickel oxide nanocellulose hybrid nanocomposites were synthesized by mixing nanocellulose with nickel chloride hexahydrate (NiCl ₂ .6H ₂ 0) in alcoholic solvent-propanol and stirred the reaction for a period of 3 hrs at 43 °C to get transparent solution with weight ratio of NiCl ₂ .6H ₂ 0 : nanocellulose as 1:2. After that sodium carbonate solution was added dropwise to the above reaction mixture with gentle stirring for 14 hrs at 82 °C and allowed to settle down. Centrifuging the reaction mixture to separate nickel oxide nanocellulose nanocomposites (NiO-NC) and washing with distilled water for several times to remove the byproducts. Sample was dried for 3 hrs at 92 °C to get the compound 5 with yield 65%. Example 4

Step A: Preparation of nanocellulose :

Step I: Dry powdered RS sample was extracted with non polar solvent - petroleum ether and polar solvent - propanol with the help of Soxhlet apparatus for 20 hrs at 56 °C to remove oil/wax etc.

Step II: After solvent extraction rice straw was dried in an oven for 1 hrs at 60 °C. Alkaline hydrolysis was done using mixture of 2% NaOH of 150 ml and 5% Na ₂ C0 ₃ of 150 ml and stirred the mixture for 12 hours. The reaction mixture was then cooled and neutralized with 10% mineral acid -10 ml HC1 per 10 gm of pretreated rice straw. Filtered the biomass and treated with 10% H ₂ 0 ₂ solution of 20 ml per 10 gm of pretreated rice straw and stirred for 10 hrs at 45 °C. The reaction mixture was then cooled and filtered and then washed with distilled water for several times to get the desired product cellulose with the yield of 55%. Step III: Cellulose was acid hydrolysed with 10% mineral acid -10 ml HC10 ₄ per 10 gm of cellulose. Reaction mixture was refluxed for 8 hrs at 85 °C. After that reaction mixture was filtered and washed with distilled water for several times to get the desired product nanocellulose with yield 50%.

Step B: Synthesis of NiO-Nanocellulose (NiO-NC) Hybrid Nanocomposites :

Nickel oxide nanocellulose hybrid nanocomposites were synthesized by mixing nanocellulose with nickel chloride hexahydrate (NiCl ₂ .6H ₂ 0) in alcoholic solvent and stirred the reaction for a period of 2 hrs at 42 °C to get transparent solution with weight ratio of NiCl ₂ .6H ₂ 0 : nanocellulose as 1:2. After that sodium carbonate solution was added dropwise to the above reaction mixture with gentle stirring for 12 hrs at 72 °C and allowed to settle down, centrifuging the reaction mixture to separate nickel oxide nanocellulose nanocomposites (NiO-NC) and washing with distilled water for several times to remove the byproducts. Sample was dried for 2 hrs at 98 °C to get the compound 5 with yield 55%.

Biological Activity of NiO-Nanocellulose (NiO-NC) Hybrid Nanocomposites of formula 5:

In Vitro Antimicrobial Activity Determination:

In a preferred embodiment of the present invention : the new and novel class of

NiO-Nanocellulose (NiO-NC) Hybrid Nanocomposites of the formula 5 has shown good antimicrobial activity in general, which was determined by disc diffusion method as shown in Figure 1. a. Test microorganisms:

Three gram positive and two gram negative bacterial strains were selected for antimicrobial activity study. Gram positive bacterias are Staphylococcus aureus MTCC

6908, Bacillus toyonensis BCT7112 and Bacillus cereus MTCC 1272; gram negative bacterias are Pseudomonas putida ATCC 17642 and Escherichia coli MTCC739. Fungal strain Candida albicans MTCC3017 was also used to study. The bacterial strains were grown in nutrient broth at 30°C for 24 hours and the fungal strain was grown on potato dextrose broth at 30°C for 24 hours. Stocks were prepared by transferring 300μ1 of liquid culture into test tubes containing 700μ1 of 50% sterile glycerol and stored at -20-30°C.

b. Disc diffusion method:

Antimicrobial activity was determined by disc diffusion method. NiO-Nanocellulose

Nanocomposites were suspended in DMSO to make a concentration of 50μg/ml and then diluted to 10μg/ml and 5μg/ml.20 μΐ of these test samples were impregnated to 6 mm diameter sterile filter paper discs and allowed to air dry. Dried discs were placed under UV light for 30 mins. 100 μΐ of the test bacteria were grown in 10 ml of nutrient broth at

30+2°C for 24 hours. Nutrient agar plates were prepared and 100 μΐ of the microbial suspension was spread onto it with a sterile spreader. For fungal strain potato dextrose broth and potato dextrose agar media were used. The discs were then placed onto the inoculated plates and incubated at 30+2°C for 24 to 36 hours. Standard disc of ciprofloxacin 5mcg was used as standard antibiotic. Zones of inhibition were measured in millimeters. All the tests were performed in triplicates and datas were expressed as Mean + standard deviation.

c. Minimum inhibitory concentration determination:

To determine the MIC six different MNC concentrations of 2, 1, 0.5, 0.1, and 0.05 mg/ml respectively were used. Bactrias and fungi were grown in the irrespective broth along with the MNC of different concentrations for 24 hours at 30+2°C. The MIC value was determined at the lowest concentration of MNC in the broth medium that inhibit the growth of the test microorganism visually.

d. Disc diffusion method:

In the disc diffusion method shown in Figure 1 , MNC showed inhibition zone against all tested microorganisms with the highest inhibition against Candida albicans (Fig.lA) of 15+lmm at concentration 50μg/ml followed by bacillus toyonensis (Fig. IB) of 7+1 mm. In Pseudomonas putida (Fig. lC) and Staphylococcus aureus inhibition zones were 4.67+lmm and 5.33+lmm respectively at concentration of 50μ§/ιη1. A little inhibition zones were detected against Escherichia coli and Bacillus cereus. Zone of inhibition in millimeters is tabulated in Table 1.

e. MIC:

The minimum inhibitory concentrations are tabulated in Table 2. Fungal strain

5 Candida albicans showed inhibition to all the tested concentrations. MIC for Bacillus

toyonensis and Bacillus cereus were 0.5 mg/ml and 2mg/ml. The remaining two bacteria under study showed inhibition at concentration of 1 mg/ml.

Table 1: Zone of inhibition in millimeters of the product in disc diffusion method.

10

Table 2: Minimum inhibitory concentration and sensitivity test against the strains.

Strains Pseudomo Escherichi Bacillus Staphylococc Bacillus Candida nas putida a coli toyonensis us aureus cereus albicans

2 + + + + + + mg/ml

1 + + + + mg/ml

0.5 + + mg/ml

0.1 + mg/ml

0.05 + mg/ml

Sensitive Sensitive Sensitive at Sensitive at Sensitive Resista

Result at at 0.1 mg/ml 0.5 mg/ml at nt

0.5 mg/ml 1 mg/ml 1 mg/ml

Antifungal Activity

The new and novel class of NiO-Nanocellulose (NiO-NC) Hybrid Nanocomposites of the formula 5 has also shown potent antifungal activity against plant pathogenic fungus Colletotrichum gleosporioides determined by using the poisoned food technique as shown in Figure 2. Colletotrichum gleosporioides with the inhibition percentage 98% as depicted in the Figure 2 which was determined by the standard antifungal activity test, viz., Poison Food Technique.

The inhibitory effects of the sample tested in vitro on mycelia growth of the fungus Colletotrichum gleosporioides. Poisoned Food Technique was used to test the antifungal activity of the product of the formula 5 with six different concentrations of sample at 100, 200, 300, 400, 500, 600 ppm prepared in the particular solvent and used 1ml of each concentration was mixed with the freshly prepared, sterilized potato-dextrose agar media in petriplates (each 1ml sample was added to 20 ml of media), allowed them to mix homogeneously and to be solidified. Control growth medium contained equivalent amounts solvent and sterilized distilled water. Fungal disks of 5 mm in diameter from a 8-day-old pure culture were placed in the center of the Petri dish containing medium under aseptic condition, incubated at 27 +1 °C for 7 days. The experiments were carried out in three replicates per treatment. Fungal growth was observed at every 24 hrs. interval. At the end of the incubation period the mycelial growth was measured. The fungicidal nature of the sample was determined by testing revival of growth following transfer of the mycelial disk to an appropriate agar plate without sample. Percentage inhibition of mycelial growth was evaluated using the poisoned food techniques .

Table 3: The Inhibitory effects of the samples on mycelial growth of

C. gleosporioides 7days after inoculation (percent inhibition)

The main advantages of present invention are :

1. The process for synthesis of a new and novel class of NiO-Nanocellulose (NiO-NC) Hybrid Nanocomposites. A novel one pot process for the synthesis of a new class of NiO-Nanocellulose (NiO-NC) Hybrid Nanocomposites. No further steps are required for synthesis of the NiO-Nanocellulose (NiO-NC) Hybrid Nanocomposites. 2. Synthesized nanocomposites have been found to possess good antimicrobial activity against (03) three gram positive and (02) two gram negative bacterial strains and fungal strain Candida albicans MTCC 3017. Gram positive bacteria are Staphylococcus aureus MTCC 6908, Bacillus toyonensis BCT 7112 and Bacillus cereus MTCC 1272; Gram negative bacteria are Pseudomonas putida ATCC 17642 and Escherichia coli MTCC 739.

3. Synthesized products have been found to exhibit good antifungal activity against plant pathogenic fungus Colletotrichum gleosporioides.

4. The invention provides for the production of nanocellulose in a very cost-effective and convenient method from a renewable agriculture resource, rice straw.

5. The chemicals used are cheap and commercially available.

6. The process is reported for the first time in the most easiest and simplest way.