WO2017006345A2

WO2017006345A2 - An ultra light weight nanofiber polymer carrier for use in agricultural and industrial applications

Info

Publication number: WO2017006345A2
Application number: PCT/IN2016/000181
Authority: WO
Inventors: Chandra T.S.; Natarajan T.S.; Sairam Kavitha; Karunakaran Arvind; Sham Raheja Anant
Original assignee: Indian Institute Of Technology Madras
Priority date: 2015-07-08
Filing date: 2016-07-06
Publication date: 2017-01-12
Also published as: WO2017006345A3

Abstract

An ultra-light weight nanofiber polymer carrier for use in agricultural and industrial applications. The proposed method for preparing the ultra-light weight nanofiber polymer carrier material can be an effective solution for providing light weight water soluble polymer carrier of bioactive agents/microorganisms for use in agricultural and industrially important microorganisms and their products and related biomolecules.

Description

AN ULTRA LIGHT WEIGHT NANOFIBER POLYMER CARRIER FOR USE IN AGRICULTURAL AND INDUSTRIAL APPLICATIONS

TECHNICAL FIELD

[0001] Embodiments are generally related to bio-fertilizers/biological fertilizers. Embodiments are also related to light weight water soluble polymer carrier of bioactive agents/microorganisms for use in agricultural and industrial applications. Embodiments are particularly related to an ultra-light weight nanofiber polymer carrier of bioactive agent/microorganisms in the field of agriculture and industrial applications. Embodiments are additionally related to a method for preparing ultra-light weight nanofiber polymer carrier material for agricultural and industrially important microorganisms and their products and related biomolecules thereof.

BACKGROUND OF THE INVENTION

[0002] In the field of agriculture, it is highly important to supply essential nutrients to plants and other herbs in order to support their adequate growth and thereby achieve greater yield rate. Statistics prove that majority of the nutrients are supplied by straight, mixture and/or compound mineral fertilizers. However, prolonged use of mineral fertilizers, especially Nitrogen (N), can result in contamination of soil and ground water. Therefore, bio-fertilizers or biological fertilizers are alternatively adapted in a wide range of agricultural applications for supplying required nutrients to the plants and herbs. Bio-fertilizers are considered to be low cost, eco-friendly and harmless to environment with the ability of supplementing nutrients.

[0003] A bio-fertilizer is a product that consists of selected and different types of living microorganisms, which are known to improve plant growth by the supply of available plant nutrients through biological processes. Such bio-fertilizers are conventionally available in formations with a carrier material where the carrier material can be typically a solid and/or liquid carrier. In majority of the prior art approaches, the carrier material forms the major bulk while the bio-active agent/microorganism is present in trace amounts which render them inefficient, complex, costly and time consuming solutions.

[0004] In an alternative embodiment, microbiological processes can be carried out as a continuous process in wide range of industrial applications in which the requisite microorganisms are immobilized as being supported on a carrier. Methods and processes for immobilization of the microorganisms on a carrier have been proposed by majority of prior arts where the industrially important microorganisms are immobilized into the carrier by a variety of processes.

[0005] In the above mentioned embodiments, the prior art approaches are however unable to obtain high concentration of bio-active agent/microorganisms supported on the carrier. Secondly, the prior arts are unable to provide high activity of the bio-active agent/microorganism supported on the carrier and pertaining to the desired microbiological reaction. Based on the foregoing, it is believed that a need exists for'an improved ultra-light weight nanofiber polymer carrier with high pay load and efficient storage for bio-active agents/microorganisms. A need also exists for an improved method for preparing ultra-light weight nanofiber polymer carrier material for agricultural and industrially important microorganisms and their products and related biomolecules thereof, as described in greater detail herein.

SUMMARY OF THE INVENTION

[0006] The following summary is provided to facilitate an understanding of some of the innovative features unique to the disclosed embodiment and is not intended to be a full description. A full appreciation of the various aspects of the embodiments disclosed herein can be gained by taking the entire specification, claims, drawings, and abstract as a whole.

[0007] It is, therefore, one aspect of the disclosed embodiments to provide for an improved ultra-light weight nanofiber polymer carrier for use in agricultural and industrial applications.

[0008] It is another aspect of the disclosed embodiments to provide for an improved light weight water soluble polymer carrier of bioactive agents/microorganisms for use in agricultural and industrial applications. [0009] It is further aspect of the disclosed embodiments to provide for an improved method for preparing ultra-light weight nanofiber polymer carrier material for agricultural and industrially important microorganisms and their products and related biomolecules thereof.

[0010] The aforementioned aspects and other objectives and advantages can now be achieved as described herein. An ultra-light weight nanofiber polymer carrier for use in agricultural and industrial applications, is disclosed herein. The operating conditions for electrospinning of Poly Vinyl Alcohol (PVA) can be optimized to obtain uniform fiber dimensions. Solutions containing 5, 10, 15 and 20 Wt% of PVA are prepared dissolving PVA (50000 Mw) in double distilled water. The PVA containing solution can be heated at 95°C for 1 hour with constant stirring to obtain a homogenous fluid.

[0011] The optimized Poly Vinyl Alcohol (PVA) can be further electrospun using an applied field in the range of 1.0 to 1.5 kV/cm. The concentration of 20 wt% can be suitable to obtain bead- free uniform electrospun fibers. Apart from the above Poly Vinyl Alcohol (PVA) (Mw, 85000-1, 24,000) (SDFCL), Starch (Rankem) and Carboxy Methyl Cellulose (CMC) (Genei) can be electrospun individually as well as in combination to obtain uniform polymer nanofibers. The electrospinning was conducted at standardized conditions maintained constant in all trials to aid comparison (Voltage applied: 1 kV/cm, Flow rate: 0.3 ml/min, Distance: 15cm). The uniformity of the fibers is analyzed by Optical Microscopy (Labomed-IVu 5100) and Scanning electron microscopy (SEM). Apart from PVA, PVA:Starch (70:30) was identified as the best composition for obtaining uniform nanofibers.

[0012] The nitrogen fixing bio-fertilizer strain of Azotobacter vinelandii (a bio- active agent) can be entrapped in the suggested fertilizer carrying membrane wherein the culture suspension of A.vinelandii can be blended with Poly Vinyl Alcohol (PVA) and electrospun to obtain nano-structured fibers. Finally, the high density microbial pure culture of A.vinelandii can be mixed with the PVA solution and used for electrospinnmg in order to entrap the bacterial cells in the fibers. A.vinelandii mixed with PVA and with PVA:Starch (70:30) can be used for electrospinnmg under aforesaid conditions which resulted in entrapment of the bacterial cells in PVA fibers.

[0013] The bacterial loading capacity of the fibers was tested by Standard Plate Count (SPC), pre and post electrospinnmg. PVA: Starch had a better loading capacity than PVA. Apart from the loading capacity the other advantages of using starch are its cheaper cost and easy availability from various sources. Starch being a natural polymer is not hazardous and an increased starch concentration in the composite fiber leads to pronounced biosafety. The proposed method for preparing the ultra-light weight nanofiber polymer carrier material can be an effective solution for providing light weight water soluble polymer carrier of bioactive agents/microorganisms for use in agricultural and industrially important microorganisms and their products and related biomolecules. [0014] The agriculture input carrying membrane can be a non-woven matrix of polymer nanofibers. The individual polymer fiber diameter size is in the range of 100-200 nm. The representative polymers of the classification include, but not limited to, poly vinyl alcohol, poly ethylene oxide, poly ethylene glycol, Polyvinylpyrrolidone (PVP), sodium alginate, xanthan, carrageenan, etc. Note that synthesis of nanofibers using electrospinning proposed herein should not be limited in any sense. Alternatively, the synthesis of nanofibers can be achieved using sol-gel casting, mechanical drawing, self-assembly polymerization, etc.

BRIEF DESCRIPTION OF THE DRAWINGS

[0015] The accompanying figures, in which like reference numerals refer to identical or functionally-similar elements throughout the separate views and which are incorporated in and form a part of the specification, further illustrate the present invention and, together with the detailed description of the invention, serve to explain the principles of the present invention.

[0016] FIG. 1-3 illustrates graphical representation of SEM images showing electrospun fibers of Poly Vinyl Alcohol (PVA), Poly Vinyl Alcohol (PVA) with A.vinelandii and single cell of A.vinelandii in the fiber, in accordance with the disclosed embodiments;

[0017] FIG. 4 (a) and FIG. 4 (b) illustrates the SEM image 400 and 450 of 11% PVA and 11% PVA:Starch (70:30) at 80,000X, in accordance with the disclosed embodiments;

[0018] FIG. 5 illustrates graphical representation 500 of entrapment of A.vinelandii in PVA and PVA+Starch, in accordance with the disclosed embodiments; and

[0019] FIG. 6 illustrates a graphical representation Second derivative of ATR- FTIR spectra of electrospun membranes of Poly Vinyl Alcohol (PVA) showing the characteristic amide absorption peak of bacterial peptidoglycan in fibers containing A.vinelandii, in accordance with the disclosed embodiments.

DETAILED DESCRD?TION

[0020] The particular values and configurations discussed in these non-limiting examples can be varied and are cited merely to illustrate at least one embodiment and are not intended to limit the scope thereof.

[0021] The embodiments now will be described more fully hereinafter with reference to the accompanying drawings, in which illustrative embodiments of the invention are shown. The embodiments disclosed herein can be embodied in many different forms and should not be construed as limited to the embodiments set forth herein; rather, these embodiments are provided so that this disclosure will be thorough and complete, and will fully convey the scope of the invention to those skilled in the art. Like numbers refer to like elements throughout. As used herein, the term "and/or" includes any and all combinations of one or more of the associated listed items.

[0022] The terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. As used herein, the singular forms "a", "an" and "the" are intended to include the plural forms as well, unless the context clearly indicates otherwise. It will be further understood that the terms "comprises" and/or "comprising," when used in this specification, specify the presence of stated features, integers, steps, operations, elements, and/or components, but do not preclude the presence or addition of one or more other features, integers, steps, operations, elements, components, and/or groups thereof.

[0023] Unless otherwise defined, all terms (including technical and scientific terms) used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. It will be further understood that terms, such as those defined in commonly used dictionaries, should be interpreted as having a meaning that is consistent with their meaning in the context of the relevant art and will not be interpreted in an idealized or overly formal sense unless expressly so defined herein. [0024] FIG. 1-3 illustrates graphical representation of SEM images 100-300 showing electrospun fibers of Poly Vinyl Alcohol (PVA), Poly Vinyl Alcohol (PVA) with A.vinelandii and single cell of A.vinelandii in the fiber, in accordance with the disclosed embodiments. The operating conditions for electrospinning of Poly Vinyl Alcohol (PVA) can be optimized to obtain uniform fiber dimensions. Solutions containing 5, 10, 15 and 20 Wt% of PVA are prepared dissolving PVA (50000 Mw) in double distilled water.

[0025] The PVA containing solution can be heated at 95 °C for 1 hour with constant stirring to obtain a homogenous fluid. The optimized Poly Vinyl Alcohol (PVA) can be further electrospun using an applied field in the range of 1.0 - to-1.5 kV/cm. The concentration of 20 wt% can be suitable to obtain bead- free uniform electrospun fibers, as illustrated at 100.

[0026] Apart from the above Poly Vinyl Alcohol (PVA) (Mw, 85000-1, 24,000) (SDFCL), Starch (Rankem) and Carboxy Methyl Cellulose (CMC) (Genei) can be electrospun individually as well as in combination to obtain uniform polymer nanofibers, as illustrated in Table- 1.

[0027] _. FIG. 4 (a) and FIG. 4 (b) illustrates the SEM image 400 and 450 of 1 1% PVA and 11% PVA.Starch (70:30) at 80,000X, in accordance with the disclosed embodiments. The electrospinning was conducted at standardized conditions maintained constant in all trials to aid comparison (Voltage applied: 1 kV/cm, Flow rate: 0.3 ml/min, Distance: 15cm). The uniformity of the fibers is analyzed by Optical Microscopy (Labomed-IVu 5100) and Scanning electron microscopy (SEM). Apart from PVA, PVA:Starch (70:30) was identified as the best composition for obtaining uniform nanofibers.

[0028] FIG. 5 illustrates graphical representation 500 of entrapment of A.vinelandii in PVA and PVA+Starch, in accordance with the disclosed embodiments. The bacterial loading capacity of the fibers was tested by Standard Plate Count (SPC), pre and post electrospinning. PVA: Starch had a better loading capacity than PVA. Apart from the loading capacity the other advantages of using starch are its cheaper cost and easy availability from various sources. Starch being a natural polymer is not hazardous and an increased starch concentration in the composite fiber leads to pronounced biosafety.

[0029] The nitrogen fixing bio-fertilizer strain of Azotobacter vinelandii (a bio- active agent) can be entrapped in the suggested fertilizer carrying membrane wherein the culture suspension of A.vinelandii can be blended with polyvinylalcohol (PVA) and electrospun to obtain nano-structured fibers. Finally, the high density microbial pure culture of A.vinelandii can be mixed with the PVA solution and used for electrospinning in order to entrap the bacterial cells in the fibers, as illustrated at 200 and 300.

[0030] The proposed method for preparing the ultra-light weight nanofiber polymer carrier material can be an effective solution for providing light weight water soluble polymer carrier of bioactive agents/microorganisms for use in agricultural and industrially important microorganisms and their products and related biomolecules. The agriculture input carrying membrane can be a non- woven matrix of polymer nanofibers. Note that the individual polymer fiber diameter size is in the range of 100-200 nm. The representative polymers of the classification include, but not limited to, poly vinyl alcohol, poly ethylene oxide, poly ethylene glycol, Polyvinylpyrrolidone (PVP), sodium alginate, xanthan, carrageenan, etc. The synthesis of nanofibers using electrospinning proposed herein should not be limited in any sense. Alternatively, the synthesis of nanofibers can be achieved using sol-gel casting, mechanical drawing, self-assembly polymerization, etc.

[0031] FIG. 6 illustrates a graphical representation 600 of second derivative of ATR-FTIR spectra of electrospun membranes of Poly Vinyl Alcohol (PVA) showing the characteristic amide absorption peak of bacterial peptidoglycan in fibers containing A.vinelandii, in accordance with the disclosed embodiments. The presence of bacterial cells in the polymer fibers was indicated through the change in their IR absorption spectra. Viability of the entrapped cells can be tested by suspending the bacteria containing membrane in aqueous buffer and performing a standard plate count on nitrogen free medium, which was selective for A.vinelandii.

[0032] The viability of microbial culture remained unaffected with no significant reduction in cell counts. Further, during incubation at varied temperatures, the bacteria entrapped in the electrospun mat retained higher cell viability than those in liquid buffer formulation. To test the biological nitrogen fixing activity of the cells in the membrane, they were extracted in saline and dilution plated onto nitrogen- free agar medium. The culture plate was then flooded with Nessler's reagent (a chemical indicator that changes from yellow to dark brown in presence of primary and secondary amines) and the culture showed considerable nitrogen fixing activity. Finally, to test the PGP (plant growth promotion) activity of the loaded membrane, an in-house pot assay was performed using tomato seedlings. Seedlings treated with bacteria loaded membrane showed improved shoot length compared to untreated pots.

[0033] The proposed invention describes deployment of the proposed method of loading the payload, which herein describes in detail the loading of agriculturally important nitrogen fixing bacteria A.vinelandii. The conditions of entrapment such as pH, temperature, loading rate, viability of the organisms before and after loading on the membrane have been optimized. Alternatively, the agriculture inputs can include bio-fertilizer strains of bacteria and fungi, plant growth promoting biochemical, etc.

[0034] The results revealed that a very high loading capacity i.e. >10 cfu/g was achieved which retained viability and nitrogen fixing ability of A.vinelandii cells. This level of cell load out performs the industry standard of 10⁸ cfu/g for the conventionally produced bio-fertilizers. Further, most commercial bio-fertilizers are permitted to have a contamination of 10³ cfu/g while the designed FCMs contained no contamination since they are synthesized using pure culture of the organism.

[0035] Therefore, electrospun fibers can be used as carrier matrices for efficient loading of bacterial cells/biofertilizer strains. Use of these carrier material would significantly reduce logistical costs as only a small amount of these fiber mats needs to be used to substitute a very high quantity of the conventional bulky carriers such as peat, lignite, silica, etc.

[0036] Apart from A.vinelandii, the nitrogen fixer Azospirillum lipoferum was also efficiently entrapped in PVA with a loading capacity of 109 cfu/mg of the membrane. The process conditions were similar to that of A. vinelandii. Paracoccus denitrificans, a denitrifier was also entrapped in PVA under similar conditions. This shows that other organisms involved in plant growth promotion and bioremediation could be entrapped efficiently by the process mentioned.

[0037] The invention claims a product which is a membrane carrying a high pay load density of bacterial culture , which is stable in refrigerated conditions, and which can be easily dispersed in the field. For application, the membrane containing the agricultural input is dissolved in water to prepare a suspension, which is then dispersed in the field through drip irrigation or mechanical spraying or any other suitable process of dispersion.

[0038] It will be appreciated that variations of the above-disclosed and other features and functions, or alternatives thereof, may be desirably combined into many other different systems or applications. Also that various presently unforeseen or unanticipated alternatives, modifications, variations or improvements therein may be subsequently made by those skilled in the art which are also intended to be encompassed by the following claims.

Claims

CLAIMS I/We CLAIM:

1. A method of producing ultra-light weight nanofiber polymer carrier for use in agricultural and industrial applications, said method comprising: optimizing operating conditions for electrospinning of Poly Vinyl Alcohol (PVA) to obtain uniform fiber dimensions; preparing solutions containing 5, 10, 15 and 20 Wt% of PVA by dissolving PVA (50000 Mw) in double distilled water wherein the PVA containing solution can be heated at 95°C for 1 hour with constant stirring to obtain a homogenous fluid.

2. The method of claim 1 wherein the optimized Poly Vinyl Alcohol (PVA) can be further electrospun using an applied field in the range of 1.0 to 1.5 kV/cm wherein concentration of 20 wt% can be suitable to obtain bead- free uniform electrospun fibers..

3. The method of claim 1 wherein entrapping nitrogen fixing bio-fertilizer strain of Azotobacter vinelandii (a bio-active agent) in suggested fertilizer carrying membrane wherein the culture suspension of A.vinelandii can be blended with Poly Vinyl Alcohol (PVA) and electrospun to obtain nano-strucrured fibers.

4. The method of claim 1 wherein mixing high density microbial pure culture of A.vinelandii can be mixed with the PVA solution and used for electrospinning in order to entrap the bacterial cells in the fibers. A.vinelandii mixed with PVA and with PVA:Starch (70:30) can be used for electrospinning under aforesaid conditions which resulted in entrapment of the bacterial cells in PVA fibers.

5. The method of claim 1 wherein said uniform polymer nanofibers can be formed by electrospinning individually/in combination at standardized conditions of at least one of the following: Poly Vinyl Alcohol (PVA) (Mw, 85000-1, 24,000) (SDFCL); Starch (Rankem) and Carboxy Methyl Cellulose (CMC) (Genei).

6. The method of claim 1 wherein the agriculture input carrying membrane can be a non-woven matrix of polymer nanofibers wherein the individual polymer fiber diameter size is in the range of 100-200 nm.

7. The method of claim 1 wherein the representative polymers of the classification can be at least one of the following polymers: poly vinyl alcohol; poly ethylene oxide; poly ethylene glycol; Polyvinylpyrrolidone (PVP); sodium alginate; xanthan; and carrageenan.

8. The method of claim 1 wherein the nanofiber polymer carrier material has a capability to load >1012cfu/g of strains.

9. The method of claim 1 wherein the synthesis of nanofibers can be achieved using atleast one of the following methods: electrospinning; sol-gel casting; mechanical drawing; and self-assembly polymerization

10. The method of claim 1 wherein the ultra-light weight nanofiber polymer carrier material can be a light weight water soluble polymer carrier of bioactive agents/microorganisms for use in agricultural and industrially important microorganisms and their products and related biomolecules.