BR102017020884A2 - FORMULATIONS OF MICROBIAN INOCULANT BASED ON POLYMERIC MIXTURES CONTAINING BIOPOLYMERS, USE OF INOCULANT FORMULATION, SEEDS OR PARTS OF PLANTS TREATED WITH INOCULATING COMPOSITION - Google Patents

Abstract

The present invention relates to an inoculation vehicle comprising a mixture of an exopolysaccharide type (eps) microbial biopolymer and a cellulose-derived polymer for plant growth promoting microbial strain formulations for use in agricultural systems. The proposed formulation is an alternative to peat that is widely used in the preservation of viable microbial cells. However, peat suitable for use as inoculation vehicles are those with high organic matter content, but tropical peat generally do not have satisfactory quality and, often, it is necessary to import the input. In addition, due to its high microbial load, there are additional costs inherent to the sterilization of the input. The carboxymethylcellulose (cmc) / eps mixture, obtained by a simple and inexpensive process, was able to maintain the viability of bradyrhizobium yuanmingense strain br3267 cells in the range of 108 to 109 cfu ml-1 for up to 120 days. at room temperature. The addition of 0.5% eps is enough to guarantee cell viability. Data obtained through rheological, viscosimetric and spectrometric analyzes in the infrared region indicate the presence of interactions between eps and cmc, which results in increased dimensional stability of the polymeric mixture (avoiding phase separation), contributing to the survival of the particles. bacterial cells. These characteristics reveal the potential use of the cmc / eps polymer mixture as an inoculation vehicle.

Description

MICROBIAL INOCULANT FORMULATIONS BASED ON POLYMERIC MIXTURES CONTAINING BIOPOLYMERS, USE OF THE INOCULANT FORMULATION, SEEDS OR PARTS OF PLANTS TREATED WITH THE INOCULANT COMPOSITION

FIELD OF THE INVENTION [001] The present invention relates to an inoculant composition comprising cellulose derivatives and biopolymers of microbial origin, such as exopolysaccharides produced by bacterial strains of the rhizobic group, being used in agriculture, more specifically in the formulation of biological inoculants for agricultural application.

DESCRIPTION OF THE STATE OF THE TECHNIQUE [002] The inoculation of seeds with bacteria from the rhizobia group contributes to increase the benefits of Biological Nitrogen Fixation (FBN) in a large number of legumes, among these, soy is mentioned as the culture that in Brazil it benefits most from the application of inoculants. The use of inoculants, despite representing a small portion of the production cost, results in savings of around US $ 7 billion.year ^-1 considering the acquisition of nitrogen fertilizers (HUNGARY. M. et al., 2013).

[003] Optimizing the contribution of FBN and other activities that promote plant growth induced by microorganisms, including biological control, in plants grown from different leguminous and non-leguminous species, demands a strategy capable of meeting two main aspects: The. selection of efficient plant / rhizobia associations and, b. development of inoculant formulations compatible with the conditions of use (BASHAN, Y. et al., 2014). The first strand aims at the prospecting of native microorganisms and areas of production of the culture of interest and the selection of efficient strains; the second, depends on studies on the compatibility of the properties of the inoculation vehicle with the microbial agent

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2/12 in order to obtain a product that has stability and validity in line with current regulations.

[004] Peat is widely used as an inoculation vehicle given its ability to maintain the viability of rhizobial cells due to the high moisture content associated with a high surface area (KALJEET, S. et. Al., 2011). However, high-quality peat deposits, which are desirable for the production of inoculants because they have a high content of organic matter guaranteeing the viability of rhizobian populations, are rare, especially in tropical regions. It is also added that the processing of peat represents an additional cost that requires significant capital investments limiting its use by small producers (SINGLETON, P. et. Al., 2002). Peat, being rich in organic matter, naturally has a high amount of microbial cells, which impairs the survival of the target microorganism. Peat sterilization is a fundamental process, however, it represents another stage in processing, increasing production costs (DEAKER, R. et. Al., 2004).

[005] Different inoculation vehicles have been tested as an alternative to peat, among these, the use of polymers has been gaining prominence in recent years. Polymers have some advantages for this purpose, such as longer shelf life, adequate viability to the place of application, ease of production and high performance (BASHAN, Y. et. Al., 1998). Several techniques for the delivery of microbial cells using polymers have been developed, however, few have been tested in field conditions (SCHOEBITZ, M. et. Al., 2013).

[006] US patent 8592343 describes a vehicle based on a polymeric CMC / starch mixture to which a compatibilizing agent is added to ensure stability avoiding phase separation and reducing the number of viable cells. This is a critical point of the CMC and starch vehicle, as it requires the development of a careful protocol considering different origins and methods of obtaining the

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3/12 starch. In patent BR1020150254970, starch was replaced in the polymeric mixture with CMC by the spirulina biomass. Also positive points are the fact that the polymeric vehicle is biodegradable, uses materials that are easily available and contains a reduced amount of water, which facilitates transportation (Freitas, C.S., 2015).

[007] In the present invention there is described a product composed of biopolymer, an exopolysaccharide of bacterial origin, used as an integral part of the polymeric inoculation vehicle in conjunction with the CMC. A characteristic of microbial exopolysaccharides is the ability to change the environment in which they are found, which gives them a wide range of applications in numerous technological areas. (ARANDA-SELVERIO, G. et. Al., 2010).

[008] Patents have been found that use an exopolysaccharide (EPS) biopolymer produced by microorganisms in order to preserve bacterial cells and assist in the aggregation of degraded soils. In document CN 106164247 A, EPS is not a vehicle, as it has a role in the formulation as an aggregation agent for soil particles contributing to the improvement of its quality. The formulation contains strains that produce EPS together with strains with characteristics that induce the promotion of plant growth, its use being recommended to increase the plant's tolerance to environmental stress conditions. In UA54847 (U), EPS of Bacillus mucilaginosus is added to bacterial formulations in order to guarantee the preservation of cell viability. The two formulations described above are liquid. Currently liquid formulations are used mainly in mechanized planting systems and do not constitute a good alternative to peat inoculants. These have reduced shelf life requiring special storage conditions, for example, refrigeration. They are more bulky, which makes transportation more expensive.

[009] The use of polymers as inoculation vehicles was proposed in JP 8143410 which uses natural polymers (cotton, linen, paper, cork)

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4/12 or synthetic (poly (vinylidene chloride), polyacrylates, poly (vinyl chloride), polyamide or polyester). These materials form a network or a film, but are not able to guarantee the survival of microbial cells for relatively long periods of storage. Another solution for formulations of bacteria of the rhizobic group, document US 2002050096, uses a polymer that contains amides, amines and imines in order to neutralize the exudates of the seeds, such as vinylpyrrolidone, vinyl acetate copolymers and styrene copolymers; and adjuvants such as carboxymethylcellulose, gum arabic, sodium alginate and the like. The polymer is part of a liquid formulation that also includes a compound containing molybdenum. Liquid formulations have some limitations already described. US 9580363 B2 aims to increase the rates of biological nitrogen fixation in plant species defined concomitantly with the reduction of nitrous oxide emission. It uses as an inoculant, bacterial species of the genera Enterobacter, Methylobacterium, Sphingomonas, and Pleomorphomonas to be used in formulations with polymeric vehicles such as CMC and EPS, among others, used in isolation. The focus of the proposal is the composition of the strains and not the vehicle itself.

[010] Flocculation and encapsulation methods have been proposed to obtain polymeric formulations with plant growth promoting microorganisms that have reduced volume. EPS produced by strains of the genera Azospirillum, Rhizobium and Zooglea during the cultivation of bacterial biomass is used in WO 1994019924 A1. Under specific culture conditions, the bacterial cells differentiate into an encysted form and form floccules that are easily separated from the rest of the culture broth. High molecular weight polymer, based on cellulose or polysaccharides, produced by certain bacterial strains are capable of promoting the encapsulation of bacterial cells during the cultivation of biomass in liquid medium in a single step (document JPH11171716). JP 1030585 proposes an inoculant in the form of

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5/12 microcapsules of sodium alginate or carrageenan gum that immobilizes the microorganism in a suitable nutrient solution. Encapsulation can be done using the ethyl cellulose polymer precipitation method (used as a temporary support for the formation of microcapsules) and consolidated by curing using the interface polymerization method in a material such as nylon-6 or nylon-10 . Flocculation or encapsulation obtained during the cultivation of bacterial biomass is restricted to bacterial strains that produce biopolymers. The encapsulation with alginate or carrageenan gum is a complex process that involves several steps that can lead to reduced cell survival. Due to its complexity, the addition of extra costs must also be considered.

[011] In addition to the encapsulation, vehicles based on polymers have been proposed that include drying and hot extrusion steps that also aim to reduce the volume of water. JP 6141848 uses a water-soluble polymer that goes through a drying step, obtaining a granular form with adjusted humidity in the range of 25 to 45%. In US 4755468, on the other hand, a carbohydrate source in a partially cross-linked polysaccharide gel is used, such as xanthan gum for formulations of cells of the rhizobic group. After mixing, the formulation goes through a drying step where the humidity is reduced to about 10%. The maintenance of low water content in formulations composed of polymers that have the capacity to absorb large amounts of water is a factor that requires strict control. Additionally, during the drying period, cell viability may be reduced. Similar to what has already been described above, extra steps during the production process can incur additional costs. A biocomposite obtained by the hot extrusion process from biopolymers and mineral nutrients was proposed in document BR 10 2012 029862 7 in order to impart new properties to the materials. It is a complex process that can also lead to increased production costs.

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6/12 [012] The use of two polymers for the production of inoculation vehicles has also been the subject of some patent deposits, however, in general, more complex processes are employed for the production of these vehicles. US 4434231 describes a process for immobilizing microorganisms within a polymeric matrix comprising a polymer gel based on one or more polymers selected from the group of polysaccharides, in which said polymer is at least partially cross-linked. The polymer is selected from natural gums produced by strains of the genera Xanthomonas Arthrobacter or Sclerotium, or biosynthetics, such as alginates, carrageenan, agar, Karaya gum, tragacanth gum, arabic gum, carob gum and guar gum. The crosslinking of the polymer can be promoted by heat treatment, treatment with a metal salt or by synergism with another polymer, preferably another polysaccharide. The fact that it is partially cross-linked, hinders the biodegradability of the formulation. The amount of these polymers in the vehicle that requires the production of significant amounts of gums. In document UA89120 (C2), two polymers are used to form a lipcogen composed of 30% acrylamide exopolysaccharide (EPPA) and 70% xanthan. EPPA is obtained by means of an EPS polymerization reaction with acrylamide. In the document BR 102013019740-8 A2, biopolymers can be copolymerized with other polymers, such as chitosan, alginate, cellulose derivatives or starches through nanotechnology reactions such as electrospinning or ectrospraying. The method produces a solid polymer in the form of nanofibers where the bacteria remain trapped.

[013] US 8592343 B2 describes a formulation obtained from a polymeric mixture carboxymethylcellulose and starch capable of maintaining viable rhizobial cells for up to six months. The process is simple and low cost, however, there is a need to make the mixture compatible using Zn or Mg oxides. Compatibility depends on the type and origin of the starch, which requires adjustments to the formulation. The replacement of starch by small amounts of EPS as

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7/12 described in this deposit resolves this limitation and dispenses with the use of the compatibilizing agent.

[014] The inoculant of the present invention has a number of advantages over the prior art inoculants, such as: (i) being a gel that maintains a sufficient water content to guarantee a longer survival of the cells and not be in excess to hinder the pellet processing of the seeds; (ii) be biodegradable, reducing the possibility of environmental impact and facilitating the release of rhizobial cells in the environment; (iii) present polymeric mixture with dimensional stability as a consequence of interactions between the biopolymer and the CMC, which reduces the chance of phase separation and guarantees cell viability and vehicle quality; and (iv) simplicity of the starting materials, ease of production of the polymer which, in addition to reducing production costs, contains a reduced amount of water, resulting in reduced weight and volume of the material, which facilitates transport to the agricultural area where it will be applied.

SUMMARY OF THE INVENTION [015] The present invention features an inoculation vehicle that comprises microbial cells and polymeric mixture containing biopolymers and cellulose polymers, and its use in the formulation of microbiological inoculants for agricultural application.

[016] Another object of the present invention is the use of the composition as a biological inoculant applied to the seeds or parts of plants intended for planting.

[017] The present invention also refers to the use of the composition as a biological inoculant applied to the soil or in the vicinity of plant roots.

[018] Still as an object of the present invention are provided seeds or parts of plants treated with said inoculant composition.

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DETAILED DESCRIPTION OF THE INVENTION [019] The present invention features an inoculant composition comprising microbial cells, polymer and EPS and their use in the formulation of biological inoculants for agricultural application.

[020] According to the present invention the inoculant composition comprises:

a) Microbial cells;

b) Polymer;

c) Biopolymer produced by microbial cells.

[021] The inoculation vehicle preferably comprises 0.5% to 8% (by weight) of biopolymer in relation to the polymeric mixture. The microbial cells of said inoculant composition preferably comprise bacteria and fungi that promote growth and have biological control properties. In a preferred embodiment, growth-promoting bacteria are nitrogen-fixing bacteria, more preferably bacteria of the rhizobic group, such as those of the genus Bradyrhizobium, other bacterial species capable of forming nodules in legumes, associative and endophytic diazotrophic bacteria, such as those of the genera Nitrospirillum, Gluconacetobacter, Paraburkholderia and Herbaspirillum. The inoculation vehicle uses polymeric mixtures based on cellulose derivatives, preferably cellulose esters, and biopolymers of microbial origin in order to obtain a product with good quality and dimensional stability (reduced chance of phase separation), lower levels of contamination and high maintenance capacity of viable cells. According to the invention, the inoculant carrier is prepared from polymers based on cellulose derivatives and microbial biopolymers, preferably exopolysaccharides produced by bacterial cells in the rhizobia group, and compatibilizing agents can be used. The polymer of

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9/12 cellulose derivative, soluble in water, can be selected from the group consisting of carboxymethylcellulose or esters thereof or its alkali metal salts, hydroxypropyl cellulose, hydroxypropylmethyl cellulose, hydroxyethyl cellulose and mixtures thereof. EPS can be produced by bacteria and fungi. In addition, the vehicle may contain adjuvants such as nutrients or growth factors such as sugars, amino acids, proteins, salts and the like, or mixtures thereof. The vehicle may also contain osmotic-regulating agents, buffering or pH-adjusting agents and others.

[022] The present invention also comprises the use, of the described composition, as a microbiological inoculant. Preferably, the use of the composition of the invention occurs through its application in seeds, in the stem, other reproductive organs intended for planting or parts of the plant. Even more preferably, the use of the composition of the invention occurs through its application in the soil or in the vicinity of the roots of plants. Additionally, an inoculation can be carried out before sowing for a few days, including a pelletizing process with the formulation of inoculants.

[023] The inoculation vehicle based on carboxymethyl cellulose and exopolysaccharides of microbial origin is able to guarantee greater competitiveness to the agricultural sector by providing a low-cost product, easy to be standardized, enough shelf time to accommodate storage and transportation period during agricultural crop meeting the need for distribution throughout the national territory. Formulations containing the proposed inoculation vehicle can be used successfully for areas with large crops that already use other inoculants, but also for family farmers who do not usually have access to inoculants.

EXAMPLES

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10/12 [024] Example 1: Use of an inoculant vehicle composed of a polymeric mixture of carboxymethyl cellulose and exopolysaccharides in formulations with the BR3267 strain of Bradyrhizobium yuanmingense. Mixtures of CMC and EPS were obtained, obtained from the strain of Bradyrhizobium yuanmingense, BR3267 in the concentrations shown in Table 1.

Example 1

Preparation of the CMC / EPS mixture

Table 1. Composition of polymeric mixtures produced in a mass concentration of 6.4 grams of mixture in 100 ml of distilled water.

Quantity of CMC (g) Quantity of EPS (mg) EPS content in the mixture (%) 6.40 0 0.0 6.37 32 0.5 6.34 64 1.0 6.27 128 2.0

[025] The mixtures followed the proportion of 64 g / L (polymer / water). The mixtures were prepared with the aid of a mixer to form a visually recorded homogeneous preparation. After preparation, the polymeric vehicle was subjected to an autoclaving process, at 1 atm of pressure and 120 ° C of temperature for 30 minutes. The gel-like polymer, before autoclaving, maintains this shape after treatment, however, acquiring a clearer appearance. Autoclaving is a classic form of sterilization to ensure the absence of microorganisms and fungi, providing adequate conditions for survival of the target microorganism that is inoculated into the polymeric base (inoculation vehicle) in the form of a microbial suspension, which can be a rhizobial suspension .

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Preparation of the microorganism [026] The rhizobia BR 3267 strain of B. yuanmingense was grown in YMA medium in order to guarantee cultural purity and, subsequently in YM medium (FRED, E.B., 1928). After growth, the cells were centrifuged at 2,500 g and then resuspended in sterile distilled water.

Preparation of the polymeric vehicle with the microorganism [027] The bacterial suspension prepared according to paragraph [23] was added to the polymeric vehicle described in paragraph [21], under manual agitation in a laminar flow hood at room temperature.

Determination of cell viability [028] In the rhizobial inoculant prepared in paragraph [25], the survival of the bacterial suspension for about four months at room temperature was evaluated. Periodically, an aliquot was collected, subjected to serial dilution and inoculated in YMA culture medium, in order to determine the number of viable cells in the product. The results transformed to log the number of colony forming units. ml ^-1 are shown in Table 2 after inoculation for up to 120 days at room temperature.

Table 2. Evaluation of cell viability (log UFC ml ^-1 ) of strain BR3267 maintained at room temperature in formulations containing CMC and EPS mixtures extracted from culture medium with volumes of 50, 150 and 250 mL and in mass concentration of up to 8 , 0%. UFC: Colony Forming Units.

Cell viability (log UFC ml ^-1 ) Concentration

EPS

Storage period (days) (%) Average value

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7 30 90 120 0.0 8.29 aA * 8.25 aA 7.10 cC 7.62 bB 7.82 b 0.5 8.50 to B 8.32 aB 8.43 bB 8.79 aA 8.51 a 1.0 8.62 aB 8.32 BC 8.22 bC 8.93 aA 8.52 a 2.0 7.78 bB 8.48 aA 8.68 aA 8.80 aA 8.43 a

[1] * Averages in the column followed by the same lowercase letter and, in the line by the same uppercase line, except the average value, do not differ by the Scott-Knott Test at a level of 5% probability.

[029] The number of cells in the initial inoculum was 2.5x10 ⁹ cfu. mL ^-1 of inoculum, of which 5 mL were added to 25 mL of the vehicle, estimating an initial concentration of 4.1.10 ⁸ ufc.ml ^-1 in the inoculant that corresponds to the log of 8.61. from the 0.5% concentration increases cell viability compared to pure CMC in the 90 and 120 day readings. On the average of the three formulations containing EPS (0.5, 1.0 and 2.0%) after 120 days of storage at room temperature, a number of cells similar to that inoculated at the beginning of the storage period were maintained. The formulation without adding EPS after 120 days, showed about 10% of the number of cells compared to the one that was inoculated at the beginning.

[030] Data obtained from rheological, viscosimetric and spectrometry analyzes in the infrared region (FTIR) indicated the presence of interactions between the biopolymer (EPS) and the CMC, which leads to an increase in the dimensional stability of the polymer mixture (prevents phase separation), contributing to the survival of bacterial cells. These characteristics corroborate the results obtained for maintaining the number of viable cells and indicate the potential for using the CMC / EPS polymeric mixture as an inoculation vehicle.

Claims (9)

1 - FORMULATIONS OF MICROBIAL INOCULANT BASED ON POLYMERIC MIXTURES CONTAINING BIOPOLYMERS, USE OF THE INOCULANT FORMULATION, SEEDS OR PARTS OF PLANTS TREATED WITH THE INOCULANT COMPOSITION characterized by the fact that it comprises a) microbial cells, b) polymer and c) biopolymer of microbial origin. 2 - MICROBIAL INOCULANT FORMULATIONS, according to claim 1, characterized by being able to guarantee viability of microbial cells that comprise plant growth promoting microorganisms including biological control agents characterized as a biological inoculant. 3 - FORMULATIONS OF MICROBIAL INOCULANT, according to claims 1 and 2, characterized by containing microorganisms that promote plant growth, comprise nitrogen-fixing bacteria including, among these, those of the rhizobia group. 4 - MICROBIAL INOCULANT FORMULATIONS, according to claims 1 to 3, characterized by the fact that the polymer of the inoculation vehicle is derived from cellulose, selected from the group consisting of carboxymethylcellulose, hydroxypropyl cellulose, hydroxypropylmethylcellulose, hydroxyethylcellulose and mixtures thereof; carboxymethyl cellulose esters and alkali metal salts of carboxymethyl cellulose. MICROBIAL INOCULANT FORMULATIONS, according to any one of claims 1 to 4, characterized in that the biopolymer is of microbial origin, including exopolysaccharides and comprises at least 2/2 a microorganism selected from the group of bacteria promoting plant growth, including those from the rhizobic group. MICROBIAL INOCULANT FORMULATIONS, according to any one of claims 1 to 5, characterized by comprising a mixture compatibility agent including at least one compound selected from zinc, magnesium, molybdenum compounds, calcium salts and modified polymers, among those, zinc oxide and magnesium oxide. MICROBIAL INOCULANT FORMULATIONS, according to any one of claims 1 to 6, characterized by comprising at least one nutrient additive or growth factor selected from sugars, amino acids, proteins, salts; and, at least one adjuvant selected from osmotic-regulating agents, buffering or pH adjusting agents. 8 USE OF MICROBIAL INOCULANT FORMULATIONS, according to claim 2, characterized in that said biological inoculant is applied to the seeds or parts of plants intended for planting, comprising stalk or other reproductive organs, or to be applied directly to the soil or in the vicinity of the roots. USE OF MICROBIAL INOCULANT FORMULATIONS, according to claim 8, characterized by being an inoculation that precedes sowing by a few days, including a pelletizing process with the inoculation formulation, defined in any one of claims 1 to 7.