RO131931B1 - Process for manufacturing pellets bioactivated with microorganisms from depleted mushroom-cultivation substrate - Google Patents

Description

The present invention relates to obtaining bioactivated pellets with microorganisms, from a depleted substrate from lignocellulosic mushroom culture, intended for soil treatment and / or soil debris covering the soil, in the case of conservative agriculture systems.

Uses of the depleted substrate from mushroom cultivation in plant cultivation technologies are known, especially for improving soil / crop substrate characteristics. The enzymatic systems involved in the degradation of lignocellulose, laccases, xylanases, lignin-peroxidases, cellulases and hemicellulases, present in large quantities in the depleted substrate from the cultivation of lignocellulosic fungi, have effects of accelerating the formation of humus and degradation of some aromatic contaminants or hydrocarbons. pesticide residues (Phan and Sabaratnam, 2012, Applied Microbiology and Biotechnology, 96: 863-873). Chitin from the depleted substrate causes an increase in the activity of chitinolytic microorganisms in the soil, with suppression of plant diseases (Parada et al., 2012, Journal of Phytopathology, 160: 390-396). Chitosan formed by deacetylation of chitin has growth-enhancing biostimulant effects, with activation of resistance systems and stimulation of plant growth (Kwak et al., 2015, Mycobiology, 43: 311-318). The depleted substrate from the mushroom culture has also been used for the production of biopreparations for agricultural use, by cultivation under (semi) solid fermentation of the entomopathogenic strains of Bacillusthuringiensis (Wu et al., 2014, Journal of Economic Entomology, 107: 137- 143), Trichoderma viride antagonists (patent RO 126363 B1, with the formation of compost / suppressive soils for mycotoxigenic fungi in the Fusarium graminearum group), phosphorus solubilizers Pichia farinose (Zhu et al., 2012, Bioresource Technology, 111: 410-416 ).

A technical problem associated with the use of the depleted substrate from the cultivation of lignocellulosic fungi for soil treatment is determined by the difficulty of uniform distribution of a high heterogeneous material (partially degraded straw, randomly embedded in mushroom mycelium) on the soil surface. A technical solution to this problem is compacting by different techniques (pelletizing, tabletting, briquetting), to generate products that can be easily managed on the ground (or on plant debris, which cover the soil in conservative agriculture systems). The high protein and chitin content of the depleted substrate determines a high adhesion to the molds used for compaction, generating blockages of the equipment used, and risks of breaking the kinematic components involved in the transfer of compressive forces.

Another disadvantage of using the depleted substrate from lignocellulosic mushroom culture as a soil treatment is due to the high variability of the final results, which is significantly dependent on the microflora specific to each soil. In order to increase the reproducibility of the results of the application of the depleted substrate of lignocellulosic fungi as a soil treatment in agricultural technologies (or as a treatment of plant residues in conservative agricultural technologies), a solution is the bioactivation with strains of microorganisms beneficial to crop plants (including biostimulant microorganisms). ), with recognized saprophytic competence.

Different conditioning processes in the form of compacted products of microorganisms that are beneficial to plants are known.

US 2013/0324407 A1 describes methods for obtaining large quantities of bacteria of the genus Methylobacterium, which involve the inoculation of biphasic media comprising a liquid phase and a solid phase, the solid phase in some cases being suspended in the liquid phase. The incubation of the culture is carried out under conditions that ensure the development of microorganisms. Two-phase fermentation media can be axenic cultures. Further, cultures of microorganisms can be harvested, for example, by filtration. The microorganisms harvested can be adhered to the solid substance. The drying of the solid substance to which microorganisms have adhered can be achieved either by lyophilization or by drying substances. The solid may be among others

RO 131931 Β1 diatomite, silica, fungal spores, particles from any part of a plant, etc. The 1 fermentation media, or the products of the fermentation media or other compositions comprising solid substances, such as dialomite, with microorganisms adhered to them, can be used in 3 bioremediation. Compositions that are used to treat plants and parts of plants that contain solids, such as diatomite, with microorganisms adhering to them, may also contain an agriculturally acceptable additive, such as lignosulfonates.

CA 2860848 A1 presents microbial strains, cultures of microbial strains, compositions 7 and methods of using them to stimulate the development and or production of plants. The compositions described may contain isolated strains or a culture of microorganisms, and may be 9 liquids or solids. These may also include a carrier suitable for agricultural use, which may be solid, for example, diatomite, silica, or combinations. The formulations may also comprise 11 and other biological substrates, such as cereals, parts of plants, etc. Solid compositions may be prepared by dispersing the microorganisms of the invention in or on the solid carrier which may be, for example, diatomite, pasteurized soil, etc. Depending on the mode of use, the compositions may also comprise other additives, such as lecithin. This document also presents microbial compositions formulated for use in the treatment of seeds, which can also be liquid or solid. These compositions may also comprise a variety of additives, including 17 calcium lignosulfonate. The composition which is deposited on the seeds may comprise diatomite, silica or the like. 19

Patent document RO 122588 B1 refers to a composition for plant growth, and to a process for obtaining it. The process comprises the passage of the exhausted substrate 21 from the cultivation of Pleurotus mushrooms in a mixer with the mantle, addition of K ₂ PO ₄ for normalization of pH and potassium content, heating at 55 ... 57 ° C and holding 23 for 30 by min, mixing at a speed of 2 to 3 rpm sigma pallets, cooling to ambient temperature, inoculation of biopreparations with controlled release based on 25 Bacillus subtilis and Trichoderma sells, maintaining for 7 ... 8 days, mixing with a frequency of one rotation per hour. 27 In the article Pelleted organo-mineral fertilizers from composted pig slurry solids, animal wastes and spent mushroom compost for amenity grasslands - Waste 29 Management, JR Rao et al., 2007, a process for transforming residues from farms into organo-mineral fertilizers in the form of pellets. The compost used 31 consists of pig manure, bird litter, depleted substrate of mushrooms, cocoa shells and ground newspaper. The compost mixes during pelletizing with dry blood and feather dust with 33 mineral supplements.

Patent application WO 2009093261 A2 refers to a biopesticide made on the basis of one or more entomopathogenic microscopic fungi, which are in the form of highly compressed tablets. The entomopathogenic fungi are selected from group 37 represented by the genera Beauveria, Metarhizium, Paecilomyces, Verticillium and Nomuraea, and the tablets contain the conidia of one or more entomopathogenic microscopic fungi, 39 UV protectors, anti-saprophytic agents, desiccants, lubricants, lubricants, lubricants and thinners. 41

EP-Patent 0929215 B1 claims the use of the cellular residue mass, remaining after extraction from soybeans of oil and proteins, as a bioporter for making 43 effervescent tablets with useful microorganisms (exemplified by Lagenidium, an entomopathogenic oomycete). One of the compositions for making the patent consists of 60 45 parts by weight of soybean cell residue, 30 parts (biomass of) Lagenidium, 5 parts sodium pyrophosphate acid, 5 parts sodium bicarbonate. Lagenidium biomass is incorporated into the biopurpose material (soybean cell mass residue), dried, mixed with the effervescent pyrophosphate / bicarbonate system and then tablet. 49

RO 131931 Β1

US Patent No. 8940074 B2 discloses a process for the manufacture of pellet biofertilizers, which includes the following steps: mixing a biodegradable material with a water soluble polymeric material to form a first mixture; mixing a polyol with water and with saline nutrients to form a second mixture; mixing the first mixture and the second mixture to form a granular conglomerate, representing the third mixture; spraying some endospores of beneficial bacteria on the granules of the third mixture, to form granules of biofertilizer; extrusion of biofertilizer granules to form compact pellets.

The common disadvantage of the compaction conditioning processes is given by the reduced survival rate of the microorganisms subjected to the conditioning by these compaction processes, which implies for both the drying and the compression stresses for the microorganisms.

The application in the conditioning environment of microorganisms of stabilizing compositions increases the survival rate, but not to a sufficient extent. Procedures are needed to stimulate the internal systems of protection of microorganisms, in order to increase their intrinsic resistance to subsequent conditioning in the form of compact pellets, in which the microorganisms are subjected to both the stress of drying and the result of the compression with high forces.

The authors established that orthosilicic acid, known as a biostimulant that increases plant resistance to biotic and abiotic stresses (Sawas and Ntatsi, 2015, Scientia Horticulturae, 19: 66-81) and as having a stimulating effect on the growth of microorganisms (Wainwright et. al., 1997, Mycological Research, 101: 933-938), also has an effect of stimulating the internal systems of protection of microorganisms against the adverse environmental factors.

Orthosilicic acid is a very weak acid, with four acid functions, at which the lowest pKa value is 9.8 (ller, The Chemistry of Silica, John Wiley & Sons, New York, 1979, p. 207). This means that at pH 9.8, orthosilicic acid is present 50% in the undissociated state and 50% in the dissociated state. between pH 2 and 8, orthosilicic acid is a neutral molecule, completely undissociated. At concentrations greater than 2 mM it begins to polymerize, through polycondensation reactions, with water release (Mcintosh, 2012, Physical Chemistry Chemical Physics, 14: 996-1013). Due to this tendency of polycondensation, the orthosilicic acid cannot be included in the culture media of microorganisms in high concentrations, but it must be constantly released in low concentrations, biologically active, from precursor compounds. The resulting biomass must then be easily conditioned in effervescent tablet formulas, ensuring the high survival of microorganisms.

The technical problem solved by the invention is to describe an easy process to be performed, obtaining pellets from the exhausted substrate from the cultivation of lignocellulosic fungi, bioactivated with microorganisms beneficial to plants, in particular microorganisms with biostimulation activity of crop plants, by to ensure a high survival rate of microorganisms and a production of pellets. It is another object of this invention to describe a process for obtaining the biomass of microorganisms with high resistance to conditioning, by compression in effervescent structures, by cultivation in environments where low concentrations, biologically active, of orthosilicic acid are constantly released.

The process according to the invention consists of the following steps:

- Axenic cultivation on minimal liquid media, including 2% diatomite, at optimum pH and aeration up to 50% oxygen saturation, with incubation temperature variation with an interval of 10 ° C, 12 h at 20 ° C and 12 h at 30 ° C for 3 to 5 days;

RO 131931 Β1

- harvesting the biomass of microorganisms and the diatomite by filtration under vacuum of at least 0.5 bar;

- drying of the biomass of microorganisms and of the diatomite, collected by filtration, up to 3 maximum 5% residual humidity;

- homogenization of 9 ... 11 parts biomass of microorganisms and diatomite, with 5

1.5 ... 2.5 parts sodium lignosulfonate, 2.2 ... 2.4 parts lecithin, 84.3 ... 87.1 parts of exhausted substrate from Pleurotus mushroom cultivation, dried up to 14% residual moisture, and 7 milled to a granulation of 1 ... 2 mm, the parts being expressed in mass units;

- compaction of the micro-organism biomass mixture - diatomite - 9-sodium lignosulfonate - lectin - depleted substrate from Pleurotus mushroom cultivation, by pressing in a pellet press with horizontal molds, to form pellets with a length of about 11 15 mm and a diameter of 5 ... 8 mm.

The process favors the controlled release, in the step of axenic cultivation on minimal liquid media, of orthosilicic acid in concentrations that are below 1 mM.

Drying of the biomass of microorganisms and of the diatomite is done by spraying 15 standardized suspensions at 10% dry matter, under mild conditions, at 13O ... 14O ° C inlet temperature and 75 ... 80 ° C outlet temperature, when The cultivated microorganisms 17 are gram-positive bacteria, which form endospores or microscopic fungi that form conidia, and by lyophilization, by gradually increasing the temperature from -25 ° C to 25 ° C, 19 to 0.9 mbar pressure, for 48 h. , when microorganisms are gram-negative bacteria. 21

This invention has the following advantages:

- ensures the constant release of low concentrations, biologically active, of orthosilicic acid 23 from diatomite, due to the cultivation of microorganisms on a minimal environment, which stimulates the production by microorganisms of the biocomposites involved in solubilizing the orthosilicic acid;

- determines an advanced survival rate of microorganisms that are cultivated 27 under conditions that favor the expression of internal resistance mechanisms to external factors, due to the protective effect of silicic acid, combined with temperature shocks; 29

- it significantly reduces the adhesion of the depleted substrate to the pelletizing matrix, due to the combined lubricating effect of lignosulfonate and lecithin. 31 below are exemplary embodiments illustrating the invention without limitation.

Example 133 in a bioreactor (Biostat® B, Goettingen, Germany) fitted with a pH sensor and a dissolved oxygen sensor (DO) (lnPro6800; Mettler-Toledo AG, Greifensee, Switzerland), and with a 35 L vessel, bring 2 L minimum M9 medium containing 1 L: Na ₂ HPO ₄ (anhydrous) 6 g; KH ₂ PO ₄ 3 g; NaCl 0.5 g; NH ₄ Cl 1 g, 10 g lactose. 40 g of 37 diatomite, a content of silicon dioxide of at least 91.5%, is suspended in the resulting medium. The resulting medium is sterilized by in situ autoclaving, and then nine microelements are added, at the following final concentrations: 39 MgSO ₄ 1 mM; CaCl ₂ 0.1 mM; (NH ₄ ) ₆ Mo ₇ O ₂₄ · 4H ₂ O 3 x 10 ' ⁹ M; H ₃ BO ₃ 4 x 10 ' ⁷ M;

CoCI ₂ · 6H ₂ O 3 x 10 ' ⁸ M; CuSO4 · 5H2O 1 x 10 ' ⁸ M; MnCI2 · 4H ₂ O 8 x 10. ₈ M; 41

ZnSO ₄ · 7H ₂ O 1 x 10 ' ⁸ M; FeSO ₄ · 7H ₂ O 1 x 10 ' ⁶ M, results from sterilized stock solutions by ultrafiltration. Check the pH and bring to pH 5.5 with 1 M HCl or 1 M. NaOH 43

All reagents used are sourced from Merck-Millipore, Darmstadt, Germany, except for colloidal silicon dioxide, which is Celite® 545 (Imerys Filtration Minerals, 45 San Jose, CA, USA). Any other reagents having the same technical characteristics may be used.

RO 131931 Β1

The medium was inoculated with 100 mL of Trichoderma asperellum Td36b conidia suspension, NCAIM P (F) 001434, normalized to 108 propagules per ml by counting on the cytometric lamella. The T. asperellum Td36b strain is known to have biostimulatory effect on crop plants (Raut et al., 2015. Journal of Biotechnology, 208, S62). The Td36b strain is cultivated for 5 days, at an aeration rate of up to 50% oxygen saturation, with the variation of incubation temperature with an interval of 10 ° C, 12 h at 20 ° C and 12 h at 30 ° C.

From time to time, aseptic samples of 2 ... 2.4 ml culture medium with microorganisms are taken in HDPE vessels (Nalgene, Thermo Scientific, Waltham, MA, USA). The supernatant is separated by centrifugation from the microbial sediment and the silica gel, and samples of 1 ml of supernatant, which is diluted with 4 ml of ultrapure water, are taken in 15 ml conical Eppendorf tubes (Eppendorf, Hamburg, Germany). The content of free orthosilicic acid is determined with a Merck kit (Merck Silicate Assay, 1.14794, Merck-Millipore). This colorimetric test is based on the reaction between silicate and molybdate ions, to form a colored complex of blue silicomolibdate, which can be detected spectrophotometrically at 810 nm. The absolute concentration of silicic acid is calculated after the construction of a calibration curve, using a silicon standard (Merck 170236, Merck-Millipore). in culture medium a concentration of orthosilicic acid is determined which is permanently below 1 mM, being the consequence of two concomitant processes - solubilization of silica under the effect of microbial metabolism, and assimilation of orthosilicic acid. In the sediment of microorganisms, separated from the diatoms and washed, the total silica, after mineralization, is determined by ICP-OES (Georgiadis et al., 2013, Geoderma, 209: 251-261). There is a continuous increase of the silicon content in the biomass of microorganisms, an increase that proves the assimilation of the orthosilicic acid by the microorganisms.

After the end of the cultivation period, the biomass of microorganisms is collected and the diatomite is filtered by vacuum filtration of at least 0.5 bar, using a Sartolab® unit (Sartorius, Goettingen, Germany). The suspension resulting from filtration is resuspended in milli-Q pure water (produced in a Milli-Q® Integral, Merck-Millipore) up to 5% dry matter. The resulting suspension is dried to a maximum of 5% residual moisture, on a spray drying plant with atomizing disc and with heated air as a drying agent, at a speed of at least 20000 rpm of the atomizing disc, at an inlet temperature of the agent drying of 13O ... 14O ° C and at an outlet temperature of the drying agent of 75 ... 80 ° C. A spray drying facility that can be used for this purpose is, for example, Niro Production Minor Unit, produced by Niro Gea (Soeborg, Denmark), or Laboratory spray dryer, produced by ICF Cibec (Maranello, Italy). Any other type of spray drying plant with similar technical characteristics can be used.

Take 10 parts biomass of dry microorganisms and colloidal silicon, which are brought into a fluidized bed mixer (MiniGlatt, Glatt, Binzen, Germany), together with 9 parts biomass of microorganisms and diatomite, with 1.5 parts sodium lignosulfonate, 2.4 parts lecithin, 87.1 parts of exhausted substrate from the cultivation of Pleurotus mushrooms, dried up to 14% residual moisture, and ground to a granulation of 1 ... 2 mm, the parts being expressed in mass units.

The sodium lignosulfonate used is Borresperse NA (Borregarrd, Sarspborg, Norway), with the following characteristics: dry matter minimum 93%; maximum calcium 0.6%, pH (10% solution) of 8.3 ± 0.8, but any other lignosulphonate with the above characteristics can be used.

Lecithin used is Thermolec® WFC, Archer Daniels Midland (Decatur, IL, USA), with a hydrophilic-lipophilic balance greater than 8, but any other lecithin modified with the above characteristics can be used.

RO 131931 Β1

The mixture resulting by homogenization in fluidized bed is pelletized using a press 1 (mill) of pellets with horizontal dies, model Kahl 14-175 (Amandus Kahl, Reinbek / Hamburg, Germany), at a specific power of 1 kW for 0.015 ... 0.02 m ² , with 3 maintaining the temperature of the pelletized mixture at about 65 ° C, to form pellets with a length of about 15 mm and a diameter of 5 ... 8 mm. 5

Any other horizontal pellet press, which provides similar conditions of densification by pressing, can be used. 7

The resulting pellets are stable, with a breaking resistance of about 4 kP.

At the end of the process of obtaining, the content of propagules of 9 Trichoderma is analyzed by cultivation on selective media. This content is at least 5 x 10 ⁷ cfu / g, and does not decrease by more than 10% by storing bioactivated pellets, at room temperature, 11 for 6 months.

Example 2 13

We proceed as in example 1, with the following differences. Glucose is used as a source of carbon and energy in the minimal environment, Brevibacillus parabrevis 15 B50 strain is used, NCAIM (P) B 001413 (strain known as biostimulant for plants, patent application RO 128931), cultivation is carried out for three days , and the homogenization step of 17 micro-organism biomass and dry diatomite is performed in the following proportions: with 10 parts micro-organism biomass and diatomite, with 2 parts sodium lignosulfonate, 19 2.3 parts lecithin, 85.7 parts of exhausted substrate from the cultivation of Pleurotus mushrooms, dried to 14% residual moisture, and ground to a granulation of 1 ... 2 mm, the parts being 21 expressed in mass units.

The resulting pellets are stable, with a breaking resistance of about 4 kP. 2. 3

At the end of the production process, the propagule content of Brevibacillus is analyzed by cultivation on selective media. This content is at least 10 ⁸ cfu / g, and 25 does not decrease by more than 10% by storing tablets at room temperature for 6 months.

Example 3 27

We proceed as in example 1, with the following differences. Glucose is used as a source of carbon and energy in the minimal environment. The Pseudoxanthomonas 29 mexicana P32 strain is used, NCAIM (P) B 001414 (known as biostimulant for plants, EP patent 2738267 B1), and the cultivation is done for 3 days. Drying is done by freeze-drying, 31 on a Christ Alpha 1-2 LD freeze-dryer (Martin Chist, Osterode am Harz, Germany), by gradually increasing the temperature from -25 ° C to 25 ° C, at 0.9 mbar pressure, for 48 hours. 33

The homogenization step of the biomass of microorganisms and of the dried diatomite is performed in the following proportions: with 11 parts biomass of microorganisms and diatomite, with 35 2.5 parts sodium lignosulfonate, 2.2 parts lecithin, 84.3 parts of exhausted substrate. in the cultivation of Pleurotus mushrooms, dried up to 14% residual moisture, and milled to a 37 grain size of 1 ... 2 mm, the parts being expressed in mass units.

The resulting pellets are stable, with a breaking resistance of about 4 kP. 39

At the end of the production process, the propagule content of Pseudoxanthomonas is analyzed by cultivation on selective media. This content is at least 41 5 x 10 ⁷ cfu / g, and does not decrease by more than 10% by storing tablets at room temperature for 6 months. 43

This work was realized through the Partnerships program in priority areas - PN II, carried out with the support of MEN - UEFISCDI, project PN-ll-PT-PCCA-2013-4-0846, contract 45 159/2014 CERES.

Claims (3)

claims 1. Process for obtaining bioactivated pellets with microorganisms from the exhausted substrate of the mushroom culture, according to the invention, characterized in that it consists of the following steps: axenic cultivation on minimal liquid media, including 2% diatomite, at optimum pH and aeration. up to 50% oxygen saturation, with the variation of incubation temperature with an interval of 10 ° C, 12 h at 20 ° C and 12 h at 30 ° C, for 3 to 5 days; harvesting of micro-organism biomass and diatomite by vacuum filtration of at least 0.5 bar; drying of the biomass of microorganisms and of the diatomite collected by filtration, up to a maximum of 5% residual humidity; homogenization of 9 ... 11 parts biomass of microorganisms and diatomite, with 1.5 ... 2.5 parts sodium lignosulphonate, 2.2 ... 2.4 parts lecithin, 84.3 ... 87.1 parts of exhausted substrate from the cultivation of Pleurotus mushrooms, dried up to 14% residual moisture, and milled granules 1 ... 2 mm, the parts being expressed in mass units; compaction of the biomass mixture microorganism - diatomite - sodium lignosulfonate - lecithin - depleted substrate from Pleurotus mushroom cultivation, by pressing in a pellet press with horizontal molds, to form pellets with a length of about 15 mm and a diameter of 5 ... 8 mm. 2. Process for obtaining bioactivated pellets with microorganisms from the fungus-depleted substrate, according to claim 1, characterized in that it favors the controlled release, in the step of axenic cultivation on minimal liquid media, of orthosilicic acid in concentrations below 1 mM. . 3. Process for obtaining bioactivated pellets with microorganisms from the fungus-depleted substrate, according to claim 1, characterized in that the drying of the biomass of microorganisms and of the diatomite is done by spraying a suspension normalized to 10% dry substance, under mild conditions, at 13O ... 14O ° C input temperature and 75 ... 80 ° C exit temperature, when the cultivated microorganisms are gram-positive bacteria, which form endospores, or microscopic fungi that form conidia, and by lyophilization, by gradual growth of the temperature from -25 ° C to 25 ° C, at 0.9 mbar pressure, for 48 hours, when the microorganisms are gram-negative bacteria.