FR2602789A1 - Microbiological products for moistening organic materials and for fertilising soils and processes for their preparation - Google Patents

Abstract

The subject of the invention is microbiological products for moistening organic materials and for fertilising soils and processes for their manufacture. A starter according to the invention consists of a mixture of a first culture of cellulolytic microorganisms which have been selected from a sample of fermented organic materials, and of a second culture of nitrogen-fixing microorganisms which have been selected from a sample of nodules of leguminous plants, each culture containing a specific medium, and both cultures are attached to an absorbent support after incubation and are mixed before delivery to the users. One application is the enrichment of the microflora of soils which are naturally poor or which are artificially depleted (disinfection) and the biodegradation of organic substrates which are more or less difficult to ferment such as straw, grape marc and vegetable wastes.

Description

 Microbiological products to humidify organic matter and to fertilize soils and preparation processes.

 The present invention relates to microbiological products for humifying organic matter and for fertilizing soils and methods for preparing these products.

 The technical sector of the invention is that of biotechnologies applied in agriculture.

 The fertility or activity of a soil is linked both to microorganisms and to all the available foods.

 The main phases of the biodegradation process of organic matter are recalled below in order to transform them into humic products assimilable by plants.

 The vegetable organic materials are, in a first phase, degraded by fungi, which can develop jointly with bacteria specialized in the fixation of 1 atmospheric nitrogen (Rhizobium for example).

 They are then transformed by cellulolytic microorganisms which break down organic matter. The microorganisms which intervene in this second phase are generally Streptomycetes or Actinomycetes according to the agricultural classification.

 In a third phase, organic matter is transformed into humic compounds containing amino acids which can be assimilated by plants. To achieve this last transformation, microorganisms need nitrogen.

 There are cultivated soils where microbiological activity is weak. There are also organic substrates whose biodegradation above ground or after incorporation into a soil, is slow or does not progress until the production of humus assimilable by plants. This poses significant agronomic problems.

 An objective of the present invention is to improve and reactivate the microbiocenosis of soils or organic substrates during fermentation by making available to users a microbial preparation - or leaven - which is intended to be spread in the soil or on organic substrates, in order to activate the biodegradation of vegetable organic matter and to lead to the transformation of this into humus assimilable by plants.

 Agronomists have noted a gradual decrease in soil fertility due to the abandonment of traditional cultivation techniques comprising inputs of fermentable organic matter (manure, compost, animal waste) and also to the excessive use of purified mineral fertilizers as well as pesticides. These factors, associated with natural elements (asphyxiation or leaching for example) lead to a weakening of the microbial activity of the soil.

 The organic fertilization technique is becoming more and more difficult, both from the supply point of view and from the economic point of view.

 An objective of the present invention is to provide users with a microbial preparation which can easily be spread on the ground by spraying in the liquid state, after resuspension. It allows to recreate a very active microbism, because it is composed of strains from the natural environment and selected for their vigor, their enzymatic activity and their capacity for assimilation and transformation.

 Another objective of the invention is to provide a leaven which allows the biodegradation of fermentable plant substrates such as straw, grape marc or plant waste and thus to enhance the residues which remain on the soil after harvest (straw, waste of but, stubble) or by-products such as grape marc.

 Although the products concerned by the present invention are microbial preparations, it is specified that these contain well-known microorganisms, present in natural environments.

 The methods known to microbiologists, which allow anyone to isolate microorganisms from a natural environment and to select vigorous strains from which the methods according to the invention can be implemented, will be described below.

 The microbiological products according to the invention are microbial preparations called leaven which are composed of a nutritive support and living microorganisms intended for the biodegradation of organic substrates.

 The objectives of the invention are achieved by means of microbiological products which are characterized by the fact that they comprise a mixture containing, on the one hand, cultures of cellulolytic microorganisms selected from samples of fermented organic matter and, on the other hand, cultures of nitrogen-fixing microorganisms selected from samples of legume nodules, each culture comprising a specific nutritive medium prepared from a common nutritive medium and comprising, in addition, a pulverulent support in which said culture media are incorporated after incubation.

The methods according to the invention comprise, in known manner, the following series of operations
- obtaining strains by isolation from a sample in a natural environment;
- creation of culture environments;
- culture of the strains isolated on a culture medium;
- development of the marketable product.

The methods according to the invention are characterized by the following operations
- we cultivate separately
on the one hand, cellulolytic microorganisms, preferably of the genus Streptomycetes or Actomycetes, in a culture medium having a pH of between 6 and 7 and preferably around 6.5;
on the other hand, nitrogen-fixing microorganisms, preferably of the genus Rhizobium, in a culture medium having a pH between 7.2 and 8 and, preferably, around 7.5;
- After incubation, each culture medium is absorbed on a pulverulent support in order to stabilize said cultures and dried in a current of hot air;
- Mixing, in equal parts or not, the powdery supports containing said cultures and homogenizing the mixture;
- And we deliver to the customer a powdery product containing a mixture of the two cultures.

According to a preferred embodiment, a method according to the invention comprises the following series of operations
- A common nutrient solution is prepared in which the following trace elements are added: Cobalt, of the order of 4.2 mg.l 1. Molybdenum, of the order of 0.11 gl; Boron, of the order -1 -l of 4 mg.l 1 and optionally copper, of the order of 1.2 mg.l;
- A first specific culture medium is prepared by adding to a part of said common nutritive solution a buffer which maintains the pH thereof between 6.2 and 6.8 and, preferably, in the vicinity of 6.5;
a second specific culture medium is prepared from another part of said common solution by adding to another part of said solution a buffer which maintains the pH thereof between 7.2 and 8 and, preferably around 7.5;
- Successive cultures are produced separately, on the one hand from cellulolytic microorganisms and, on the other hand, from nitrogen-fixing microorganisms respectively in increasing volumes of said first and said second culture medium;
- Said cultures are stabilized after incubation and before delivery to users, they are mixed, the mixture is homogenized and it is absorbed on a pulverulent support.

 The invention results in new products intended for agriculture which make it possible to reconstitute the microflora of the soils and to increase the fertility of the latter.

 A more detailed description of the various steps involved in the methods according to the invention is given below, without any limiting character.

 I - OBTAINING STRAINS.

We take humus products (soil, manure, compost)
Streptomycetes or those having a good fermentative power containing Actinomycetes.

 Nitrogen-fixing microorganisms (Rhyzobium) are obtained, either from samples of humus products or, preferably, from nodules of crushed legumes.

 The samples are suspended in sterile water to protect from contamination.

 The suspensions obtained are successively diluted each time in ten times their volume of sterile water. The purpose of these successive dilutions is to eliminate the minority strains and to promote the growth of the predominant strains sought.

From each diluted solution, inoculations are carried out on nutritive media (nutritive agar in petri dish) whose pH and composition depend on the group of microorganisms which it is desired to select. Between
For Streptomycetes or Actinomycetes, the pH is maintained / 6.2 and 6.8 and, preferably, close to 6.5 and the culture medium is nitrogenous (for example by addition of ammonia nitrate at a rate of -I 0.1 gl
For nitrogen fixers, the pH is maintained between 7.2 and 8 and preferably around 7.6 and the medium is not enriched in nitrogen.

 The selective action of the media allows the development of the microbial groups sought.

After incubation for a few days, the best-developed colonies are taken from each dish. Microscopic observation allows more precise sorting of Actinomycetes strains,
Rhizobium and nitrogen fixers.

 From the most vigorous colonies selected, subcultures are carried out in separate cultures in the selective media, the compositions of which are defined below.

 After developing the strains during an incubation at room temperature, the tubes of inclined nutrient agar are kept at 4. They constitute the reserve collection from laqueile, we prepare the industrial leaven.

 II - CULTURE MEDIA.

Culture media include:
- on the one hand, a common nutritive solution comprising trace elements;
- on the other hand, a specific additive fraction from each group of microorganisms.

 1) Common nutrient solution with trace elements.

 The industrial process according to the invention comprises a first step which is the preparation of a nutritive solution common to the two cultures. This common nutrient solution comprises, on the one hand a conventional nutrient solution, for example the so-called Winogradsky solution. This solution also contains carbohydrates such as glucose and starch.

Instead of a Winogradsky solution, it is possible to use other equivalent culture media well known in microbiology, such as, for example, Young medium, Koser medium,
Gardon and Smith>Ringer's solution, the Stephenson medium, etc.

Optionally, a brewer's yeast extract is added to these culture media at a rate of 2 g / l.

According to a characteristic of the invention, the common nutritive solution also comprises several trace elements, among which in all cases
- Cobalt between 3 and 5 mg.l and preferably of the order of 4.2 mg.l, under different radicals and, preferably, in the form of sulfate;
- Molybdenum between 0.05 and 0.15 gl 1 and, preferably, of the order of 0.11 gl 1, under different radicals and, preferably, in the form of ammonium molybdate.

 - Boron between 3 and 5 mg.l 1 and, preferably, of the order of 4 mg.l 1, under different radicals and, preferably; in borate form.

As well as
- copper between 1 and 1.5 mg.l and preferably of the order of 1.2 mg.l 1, under different radicals and, preferably, in the sulphate form;
- Manganese between 2 and 4 mg.l, under different radicals and, preferably in sulfate form;
- iron between 10 and 20 mg.l 1 under different radicals and, preferably, in chelate form.

Application example
By way of illustration, the composition of a preferred nutritive solution common to the two culture media is given below.

This aqueous solution contains: * A solution A composed of conventional components, the quantities of which are given in gl 1 of solution
- calcium sulfate = 0.13 to 0.25.

 - magnesium sulfate = 0.13 to 0.25.

 - monopotassium phosphate = 0.25 to 0.5.

- starch = 2.5 to 5
- ammonium molybdate = 0.2 to 0.3.

 - glucose = 1.5 to 3.

 - yeast = 1 to 2.

* A solution B of trace elements which contains the following mineral components
- copper sulphate = 5 mg.l.

- sodium octaborate = 20 mg.l
- manganese sulfate = 10 mg.l
- cobalt sulfate = 20 mg.l
- ammonium molybdate = 0.2 gl
- EDTA sodium iron salt = 0.1 g
The nutrient solution also contains humic compounds (from 6 to 45 ml of a 10% solution) obtained from manure or compost, for example. To this end, the product is boiled and the solution which contains the humic acids and their derivatives is then recovered.

 2) Obtaining specific nutrient media.

From the common nutrient solution defined above, two different nutrient media are prepared
a) First environment.

It is intended for the culture of microorganisms of the actinomycete group. I1 contains
- the common solution;
- complex buffer which maintains the pH between 6.2 and 6.8 preferably, 6.5, which was made by ailleutsl'bjet du patent FR.

No. 76 / 23.114;
- ammonia phosphate of the order of 2.5 gl
- a 10% aqueous solution of grape seed cake = 13 ml.l 1 *
By way of nonlimiting example, a culture medium for actinomycetes contains, for one liter
- 970 ml of solution A;
- 10 ml of solution B;
- 20 ml of extracts of humic acids and of grape seed cake;
- 2.5 gl 1 of ammonia phosphate;
- a complex buffer in sufficient quantities to maintain the pH between 6.2 and 6.8.

 b) Second environment.

It is intended for the cultivation of microorganisms of the rhizobium group. I1 contains
- the common solution;
- a complex buffer in sufficient quantities to maintain the pH between 7.2 and 8, preferably around 7.5;
- bipotassium phosphate = 0.2 gl
- potassium sulphate = 1 gl
By way of nonlimiting example, a culture medium for rhizobium contains per liter
- 945 ml of solution A;
- 10 ml of solution B;
- 45 ml of extracts of humic acids;
- 0.2 g of bipotassium phosphate;
- 1 gl 1 of potassium sulphate
III - PREPARATION OF THE PRODUCT.

Product preparation includes the following five distinct phases
- culture in test tubes on agar medium;
- culture in boxes of roux in liquid nutritive medium;
- industrial culture in liquid nutrient medium and put on a powder support;
- additional fraction;
- quality control.

 1) Culture in test tubes on agar medium.

 From the strains of the collection, subcultures are carried out on inclined tubes in separate cultures which; after incubation for 10 days between 10 and 200 and suspension, will be used to inoculate the liquid medium of the boxes of roux.

 2) Culture in boxes of roux in liquid nutritive medium.

 The sterile contents of the boxes of roux are then inoculated, which contains about 100 ml of specific nutritive liquid medium.

It is incubated for 8 to 10 days at an appropriate temperature.

 3) Industrial culture in liquid nutrient medium and put on a powder support.

 This operation is carried out using pasteurized nutritional medium which is inoculated massively by means of the contents of the boxes of roux. Development into separate cultures by incubation for five days. The tanks are dried under a stream of hot air after incorporating a powder support, for example kaolinite.

The purpose of this fixing on a powder support is to stabilize the product (the microorganisms are thus kept in latency and checks over time have revealed that after a period of five months, the microbial count was still close to germs per gram.

 4) Additional fraction.

During the grinding thereof, an additional dehydrated nutritive fraction is incorporated into the pulverulent support which is intended to
- protect the microbial flora when the product is suspended;
- stimulate the biological activity of the strains present, in order to reduce the latency phases.

This nutrient fraction can only be mixed at the time of use. This mainly organic fraction consists in particular of vitamins and growth factors, incorporated in a support based on carbohydrate and vegetable hydrolyzate accompanied by a limited amount of complex buffer to control the hazards due to the setting water. suspension and any risks that may arise
- unfavorable influence of pH;
- bacteriostatic or bactericidal effect with respect to the selected active flora carried by the product thus produced.

 After homogenization of the mixture, the product is packaged in an airtight package for delivery. For use, the powder thus obtained is suspended in water and is spread over the soil or over the organic mass to be reactivated.

 5) Quality control.

During all of the phases mentioned, aliquots are regularly sampled for quality control of the batches produced. The latter is carried out by means
- enzymatic tests: phosphatase and dehydrogenase in particular, as well as, optionally, cellulase, pectinase, amylase;
- the microbial count of the different cultivated groups.

 This makes it possible to obtain quantified values representative of the importance and the activity of the flora present, as well as a reflection of its enzymatic capacities.

 All the checks carried out regularly reveal populations of the order of 5.108 germs per gram as well as enzymatic activities of the order of 1500 CU for phosphatase and 4500 CU for dehydrogenase, with stability over time during a prolonged storage (1 CU = 1 conventional unit of enzymatic measurement).

 IV - TESTS.

 The tests were carried out by adding 5 ml of leaven according to the invention in tanks each containing 10 kg of organic substrate, the substrates chosen being straw, vegetable compost, manure in the course of fermentation and peat.

 The tests were carried out outside in hot weather and the humidity of the tanks was regularly maintained.

The samples taken periodically every month after the 60 days of incubation, were subjected to the following biochemical analyzes
- determination of ammonia, nitrites, nitrates, dry matter, organic matter and humus;
- determination of amylase, asparaginase, urease, nitrite reductase, catalase, dehydrogenase, alkaline phosphatase and sulfatase.

These chemical and enzymatic analyzes were carried out by analytical methods well known to biochemists (CF. BRISOU "Techniques of bacterial enzymologies" published by
MASSON). The measurements on the seeded tanks were compared with measurements on unseeded control tanks.

 Tests have shown that non-weeded straw, which is a fermentable material, reacts very well to sowing with sourdough. Its influence leads to faster mineralization and a greater release of nitrogen compared to the control, with better regulation, of its cycle.

 The enzymatic activities: amylase, urease, nitrate reductase, phosphatase and sulfatase are much greater on the seeded sample than on the control.

 With regard to the tests on vegetable compost (at the start of fermentation), we could expect a weak reaction to a new sowing with a leaven according to the invention. Analyzes reveal that the leaven did not have a significant influence on the nitrogen cycle. On the other hand, the enzymatic activities of phosphatase and sulfatase are greater in the treated sample than in the control.

 For peat, an organic material which is not very fermentable in its natural state, tests have shown that a strong release of ammonia is quickly obtained, followed by stabilization. The release of nitrates is greater than that observed on the control.

 The enzymatic activities of urease, nitrate reductase and phosphatase are significantly higher on the seeded sample than on the control.

 Manure, an organic material that is very easily and very quickly fermentable, used for the tests, was in the process of evolution.

 The tests show a significant mineralization of nitrogen at the start, then a slightly greater release compared to the control. The phenomenon is more significant on the seeded manure than on the control.

 The enzymatic activities of urease, phosphatase, sulfatase and nitrate reductase are improved.

 The tests carried out have shown that by using only 5 ml of liquid leaven according to the invention per 10 kg of substrate, a significant improvement in nitrogen mineralization and certain enzymatic activities is quickly obtained, even in the case of substrates. slowly fermentable like straw or peat. On manure, which is however easily fermentable, an improvement in certain enzymatic activities is obtained. On a plant compost seeded during industrial manufacture, only the activities of sulfatase and phosphatase are improved.

 The tests compared on seeded samples and on manure and compost controls containing a large natural microflora have shown that the addition of leaven according to the invention enhances the action of natural microflora and improves the release of elements and regularity of the mineralization cycles while ensuring a notable increase in the enzyme capital of fermentative origin

Claims (9)

 1. Microbiological product intended to humect organic matter and to fertilize soils of the type comprising living micro-organisms and a culture medium, characterized in that  cellulolytics which it comprises a mixture, on the one hand, of cultures of microorganisms \ and, on the other hand, of cultures of nitrogen fixing microorganisms, each culture comprising a specific culture medium prepared from a common nutrient medium and it further comprises a pulverulent support in which said culture media are absorbed after incubation, which has the effect of stabilizing said products.  2. Microbiological product according to claim 1, characterized in that it is composed of a mixture substantially in equal parts, on the one hand, of cultures of microorganisms of the genus ActinomycetesPtiStrptDmycètese, on the other hand, of cultures of microorganisms of the genus Rhizobium.  3. Microbiological product according to any one of claims 1 and 2, characterized in that said common nutritive medium mainly contains the following trace elements  - Cobalt: between 3 and 5-mg.l -1 and, preferably, of the order of 4.2 mg.l;  - Molybdenum between 0.05 and 0.15 g.l and, preferably, of the order of 0.11 g.l;  -1  - Boron: between 3 and 5 mg.l and, preferably, around  1-1 of 4 mg.  4. Microbiological product according to claim 3, characterized in that said common nutritive medium contains, in -l and, -1 addition, copper between 1 and 1.5 mg.l 1 and, preferably, 1.2 mg. l 1.  5. Product according to any one of claims 1 to 4, characterized in that said common nutritive medium contains, in addition, extracts of humic compounds.  6. - Process for the preparation of a microbiological product intended to moisten organic matter and fertilize the soil, characterized by the following operations  - we cultivate separately:  on the one hand, cellulolytic microorganisms,  or Streptomycetes preferably of the genus Actinomycetes in a culture medium having a pH of between 6 and 7 and preferably. close to 6.5;  on the other hand, nitrogen-fixing microorganisms, preferably of the genus Rhizobium, in a culture medium having a pH of between 7.2 and 8 and, preferably close to 7.5;  - After successive incubations, each culture medium is absorbed on a pulverulent support in order to stabilize said cultures and dried in a current of hot air;  - Mixing in equal parts or not, the powdery supports containing said cultures and homogenizing the mixture;  - And we deliver to the customer a powdery product containing a mixture of the two cultures.  7. Method according to claim 6, characterized by the following series of operations  - a common nutritive solution is prepared in which the following trace elements are incorporated, in particular  Cobalt: between 3 and 5 mg.l 1 and, preferably,  mg.l on the order of 4.2 mg.l;  .Molybdenum: between 0.05 and 0.15 g.l 1 and, preferably, -1 of the order of Q, 11 g.l  -1  Boron: between 3 and 5 mg.l and, preferably, of the order of 4 mg.l;  - Preparing a first specific culture medium by adding to a portion of said common nutrient solution, a buffer which maintains the pH thereof between 6.2 and 6.8 and, preferably, around 6.5;  - A second specific culture medium is prepared by adding to another part of said common nutritive solution a buffer which maintains the pH thereof between 7.2 and 8 and, preferably, of the order of 7.6;  - Is carried out separately, in increasing volumes of the first and second culture medium, respective cultures of cellulolytic microorganisms and nitrogen fixing microorganisms;  - After incubation, each of the cultures is fixed by making each culture medium absorb separately by a pulverulent support, for example by ground kaolin and dried in a current of hot air, so that said cultures are stabilized and can undergo long-term storage;  - and before delivery to the customer, the pulverulent products containing the two cultures are mixed together and the mixture is homogenized.  8. Method according to claim 7, characterized in that said separate cultures comprise the following series of operations  - strains selected from a collection of strains grown on inclined tubes are taken;  - Said strains are put in aqueous suspension;  - a sterile nutritive medium is inoculated in a roux dish with samples of said aqueous suspensions and incubated between 10 and 200 C;  - an industrial culture is carried out on pasteurized liquid culture media;  - After incubation, each culture medium is made to absorb separately by a pulverulent support and dried in a current of hot air.  9. Method according to any one of claims 6 to 8, characterized in that one incorporates in the pulverulent support a dehydrated nutritive fraction which is composed of vitamins and growth factors incorporated in a support based on hydrates of carbon and vegetable hydrolyzate.