WO2003046156A2 - Bacterial biomasses, protocol for obtaining same, and uses thereof for bacterization of soils and crop residues - Google Patents

Abstract

The invention concerns a method for obtaining in vitro bacterial biomasses of edaphic origin comprising steps which consist in: contacting an aqueous soil suspension with a matrix substrate, so as to form a hydrated zone, or biofilm, at the surface of the substrate, similar to the one adjacent to the clay-humus complex of the soil, essentially containing the internal bacterial constituent of the soil, so as to retain at the surface of the matrix substrate, the essential part of the bacterial flora endogenous to the original edaphic media; maturing the biofilm providing oligotrophic conditions enabling the cells which constitute most of the edaphic biofilm which have colonized the matrix substrate to migrate to the supernatant liquid phase of the matrix substrate, and gradually to prevail over it; recovering and culturing the bacterial strains most prolific in rich liquid culture media, with internal character, rustic and persistent in situ, capable of being cultured in liquid environment, hence capable of industrial production. The resulting bacterial biomasses and strains are useful for microbiological bacterization of soils and crop residues, in particular cereal crop residues, including those of corn.

Description

 New bacterial biomasses, their production protocol, and their use for the bacterization of soils and crop residues

The invention relates to new bacterial biomasses, their production protocol, and their use for the bacterization of soils and crop residues.

Bacterization of arable soils consists of supplying (micro) biological means to soils and agronomic crops, part of the nutrients, in particular diatomic nitrogen from the atmosphere, necessary for obtaining economic and sustainable yields.

Traditionally, bacteria have mainly been synonymous with the use of inocula of Rhizobhi.ni strains for the coating of seeds of Leguminosea species such as soybeans, alfalfa and red clover.

It has also been proposed to use endomycorrhizae, endophytic diazotrophic acetobacter, rhizobacteria PGPB (Plant Growth Promoting Bacteria) microalgae and cyanobacteria, or non-symbiotic diazotrophic bacteria in association with crop residues.

The use of bacteria in agronomy, and more particularly in cereal production, came up against the problem of their non-persistence, once reintroduced into the soil. Therefore, they cannot ensure the achievement of the desired agronomic effect, namely the increase and the stability of the grain yields, their protein contents, and / or the reduction of the levels of inputs, in particular the mineral nitrogen.

It follows that the bacterial biomasses proposed to date do not allow a satisfactory exploitation of the compounds resulting from the in situ degradation (ie in the field) of the residues of agronomic crops, in particular the residues of intensive cereal cultivation, including that of grain corn.

The soluble and assimilable compounds resulting from the in situ degradation of cereal crop residues are poorly valued by all of the soil microflora, and more particularly its diazotrophic and non-symbiotic component called azotobacterial. This lack of valuation largely explains the realization of three phenomena, sometimes harmful, well known ; (1) the continual presence of agronomic quantities of straw residues on the soil surface at the time of the agricultural work, (2) immobilization of nitrogen by non-diazotrophic microorganisms of the soil causing a "nitrogen hunger" in the culture in place, and, paradoxically, (3) an appreciable drop in carbon contents (soluble, labile and total) and microbiotic biomass.

Another problem encountered with bacteria is linked to their isolation and their mass production traditionally carried out by serial dilution of a soil suspension and the cultivation of an aliquot of the same dilution of this aliquot on an agar medium. rich. The strains thus isolated, once sufficiently characterized, are then used to seed conventional bioindustrial fermenters. The capacity of the inocula thus produced to durably increase the in situ activity of the organisms concerned, diazotrophy in arable soil amended with crop residues for example, is therefore very weak and / or uncertain today. The use of various porous supports, solid or organic, in order to protect these bacteria once re (introduced) in the grounds, does not seem to have been able to allow a greater effectiveness of this type of inoculation.

The study of these problems by the inventors led them to develop conditions for the isolation of bacteria from the soil using protocols and isolation media, or retention, in vitro, capable of ensuring the maintenance and survival of the bacterial strains most capable of active persistence in edaphic environments, and satisfactory bacterification of soils once (reintroduced, thanks to their properties of persistence in situ.

Historically, there are three approaches to obtaining bacterial strains of edaphic origin (that is to say from arable soils) in principle best suited to active persistence in situ once reintroduced: a. isolation on abiotic matrices: with the use of media with edaphic characters (ie silica gels with or without earth extracts), without however integrating the main components (silicic, humic, carbonaceous, and hydrological) of these environments; b. pre-incubation on abiotic supports: this is a pre-incubation in an edaphic medium, or within a more or less fine fraction thereof, of bacterial cells already isolated and conventionally cultivated; vs. culture in impoverished media: a voluntary depletion, in particular in carbon, of culture media of bacterial cells isolated conventionally is carried out.

The solution provided by the invention integrates these 3 approaches, and more particularly approach a), within the obtaining protocol defined below, which leads to the obtaining of the constituent bacterial strains and biomasses, in terms of adaptation, persistence and activity in situ, and this beyond a simple a posteriori conditioning of bacteria already isolated and / or cultivated according to approaches a), b) and / or c) di1 es more conventional.

In addition, the invention takes into account the process of formation of biofilms in an aqueous medium. In this regard, the inventors have considered that the most significant edaphic zone for soil bacteria corresponds to the hydrated zone (biofilm) adjacent to the clay-humic complexes (CAH) of the soil, and that the formation and functioning of this- this is probably subject to the same mechanisms as those involved in the formation and functioning of biofilms in the broad sense.

According to Characklis (1990) and Characklis and Marshall (1990) the accumulation of a biofilm on the surface of a matrix, biotic or abiotic, is the result of eight distinct physicochemical and biological processes:

1. Conditioning of the matrix

2. Transport of bacterial cells (plankton) to the matrix

3. Adsorption of a portion of these cells

4. Desorption of a portion of these cells

5. Irreversible adsorption of complementary cells

6. Growth of the bacterial cells constituting the biofilm at the expense of the supernatant aqueous phase

7. Attachment of cells and particles in suspension in the aqueous phase

8. Release of cells constituting the biofilm by volumetric displacement, erosion, wear, etc. The realization of these processes allows in principle the formation of a biofilm with two phases (solid and liquid), and more precisely with five compartments:

1. A matrix substrate (silica gels or agar for example)

2. The basic biofilm containing most of the bacterial cells

3. The surface biofilm essentially containing complementary cells

4. The supernatant liquid phase

5. The ultimate aqueous phase

However, it turns out that the biofilm as such is mainly constituted by compartments 2, 3 and 4.

According to the most recognized edaphic model (Davies et al. 1998; Gordienko 1990; Pochon and Tchan 1948; Pochon and De Barjac 1958), the bacterial microflora is also essentially made up of three components, the most rustic and oligotrophic native component. , the rather copiotrophic zymogenic component associated with this native component, and finally the very marginal but easily isolatable component, essentially zymogenic and tιy, s copiotrophic.

The invention is based on an original conceptual comparison between the descriptions of biofilms in the broad sense and the hydrated zone added to the soil CAHs.

It therefore aims to provide a process for obtaining bacterial biomasses essentially formed from persistent and active strains, once reintroduced into an original target soil, characterized by the integration of APC (agro-pedoclimatic) characteristics, in particular by interacting with the CAHs and ISL (solignocellulosic interface) of an identifiable edaphic zone.

It also targets bacterial biomasses and new strains of these biomasses having the metabolophysiological characteristics commonly sought, such as diazotrophy, fungistatic and / or phytosanitary activity, the production and secretion of growth regulators, the catabolism of xenobiotics, etc.).

The invention further relates to the use of these bacterial biomasses and strains for the bacterization of soils and crop residues, in particular cereal crops. The process for obtaining in vitro bacterial biomass of edaphic origin is characterized in that it comprises the steps of:

- bringing an aqueous soil suspension into contact with a matrix substrate, so as to form a hydrated zone, or biofilm, on the surface of the substrate, similar to that attached to the soil HAC, essentially sheltering the bacterial component native to the soil, in order to retain, on the surface of the matrix substrate, most of the bacterial microflora endogenous to the original edaphic environments.

- maturation of the biofilm with establishment of oligotrophic conditions allowing the most constitutive cells of the edaphic biofilm which have colonized the matrix substrate, to migrate towards the liquid phase supernatant said substrate, and gradually to dominate it,

- recovery and culture of the most prolific bacterial strains in rich liquid culture media, of an indigenous character, rustic and persistent in situ, cultivable in liquid media and therefore suitable for industrial production

In this process, an inorganic and abiotic matrix substrate is used, in an aqueous medium, promoting the production of exopolysaccharides (EPS) and. other components of the bacterial glycocalyx, as well as the organization in a network of a sufficient number of cells capable of initiating the first steps of formation of a biofilm on the surface of the substrate.

Materials suitable as substrates include silica gel, polystyrene, clay, porous glass or even vermiculite.

The water in the aqueous suspension contains compounds present in the relative proportions characteristic of the original target soil, namely humic and fulvic substances, carbonaceous compounds and in particular mono-, oligo- and polysaccharides, of plant origin, animal, and bacteriofungal, nutrients.

The maturation of the biofilm is carried out by alternating a phase where the biofilm formed on the abiotic surface is not in the presence of a supernatant liquid phase, with a liquid phase comprising all of the bacterial cells supernatant the solid phase. This maturation results in principle from the migration of native bacteria to the liquid phase of the biofilm formed on the matrix substrate. The volumetric displacement takes place only during and after aging (maturation) of the biofilm (that is to say of the biofilm on the surface of the matrix substrate). This maturation allows the release, in the aqueous phase supernatant of the matrix substrate, of the most constitutive bacterial cells of this biofilm.

The maturation of the biofilm can be facilitated / increased if the supernatant liquid phase is periodically dried, or at least removed, so as to promote the growth of the bacterial biomasses constituting the biofilm, which are for the most part aerobic. It is then restored by adding aseptic water. This addition can be carried out for example by microfiltration.

The invention thus makes it possible to reconcile the microbiological diversity (especially bacterial) of the soils with the microbiological purity of the biomass to be produced in conventional bioprocesses, which has never really been attempted until now. The isolation of bacterial strains is in fact mainly carried out using relatively rich culture media and according to metabolic characteristics (eg resistance to certain antibiotics, in vitro diazotrophy, specific activities of certain enzymes, etc.) and / or genomics (eg using oligo-nucleotide probes specific to certain operons of agronomic or industrial interest) easily identifiable, but which have basically little or nothing to do with the adaptation and active persistence of these cells native bacteria rather than zymogens once (re) introduced into various edaphic environments.

The protocol for obtaining bacterial biomasses of the invention thus promotes the microbiological dominance of bacterial strains with edaphic characters, and nevertheless ensures that these also remain sufficiently copiotrophic (i. Capable of growing in rich media) to be produced in industrial quantities by conventional bioprocesses.

On the contrary, bacteria obtained according to processes which do not integrate the foregoing provisions within the same protocol are ineffective. This is so: o bacterial biomasses 1) obtained from biotic surfaces but without the presence of a biofilm or a humic complement, and / or

»Bacterial biomass 2) obtained from abiotic surfaces according to the protocol in question, but a) with a humic complement not modeled on that of the target soil and / or b) with a carbonaceous substrate provided in an amount greater than the concentrations normally present in the soil,. and / or • bacterial biomass 3) obtained from unconditioned abiotic surfaces (ie without the development of a hydrated film or biofilm).

To illustrate this demonstration, a series of control inocula not integrating at least one of the concepts envisaged above was produced, namely notion 1: adhesion to inorganic and abiotic surfaces; concept 2: oligotrophic growth from carbohydrate and humic supplements; notion 3: expression of benthic and planktonic characters, the direct comparison of the effects obtained with the biomasses of the invention and with these witnesses makes it possible to interfere that the integration of the 3 notions (1,2,3) mentioned above is possible to obtain the desired results and constitute a means of identifying the biomasses of the invention as set out in the examples.

According to a preferred embodiment of the process of the invention for the progressive isolation of rustic and persistent bacterial biomasses in situ, the following steps are advantageously used:

a) - immediate and temporary retention of most of the endogenous bacterial microflora in the original soil media:

In order to make this retention step less drastic in terms of elimination and loss of the less copiotrophic strains, a matrix substrate is pre-incubated with the soil suspension containing the bacteria.

The matrix substrate is inorganic and abiotic, such as silica gel, clay, polystyrene, porous glass, vermiculite. The surface of the substrate is conditioned by bringing it into contact with a retention medium essentially modeled on the characteristics of the original target soil. These characteristics include, but are not limited to: (1) the contents of humic and fulvic substances and their constitutions in terms of the various phenolic and benzoic compounds, (2) the contents of carbonaceous compounds, and in particular the mono-, oligo- and polysaccharides of vegetable, animal and / or bacteriofungal origin, (3) the particle size of the mineral part of the soil, and especially the finest, or at least excluding aggregates greater than 5 mm in diameter, (4) the immediate nutrient contents (nitrogen, phosphorus, sulfur, etc.) capable of influencing bacterial development in the short term.

This pre-incubation in the presence of water of the retention media allows the satisfactory formation of a hydrated film or biofilm on the surface of solid matrix substrates.

The bacterial strains, which gradually come to dominate on the surface of these substrates and within these biofilms, are thus more specifically adapted to APCs on which the constitution of these biofilms has been modeled and are therefore also more capable of actively persisting once reintroduced into these APCs.

b) - progressive dominance of the desired bacterial strains, diazotrophs and / or fungistats for example, over these specific retention media and characteristics of the original edaphic media:

The culture or retention media become de facto the source of any candidate bacterial biomasses. Bacterial cells being good producers of exopolysaccharides (EPS) used for the formation of biofilms on the surface of the substrate, they can also be brought to dominate the liquid phase restored to its surface. Indeed, the biofilms thus formed can in this sense mature. It is during this maturation process that the multiplication of biofilm cells makes it possible to gradually move the bacterial cells towards the surface of the biofilm, by a process called volumetric displacement (Characklis 90 and Characklis and Marshall 90 above). These cells are therefore eventually found free in the liquid phase supernatant on the surface of the biofilm. Insofar as this liquid phase and the biofilm substrate are almost devoid of assimilable mineral nitrogen, the cells concerned will be mostly azotobacterial.

c) - isolation, by micro-biological dominance, of the most prolific bacterial strains sought in rich and more conventional liquid culture media: The biomasses and strains of the invention, although relatively more oligotrophic than more conventional strains, must nevertheless remain cultivable in conventional rich environments, such as they are used during industrial bio-processes. However, and since the aliquots coming from the liquid phases supernatant the media for obtaining these strains are not necessarily of absolute microbiological purity, there is a risk that the desired candidate strains which they contain are displaced (ie dominated in number) by more copiotrophic contaminations and contrary to the desired goal. To avoid as much as possible such contamination, the culture medium is restored with an aliquot containing approximately 90% of cells originating from the phase supernatant the obtaining media and only approximately 10% of cells actually pre-incubated in order to promote, possibly the dominance of rather oligotrophic cells. Following the third, even fourth, pre-incubation cycle, an aliquot of approximately 1 per 1000 of this latter preculture will be transferred to a conventional rich liquid medium with nitrogen, the cells which contain this aliquot can then be produced in mass and conventionally.

It is recommended to carry out a monitoring and control of the purity of these biomasses and strains in addition to a series of non-functional genomic profiles of the RAPD type, and more functional according to the particular expression of certain most important mechanisms in terms of edaphic adequacy (eg. alg, moult, eps, pob, rhl, etc.).

The candidate prototype inocula are formulated and packaged for use in the field.

Liquid and solid formulations not only ensure a satisfactory conservation period of a minimum of 6 months, ie. 4 ° C, i.e. at room temperature (i.e. from 18 to 27 ° C), but also to further ensure pre-adaptation of the bacterial cells already selected according to the present invention.

The invention also relates to isolated bacterial biomasses, characterized in that they comprise, as dominant strains, bacterial GRAM negative strains, of edaphic character, indigenous, oligotrophic, mainly aerobic, having metabolo-physiological characteristics of diazotrophy, fungistatic and / or phytosanitary activity.

Advantageously, these bacterial biomasses are capable of growing easily in liquid media, which makes it possible to produce them in industrial quantities by conventional bioprocesses.

As illustrated by the examples given below, these biomasses are characterized by the following phenotypes expressed in vitro and / or in situ: relative to isolated controls according to all or part of the conventional protocols 1), 2) and 3) recalled above, they are more mucoid than these controls, more encysted and more durably, less copiotrophic than said controls, and produce self-induction factors ( FAI) at higher rates than controls, for example octanoyl homoserine lactone.

Compared to said controls, they are capable of maintaining the mineral nitrogen content of soils reconstituted in microcosms according to a particle size fraction> 3mm, over a period of more than 28 days of incubation.

Preferred bacterial biomasses of the invention are Azobacteriaceae, Pseudomonaceae, or Rhizobiaceae.

The genomic and molecular characterization of the isolated strains shows that they contain and express a genomic fragment analogous to characteristic fragments of algD and algU responsible for the production of EPS, as well as characteristic fragments of pobA and pobR responsible for the production of PHBH. , found in many Azoto bacteriaceae, Pseudomonaceae and Rhizobiaceae.

The bacterial biomasses obtained are particularly suitable for the microbiological bacterization of soils and crop residues, in particular residues of cereal crops, including those of grain corn.

Thanks to the persistence of their activity in situ once (re) introduced into the soil, during the period of cultivation of the agronomic crops concerned, they can use the residues in the process of decomposition as energy and structural substrates.

The invention therefore relates to their use for such bacteria by introducing them in situ, near said residues, so as to exploit the soluble carbon released as a result of their degradation.

To this end, inoculas prepared from bacterial biomasses and isolated strains are advantageously sprayed on the crop residues, especially when they are buried in the soil. Inoculation rates of 100,000 to 1 million viable cells / g of residue, or 1 g / tonne of residue are used, for example. At the edapho-agronomic level, there is observed in situ or in microcosms, greater persistence in situ and over time of the activity of nitrogenase, in the case of azotabacterial biomasses capable of diazotrophy, an increase in the accumulation of l mineral nitrogen, in particular in the ammoniacal form, an increase in plant biomass, of the ratio between the vegetative and root parts, an initial delay in the degradation of the crop residues attributable to the fungistic activity of the (re) introduced bacteria.

Other characteristics and advantages of the invention are given in the examples which follow, with reference to FIGS. 1 to 8, which represent, respectively:

- Figures 1 A and 1B, the PCR bands produced by AlgU V2 primers according to the type of matrices used;

- Figure 2, the bands produced by RAPD, according to a series of six primers (nlO) characteristic of candidate strains of the Azotobacteraceae type, compared to a strain ά'A. witness chroococcum 76,112,

- Figure 3, the measured mineral nitrogen contents, on microcosms and microparcels inoculated with various controls and candidate strains, -, _

FIG. 4, the ratio, as a function of the incubation time between the acetylene reduction activity in the presence of culture residues inoculated according to the invention or with controls, and not inoculated,

- Figure 5. the mineral nitrogen contents of 2 series of microcosms treated according to the invention or by controls, with addition of para-humic compounds;

FIG. 6, the effect of an antibiotic on the treatment of culture residues and by controls,

- Figures 7A and 7B, the decomposition of crop residues as a function of the incubation time (laboratory and in the field);

FIGS. 8A and 8B, the effect of the particle size of soils reconstituted in microcosms on the properties of bacteria, and

- Figures 9A to 9E, the effects of biomass of the invention compared to controls. Example 1: Isolation of candidate strains of the Azotobacteraceae type.

. soils used for sampling

The results obtained are reported on 2 sites (Terrefort and Groies) whose APC characters are as follows:

Table la: Physico-chemical analyzes of soils F4 and 19 (“Terrefort”) on the experimental area of Inra-Toulouse

Parcel

F4 19

Granulometry Clay 230 260 Fine silt 242 227 Coarse silt 139 165 Fine sand 167 207 Coarse cable 222 141

Organic carbon 8.5 9.2

Organic matter 14.6 15.8

Organic nitrogen 1.07 1.13 pH water 5.9 6.6

CaCO 0 0

P ₂ O ₅ 0.11 0.06

Exchangeable 1.91 2.75

My exchangeable 0.014 0.015

Exchangeable mg 0.173 0.187

K exchangeable 0.183 0.134

Humidity at 105 C 24 28

Table lb: Edaphic characteristics of Groies soils near Poitiers (86)

LANDS ... Dry o Mainly north of the department

CHARACTERISTICS o Brown color Loamy clay o Substrate: limestone

PROPERTIES o Limestone, shallow, stony soil (limestone)

• Healthy soil

»Low water reserve (irrigation opportunity) o Good to medium fertility

ALTERNATIVE: Large groie or clay-limestone o Deep soil, more clayey c good water reserve

. isolation protocol

A soil / water suspension is brought into contact with an agar or silica gel matrix substrate. The biofilm which is formed in contact with the matrix substrate meets the characteristics of the original target soil, with regard in particular to (1) the contents of humic and fulvic substances and their constitution in terms of the various phenolic and benzoic compounds, (2) the contents of carbon compounds, and in particular the mono-, oligo- and polysaccharides of vegetable, animal and / or bacteriofungal origin, (3) the particle size of the mineral part of the soil, and especially the finest, or at least the exclusion of aggregates greater than 5 mm in diameter, (4) the immediate nutrient contents (nitrogen, phosphorus, sulfur, etc.) capable of influencing bacterial development in the short term.

To obtain the maturation of the biofilm, the liquid phase is periodically restored, namely for example once a week, in order to avoid drying of the biofilm and to wait long enough between two restorations (eg around seven days), so as to give time to the most constitutive azotobacterial cells of the biofilm thus formed of migrate by volumetric displacement towards the surface of the biofilm. In order to avoid the dominance of copiotrophic azotobacterial cells at the expense of the desired azotobacterial cells, which are rather oligo- or meso-trophes, more native and persistent once (reintroduced, but also therefore easily dominated in rich in vitro environments), it is necessary to avoid to load the gels and the liquid phases which float them with carbon substrates, in particular simple sugars such as glucose, and / or mineral nutrients. Preferably aseptic distilled water is used (in particular by micro-filtration You should also avoid excessively breaking or disturbing the biofilm when restoring by using a too sudden or directed jet of water when restoring the liquid phase. In general, it is sufficient to use 1 ml of water on gels contained in 10 mm diameter petri dishes and 5 ml for 33 mm diameter dishes After approximately seven days of incubation 22-27 ° C, the final restoration, generally the third, aliquots of these liquid phases used to inoculate a series of conventional liquid culture media.

For the isolation of the candidate strains sought, the procedure is advantageously as follows. an aliquot of 1 ml is placed in 1000 ml of liquid glucose medium (with 4th Winogradsky salts including those coming from a soil extract at a level of 10% w / v) without mineral nitrogen and / or assimilable, and left pre- incubate 1 to 2 days at 22-27 ° C. This transfer is carried out from the culture thus obtained another two to three times, but using only 100 micro-liters (1/10 of ml) of this culture of "Rank I" and 9/10 of ml of a new aliquot coming directly from the liquid phase supernatant the original obtaining medium. We reduce the probability, without eliminating it, of seeing a candidate biomass rather less oligotrophic gaining the upper hand, in terms of number of cells per ml of culture medium, over the candidate biomass even more oligotrophic, persistent and active in situ .

- identity of organizations

The bacteria were isolated on selective media devoid of mineral and / or organic nitrogen thus ensuring the dominance of these diazotrophic organisms at the expense of other heterotrophic organisms in the soil. The organisms thus retained are Gram negative, aerobic, diazotrophic and in the form of rods of approximately 1 to 3 microns.

We know that A. chroococcum is recognized as being the most adapted to living conditions édaphique. This strain was therefore retained as a reference for the strains of the invention. Indeed, the macro- and microscopic observations of the strains of the invention are in agreement with the descriptions of Azotobacter chraococcum Beiferinckii 1901 of ATCC, DMS and the Pasteur Institute. A genomic and molecular characterization of these strains has therefore been established using mechanisms known in Azotobacter.

- biological properties of organisms

The bacteria of the Azotobacieracea type obtained with the technology of the invention are capable of fixing diatomic nitrogen and of using carbonaceous substrates originating from the decomposition of crop residues in the field, and in particular those originating from the decomposition of straws from cereals and corn. In principle, certain azotobacterial ecotypes are also capable of establishing rhizospheric associations and thus promoting the growth of the plant, but are not however virulent. They are not either, and unlike some Acetobacter, endophytes.

Azotobacteraceae bacteria are generally found in soils, they are Gram negative, and have a conventional and heterotrophic mode of growth and development. Under conditions of water or other stress, they can become encysted using exopolysacchanide (EPS) capsules and more particularly alginate. In this sense, this production of PS is essential for the survival and the hardiness in edaphic medium of these bacteria; the more or less fine characterization of this mechanism will therefore serve to identify these organisms as described below.

These bacteria are responsible for the fixation of diatomic nitrogen in soils by a non-symbiotic route. They are aerobic, unlike Clostridium, and are generally recognized as being unable, depending on the circumstances, to fix by diazotrophy only a few kg, even a few tens of kg of nitrogen per hectare per year. However, these biological fixation rates are above all limited by the (non) availability of carbon substrates following the (non) persistence of diazotrophic strains (re) introduced into particular soils.

It will be noted that, in accordance with the invention, these overall rates per hectare can be at least tripled, or even quintupled. It has indeed been demonstrated using experimental data obtained in the laboratory, in the greenhouse, in a culture chamber and in experimental plots in the field that the invention effectively allows greater relative efficiency of the isolated azotobacterial strains, once these (re) introduced into their agro-pedo-climate ( APC) of origin.

- methods of analysis

. establishment of the identity of organizations

The identity of the organizations was established based on their adaptation to the CPAs. The results reported below relate to the 2 APCs from which the strains were isolated, and into which they will be re-introduced in the form of bacterial inocula: one in the Toulouse region (31) (Terrefort or "TF31", see Table La) and the other in the Poitiers region (86) (Groies or "GR86", see Table lb).

For the purpose of characterization, oligonucleotides were used capable of initiating the polymerization of conserved segments of a gene recognized as essential for the synthesis of exo-polysaccharides (EPS), in particular alginate, necessary for the survival of bacteria in the grounds.

. Oligonucleotides used for the functional characterization of AlgU and AlgD segments of candidate Azotobacteraceae strains.

The two regions considered to be the most conserved of the genomic segments aUU and algD have already been chosen as belonging to the genera Pseudomonas and Azoiobacter (information on Genebank concerning the segment AVU11240. AlgD Azobacter vinelandii ATCC 9046 Campos et al. Journal J. Bacteriol. 178 (7), 1793-1799 (1996)). Seven sets of oligonucleotide primers were developed from these regions to serve to amplify, by PCR, portions of a few hundred bases of these genes which can be easily identified by electrophoresis:

PourAlsD

> AlgDl TGG GCT ATG TMG GTG CAG T primer

> Primer AJgD2 ACS ACC ACG GTG TGG CGA A Primer ÂlgD3 GGT GTA CTT GAT CAT CTC GGC For AlgU

> AlgUl TGGTYGQRC GRGTRC AGC G primer

> AlgU2 primer ACGRTAKGC TYY GAT GAA AlgU3 primer TTYTAT ACMTGGCTGTAT C

> Primer AlgU4 GAC CCYACS GTY CCMAC

The results obtained are given in FIGS. 1A and 1B which represent:

A. The PCR bands produced by the AlgU 1/2 primers according to the type of templates used. The expression of the PCR-AlgU bands in all likelihood denotes segments of different masses and therefore characteristic of the candidate strains TF31 and R86, respectively;

B. The demonstration of the difference between the AlgU 1/2 PCR bands for the candidate strains TF31 and GR86.

The primers AlgUl and U2 appear to be the most discriminating, making it possible to obtain reproducible PCR products characteristic of the two candidate strains selected, and this vis-à-vis the strain used as control, Azotobacter chroococcum 76.116 from the CNCM of l 'Institut Pasteur (see Figure 1).

. control of the microbiological purity of the candidate strain and of the preparation The so-called “RAPD” technique (Random Amplified Polymorphism DNA) is most generally used to rapidly obtain a genetic profile of a bacterial organism. A choice of oligonucleotide primers used to amplify by DNA segments of DNA, a priori randomly arranged within the genome of the organism, can therefore serve as a QA € Q tool (Quality Assurance, Quality Control) during production and experimental deployment of bacterial organisms. This choice is dictated by (i) the appearance of a “PCR band. », Firstly, (ii) the systematic re-appearance of this band during successive PCRs, and (iii) the relationship that this community of bands will establish between the various strains that we are trying to distinguish others.

The .candidate strains having been characterized as being of the Azotobacteraceae type and probably ecotypes of A. chroococcum Berjerinclâi, Gram negative, aerobic, diazotrophs, in the form of rods, sometimes ovoid, and of size from 1 to 3 microns depending on the stage of development of the culture and the composition of the medium, it was above all a question of distinguishing the various candidate strains TF31 and GR86, from a type culture strain, in particular A. chroococcum Berjerinkcii 76.116 from the CNCM of the Institut Pasteur.

From two series of nine random primers each composed of ten nucleic bases

(see Table 2), six primers were chosen according to the three selection criteria mentioned above (See Buaicoa et al. 1999).

Table 2: Synopsis of all the primers (decamers) tested within the framework of the RAPD analysis of the three strains and bio-masses azotobacterial, including the reference strain Azotobacter chroococcum 76.116 from the CNCM of the Institut Pasteur.

Production of PCR-RAPD bands

Azotobacter Sequence Strain Strains

Decamera Oligonucl eotidiques chroococcum candidates candidates TF31 GR86

Vtl-3 TCG TAG CCA A? -? -

Vt2-4a TCA CGA TGG A? - **

Nt3-4c TCT CGA TGC A? -? -

Nt4-5a CTG TTG CTA C? -? -

Vt5-8 TGG TCA CTG A? -? -

Vt6-10b GGA AGT AGT G? -? - 7-1 ACC TCG AGC A? -? -

Vt8-12b CGG CGC CTG T. . ****

Nt9-13 ATT GCG TCC A? -? -

Azrl GGC CCG ATC G? -? -

Azr2 TAC CCC GAG G? -? -

AzrS TGC GGC TCA G? # ** ***

Azr4 TCC CAG CGC G? -? -

Azr5 GCA TGC ACG G * ** **

Azrδ GCC AGT CCC G? **? -

Azr7 GCT GCC CGA G? ? -? .

AzrS GTG CGA CCA C? ** # *

Azr9 TTC GGC GCG A * ** **

Notes? : primer used but no PCR band -: primer abandoned due to the absence of a PC R .. band *: PCR band carried out **: PCR band not carried out The results obtained are reported in FIG. 2, which represents the bands produced by

RAPD. The six primers, which are decamers, ensured that the candidate strains were actually related to. AT. chroococcum 1901 / 76.116 from the Institut Pasteur (see bands a2 and a5 in Figure 2), while being distinguished from them (see bands al, a3, a4 and a6 in Figure 2). This PCR-RAPD profile also makes it possible to distinguish between the candidate strains TF31 and GR86, which corroborates the more functional analysis of these two strains as a function of the gene segments alg (see FIG. 1).

The results obtained are reported in FIG. 2, which represents the bands produced by RAPD, according to a series of six primers (n10) characteristic of the candidate strains of the Azotobacteraceae type, compared to a strain of A. chroococcum 76.1 12 witness.

Identification of biomasses of the invention in relation to controls

It is recalled that, in general, the in vitro retention protocol of bacterial biomasses with edaphic characters best suited to survival, persistence and activity during the period of cultivation of the agronomic crops concerned integrates the three ( 3) following concepts within the protocol, namely:

© Concept 1: Adhesion to inorganic and abiotic surfaces

- Concept 2: Oligotrophic growth from carbohydrate and humic supplements

- • Concept 3: Expression of benthic and p ^' anctonic characters

These biomasses can indeed be identified and distinguished in relation to a range of control biomasses produced more or less conventionally (traditionally) and / or according to a variant of the said protocol integrating only two, or even only one, of said concepts. More precisely, a series of tests in microcosms, or even in micro-plots incorporating one or more of the inoculated controls (azb and / or Azb) listed below, and the candidate biomass (AZB) can demonstrate, a posteriori, the implementation of the said protocol;

s TM1 - Azb Type I control: These biomasses are isolated from agar gels or other conventional organic and biotic matrix surfaces on which an earth extract is modeled on the edaphic situation of the original / target soil. Due to the presence of a relatively rich matrix substrate, they are therefore in principle especially oligotrophic in nature and contrary to the principle of active persistence as targeted by the invention;

»TM2 - Azb Type II control: These biomasses are isolated in the presence of silicic gels by alternating the liquid and solid phases by gradual drying of the supply of distilled water on the surface of gels which have not received any earth extract. , or having received an earth extract (ie glucido - humic supplement modeled on the target soil) from a soil of different origin from that of the target soil. o TM3 - Azb Type III control: As with the Azb Type I control, these biomasses are isolated from gels, but silicic and / or inorganic and abiotic, on which an earth extract is modeled on the soil condition of the original soil / target. An earth extract is brought to the surface of the silica gel (installation of a first and last liquid phase), but this is not restored once it has dried.

TM4 - Azb control 1: These candidate bacterial biomasses are obtained according to the protocol of Pochon (1954), Pochon and Tchan (1948), Pochon et al. (1958) and Sasson (1967). However, and according to the present definition, these biomasses are obtained without the impregnation of silicic gels, and / or other inorganic and abiotic matrix surfaces, with any soil extract. . o TM5 - Witness azb 2: These biomasses are obtained by means of protocols for obtaining edaphic characters but strictly with regard to the carbohydrate and humic complement of the original / target soil; this supplement ("soil extract") is either brought to the surface of an organic / biotic matrix surface (eg. agar gel) or in a rich and more or less conventional liquid culture medium. o TM6 - Witness azb 3: These biomasses are already listed in the various bacterial collections (ATCC, UTEX, Institut Pasteur, DMZM, etc.) and have been isolated conventionally, or at least without particular consideration seeking to reproduce in vifro, during of their isolation, the edaphic character, within the meaning of the present invention. It is also possible to distinguish two types of azb 3 cookies; (a) ^"hand Witnesses azb 3" endogenous "(azb3a), that is to say these biomasses bacteria obtained conventionally, but directly from a sample of the original / target soil, and (b) on the other hand the "exogenous" azb 3 controls (azb3b). that is to say, these bacterial biomasses already listed in the collections.

Graphical representation of the AZB effect compared to control inocula

The realization of this ".AZB" effect has been represented graphically by ordering the reports of the agronomic results obtained either in microcosms, or in a greenhouse, or in micro-plots in the following fields (see Figure 9A and B): β AZB / Witness reports Azb (I, II and III) versus AZB report / Azb controls (1, 2 and 3) o AZB report / Azb controls (I, II and III) versus AZB report under conditions favorable / unfavorable for the expression of the effect "ÀZB" from the implementation of the invention.

This presentation makes it possible to demonstrate the effectiveness of the bacterial biomasses obtained according to the invention with respect to their various control inocula. This representation also makes it possible to easily identify the relatively effective inoculating preparations which can serve as candidate strains for the manufacture of inocula intended for marketing.

By way of example, 3 sets of experimental data have been reported and are illustrated respectively in FIGS. 9C, 9D and 9E:

Figure 9C: (a) Relative efficacy of various inoculating preparations from azotobacterial biomasses isolated from two types of soil (F4 and 19);

“Figure 9D: (b) Relative effectiveness of various inoculating preparations from azotobacterial biomasses isolated from two types of soil (F4 and 19) according to the results obtained during incubation in microcosms with and without intact soil aggregates; o Figure 9E: (c) Relative efficacy of various inoculating preparations obtained and tested during a series of experimental tests (Exp 17, 18, 19, 20 and 21). Relative AZB / control efficiencies greater than 1.00 indicate inoculating preparations more effective than conventional preparations. This improvement in efficiency is necessarily attributable to the implementation of the invention.

Experimental samples including conventional Azb controls and Type 1 Azb controls:

Nitrogen content / kg of soil

Table 3 below indicates the mineral nitrogen contents (Nm) of the microcosm soils incubated in the presence of 1% w / w of corn culture residues inoculated with bacterial preparations according to the invention (AZB) and their ^' TM6 and TM1 cookies (ie internal cookies).

Table 3

, -,. 1

Tests TM6 indicator TM1 indicator AZB AZB / TM1 AZB / TM6

Val 2 54 57 71 1.25 1.31

Val 3 58 62 64 1.03 1.10

Val 4 57 55 71 1.29 1.25

Val 5 62 62 62 1.00 1.00

Val 6 60 62 63 1.02 1.05 loyenne 59 62 68 1.10 1.16

¹ Phase II: from January 98 to December 1999

² Azb Type I warning light

. accumulation of mineral nitrogen (Nm) per kg of soil according to the retention conditions on media as used in the invention.

In general, the isolation protocol of the invention promotes the in vitro retention on the surface of abiotic materials conditioned so as to house biofilms conducive to the evolution of bacterial strains (azoto) with edaphic characters. One of the main characteristics of these edaphic bacterial biomasses thus retained is in principle their oligotrophy which is associated with their greater hardiness and efficiency in situ. To highlight this rustic oligotrophy, sometimes called zymogeny, the experimentation res m ed in table 4 below was carried out: Table 4

Accumulation of mineral nitrogen (Nm) by. kg of soil according to the retention conditions on media of the invention. The conditions for obtaining bacterial biomass (azoto) in italics cancel out the effectiveness of the protocol of the invention, by establishing retention conditions which are too rich in carbon substrates.

Pre-incubation Presence of glucose Compared to soils on azb media, control With conventional cellulose retention mg Nm / kg sol ³

yes YES 1.02 26 (3) no yes 0.99 26 (1) yes no 1.14 29 (2)

no no 1.38 36 (2)

Microcosms of 25 g of Terrefort soil reconstituted over 28 days of incubation at 22 ° C

Azb Type II (TM2) control experimental data:

. Effectiveness of the protocol of the invention for promoting the production of aerobic biomass (azoto) according to the edaphic nature of the original and "target" soil.

The results obtained are summarized in Table 5 below:

FIG. 3 gives the mineral nitrogen contents (Nm) / kg of soil by inoculating various controls and candidate strains, in microcomes and on microparcels. Table 5

Biomass Edaphic nature Edaphic nature AZB / Azb Type II (TM2) ratio candidates of the soil of origin of the "target" soil First series Second series

Control TM2 Argilo-silty Clay 1.52 1.07 Control TM2 Limon Clay 0.75 0.89 Control TM2 Sandy A. clay 0.63 1.31

AZB Clay Clay 2.02 2.81

Two series of the same soil were pre-incubated, from which it is desired to isolate, according to the protocol of the invention, the candidate AZB bacteria with and without 2% P / N of cellulose.

In principle, this pre-incubation should have favored the proliferation of rather zymogenic and less oligotrophic bacteria and thus made them more easily, or at least more probably, isolable given their greater number.

Under such conditions, the strains obtained by the protocol of the invention ("AZB") should be little or no more effective than conventional azobacterial biomasses (Azb). In addition, a 1% glucose solution sufficient to destroy the oligotrophic conditions in principle present on the retention media according to the invention was added. Three negative controls canceling the action of the candidate strains could be established at the time of retention of the most oligotrophic and rustic bacterial biomass (azoto) (see table).

It should be noted that only the complete absence of carbon substrates provided makes it possible to obtain, according to the protocol described above, AZB bacterial (azoto) biomasses truly more effective than the azb azotobacterial biomasses more conventionally isolated.

This experiment perfectly highlights the roles of oligotrophy during the retention of the most rustic and effective bacterial biomass (azoto) once reintroduced in the edaphic zones as an inocula. It should be remembered that this condition of obtaining that is oligotrophy is necessary but ^" not sufficient to ensure the isolation of the most edaphic bacterial strains. Indeed, the hardiness and resistance to nutritional stress does not correspond exclusively to soil bacteria but remain nevertheless essential here, given the upsurge in oligotrophic conditions in edaphic zones.

Other experimental data:

. Study of the increase in the content of microcosms in mg of mineral nitrogen (Nm) per kg of reconstituted soil, and of the persistence of the activity of nitrogenase over time measured according to the ratio of the activity of reduction acetylene (ARA) at time t2 and time tl. The results obtained are summarized in Table 6 below.

Table 6

Trial 1 Trial 2 Trial 3

Microcosm Nm ARA ³ Nm ARA ⁴ Nm ARA ⁵

Crop residues 28 3.87 10.8 22 60 1.6

Culture residues with TM5 control 29 4.42 14.8 45 66 2.1

Crop residues with AZB 40 6.67 15.3 112 72 2.7

. Mineral nitrogen content (Nm) of microcosm soils incubated in the presence of 1% w / w of, _ corn crop residues inoculated with AZB bacterial preparations and their azb and Azb controls (ie "internal" controls)

^J t2 = 26 h after the start of incubation of the microcosms; tl = 22 h ^{^} t2 = 72 Ii after the start of incubation of the microcosms: tl = 24 h ^D t2 = 66 h after the start of incubation of the microcosms; tl - 4 p.m.

The results are summarized in Table 7 below

• Table 7

ng Nm / kg soil -

Tests Phase 1 witness TM6 witness TM1 AZB AZB / TM1 AZB / TM6

119 51 Na 56 Na 1.10

III 46 Na 61 Na 1.33

IV 53 na 54 Na 1.02

Eai 53 na 54 Na 1.02

Ea2 51 na 56 Na 1.10

Ea3F4 51 na 56 Na 1.10

Ea3I9 52 na 55 Na 1.06

Ea4 49 na 58 Na 1.18 ea4bis 52 na 55 Na 1.06

Average 51 na 56 Na 1.10

Phase II trials ⁷ Control TM6 control TM1 AZB AZB / TMl AZB / TM6

Val 2 54 57 71 1.25 1.31

Val 3 58 62 64 1.03 1.10

Val 4 57 55 71 1.29 1.25

Val 5 62 62 62 1.00 1.00

Val 6 60 62 63 1.02 1.05 ~ *

Average 59 62 68 1. 10 1.16

⁶ Phase I. from September 96 to December 97 ^η Phase II: from January 98 to December 99

Figure 5 gives the mineral nitrogen contents (mg Nm / kg sol) of two series of microcosms in parallel, with and without the contribution of the equivalent of about fifty units of mineral nitrogen per hectare, according to the type of bacterial inocula (azoto) used (azb - conventional control inocula consisting of conventionally isolated azotobacterial strains; silicic gels (GS) conditioned according to the invention plus various parahumic substances (SpH i, 2 and 3), a polycondensate catechol (PC) and SpH).

Kinetics of degradation of crop residues in the field over a period of 5 months (from October 2000 to March 2001) on "Chestnut red soil" type soil, INRA experimental domain, Lusignan (86). The results obtained are reported in table 8 below.

Table 8

⁸ According to the degradation kinetics dRC = A xe ^'1 ", where dRC represents the quantity of crop residues degraded at time t, and A the quantity of residues at time 0, ie 4.00 g of dry matter.

. Bacterization according to the invention of crop residues (CR) - effect of finer grinding on the use of substrates resulting from their degradation and in principle usable by bacterial biomass (azoto) thus brought to the surface of these residues . The results obtained are summarized in Table 9 below

Table 9

_m g N _m / kg Soil

Inocula Fine CR milling Coarse CR grinding

TM6 indicator 58 52 TMl indicator 52 48 AZB 82 55

It will be observed that the coarser grinding (3 mm) of these same residues probably does not allow the AZB biomass to contribute to the relative increase in the mineral nitrogen contents (Nm) of the microcosms compared to the inoculated controls TM6 (ie isolated azotobacterial biomass conventionally) and TMl (ie isolated azotobacterial biomass according to an ineffective variant of the invention. . Increase in biomass: the results obtained are reported in Tables 10, 11 and 12 below:

A) Increase in the production of plant biomass (aerial) by the species Lolium muliiflorum and Triticum aestivum according to the mode of cultivation in pots (greenhouse) during the incubation of residues of cereal crops (wheat straw) over a period of 28 to 56 days (Trial 5) with and without the various inocula of the invention and their controls TM6 and TM1.

Table 10

my biomass

Method of growing in pots Test'i " ¹ Test2 ^a Test3 ^b Test4 ^D Test5 ^b

Crop residues 9.1 7.1 33 37 35

Crop residues with control TWI6 8.7 5.9 39 32 37

Culture residues with TM1 control na 8.1 39 32 37

Culture residues with control T 2 na na 39 32 37 Culture residues with AZB 10.0 8.7 40 41 39

Note: a; g of biomassZ, o / πw) ffπ // ft? oπ / per pot b; mg of biomass Triticum aestivum per seedling

B) Percentage (%) of increase in grain yields (yield at 15% humidity) and of PMG (weight of a thousand grains) during agronomic field trials (1999-2000 campaign at Toulouse 31 on Terrefort soil, and 2000-2001 campaign in Lusignan on red chestnut soil) compared to non-inoculated witnesses combined with TM6, TMl and AZB.

Table 11

Yield 15% hurn PMG

Mode of cultivation in the field Toulouse Lusignan Toulouse Lusisnan

Culture residues with TM6 control 4% 0.73% 5% 0.08% Culture residues with TM1 / 2 control ^at 13% 0.86% 4% 0.02% Culture residues with AZB 20% 1.47% 6% 0.94%

Note: a; TMl witnesses for Toulouse, TM2 for Lusignan

C) Coincidence of increases in yields and PMG during the agronomic trials in the 2001-2001 campaign field in Lusignan on red chestnut soil soil, and the various indices of crop residue recovery obtained in parallel on these same plots . Table 12

Cultivation method in field Yield "PMG ^b -k dRC ^d dRC ^e

Culture residues with control TM6 63 0.08% 41 2.10 0.70 Culture residues with control T 2 71 0.02% 37 2.02 0.81 Culture residues with AZB 126 0.94% 47 2.42 1.19

Note: a; increased yields in kg grain / ha at 15% humidity b; weight thousand grains of wheat compared to the non-inoculated controls combined with the controls TM2 / 6 and AZB c, degradation coefficient x I0 ' ⁱ according to dRC = A * e- ^t d; degradation of crop residues (4.00 g at tO) over 164 days e; degradation of the culture residues compared to the non-inoculated controls combined with the TM2 / 6 and AZB controls

. Increase in the plant biomass / macitary biomass ratio attributable to the rhizospheric action of (re) introduced bacteria.

Table 13 below gives the results obtained concerning the increase in the ratio between the vegetative (aerial) and root parts (Vegetative Index, IV) of the seedlings cultivated on soils incubated in the presence of cereal crop residues (wheat straw) and not treated with the inocula according to the invention and their controls TM6 and TM2.

Treatment of crop residues - vegetation / roots report (Vegetative Index)

Trial 1 Trial 2 Trial 3 Average

TM6 tell-tales 0.85 na na 0.85 TM2 tell-tale na na na TF3i (AZB TF31) 0.90 na na 0.90

Indicator TM6 1.02 0.90 1.03 0.98 (0.03) ⁹ Indicator T 2 na 1.03 1.09 1.06 (0.04) GR86 (AZB GF86) 1.05 0.92 1.36 1.11 (0.09)

(Ccartype)

. Delay in the immobilization effect of nitrogen compared to that obtained on control culture residues treated with an antibiotic.

The effect of treating cereal crop residues (wheat straw) with an antibiotic (ampicillin) is studied at an agronomic rate (10 kg kg / 10 mg of residue / hectare (approximately 1000 mg of soil)). The results obtained are summarized in Table 14 below.

Table 14

Method of growing in pots Trial 1 Trial 2 Trial 3 Medium

Crop residues 33 37 35 35 (2) ¹⁰

Crop residue with antibiotic ¹¹ 37 39 38 38 (1)

Crop residues with AZB 40 41 39 40 (1)

The observed effect is attributable to the delay instilled in the bacteria involved in the degradation of crop residues, and therefore incidentally to the immobilization of assimilable mineral nitrogen in the soil. This reduction in the immobilization of assimilable nitrogen allows greater plant growth of the seedlings tested in place; this increase in vegetative growth is analogous to that observed in the presence of crop residues treated according to the invention. This treatment can therefore also be conceived as a good (micro) biological alternative to a possible treatment with antibiotics of crop residues.

Figure 6 gives a graphic representation of three NB5 experiments with the use of ampicillin as an antibiotic control. It will be observed that ampicillin is more favorable to vegetative growth than the inoculated controls azb and Azb, while AZB is more favorable than ampicillin.

. Degradation of crop residues

The decomposition into microcosms in the laboratory and in the field, in nylon sachets, of the cereal crop residues treated according to the invention is compared with their uninoculated twin controls.

The results obtained are given in FIGS. 7 A AND 7B.

. effect of the granulometry of soils

The results relating to the study of the relative effectiveness of the bacterial (nitrogen) biomass of the invention in maintaining the mineral nitrogen content of soils are reported in FIGS. 8A and 8B. reconstituted in microcosms according to the particle size fraction (> 3 mm in (A), and less than 1 mm in (B)) of these fractions. It will be noted that only the AZB biomasses are effective and that this efficiency with respect to the inoculated controls (azb and Azb) and non-inoculated (control) is no longer observed when the biomasses evolve within the 1 mm fraction.

Bibliographical references

Burucoa, C.; V. Lhomme and J.L. Fauchere. 1999. Performance criteria of DNA fmgerprinting methods for typing of Heliobacter pylori isolâtes: experimental results and meta-analysis. L Clin. Microbiol. 37 (12): 4071-4080.

Characklis, W.G. 1990. Biofilm processes. In: "Biofilms" (Characklis and Marshall, eds.),

John Wiley and Sons, NY, NY.

Characklis, W.G. and K.C. Marshall. 1990. Biofilm: a basis for an interdisciplinary approach. In: "Biofilms" (Characklis and Marshall, eds.), John Wiley and Sons, NY, NY.

Davies, L .; H-C. Flemming and P. A. Wilderer. 1998. Microorganisms and their roel in soil. In: "Bioremediation: principals and practice, Vol. 1" - Fundamentals and Applications (S.K. Sikdar and R.L. Irvine, eds.). Technomic Publ. Inc., Lancâster (UK), Basel (CH).

Gordienko, S. 1990. Conceptual model of the functional structure of autochtonous component in soil microbial organisais communities. Ekologia 9 (4): 429-439.

Pochon, J. and Y-T. Tchan. 1948. Summary of soil microbiology: principles, techniques, place in geo-biological cycles. Masson et Cie., Eds, Paris.

Pochon, J. 1954. Technical manual for microbiological soil analysis. Masson et Cie. Eds Paris.

Pochon, J. and H. DeBarjac. 1958. Treatise on soil microbiology: agronomic applications. Dunod, Paris.

Sasson, A. 1967. Eco-physiological research on the bacterial flora of soils in arid regions of Morocco. Doctoral thesis, Faculty of Sciences of Paris, defended on June 26, 1967.

Claims

1. Method for obtaining in vitro bacterial biomasses of edaphic origin, characterized in that it comprises the steps of:

- bringing an aqueous soil suspension into contact with a matrix substrate, so as to form a hydrated zone, or biofilm, on the surface of the substrate, similar to that attached to the soil HAC, essentially sheltering the bacterial component native to the soil, in order to retain on the surface of the matrix substrate, most of the bacterial microflora endogenous to the original edaphic environments.

- maturation of the biofilm with establishment of oligotrophic conditions allowing the most constitutive cells of the edaphic biofilm which have colonized the matrix substrate, to migrate towards the liquid phase supernatant the matrix substrate, and gradually to dominate it,

- recovery and culture of the most prolific bacterial strains in rich liquid culture media, of an indigenous character, rustic and persistent in situ, cultivable in liquid media, and therefore suitable for industrial production.

2. Method according to claim 1, characterized in that the matrix substrate is an inorganic and abiotic matrix substrate, in aqueous medium, promoting the production of exopolysaccharides (EPS) and other components of ~ ι bacterial glycocalyx, as well as network organization of a sufficient number of cells capable of initiating the first stages of biofilm formation on the surface of the substrate.

3. Method according to claim 2, characterized in that the water of the aqueous suspension contains compounds present in the relative proportions characteristic of the original target soil, namely humic and fulvic substances, carbonaceous compounds and in particular mono -, oligo- and polysaccharides, of vegetable, animal and bacteriofungal origin, nutrients.

4. Method according to any one of claims 1 to 3, characterized in that the ripening biofilm dir is carried out by alternating a phase where the biofilm formed on the surface abiotic is not in the presence of a supernatant liquid phase, with a liquid phase comprising all of the bacterial cells supernatant the solid phase.

5. Isolated bacterial biomasses, characterized in that they comprise, as dominant edaphic, indigenous, oligotrophic, mainly aerobic, strains having metabolophysiological characteristics of diazotrophy, fonti-static and / or phytosanitary activity.

6. Biomass according to claim 5, characterized by the following phenotypes expressed in vitro and in situ with respect to control bacterial biomasses obtained from biotic surfaces, but without the presence of a biofilm or a humic supplement and / or obtained from abiotic surfaces, but a) with a humic complement not modeled on that of the target soil and / or b) with a carbonaceous substrate provided in an amount greater than the concentrations normally present in the soil, and / or obtained from unconditioned abiotic surfaces, said said Bacterial biomasses are more mucoid than these controls, more encysted and more durably, less copiotrophic than said controls, and produce self-induction factors at higher rates than controls, for example 4th octanoyl homoserine lactone.

7. Bacterial biomass according to claim 6, characterized in that, with respect to said controls, they are capable of maintaining the mineral nitrogen content of the soils reconstituted in microcosms over a period of more than 28 days of incubation of Azobacteriaceae, of Pseudomonaceae , or Rhizobiaceae.

8. Bacterial biomass according to any one of claims 5 to 7, characterized in that it is Azobacteriaceae, Pseudomonaceae, or Rhizobiaceae.

9. Bacterial strains isolated from bacterial biomass according to any one of claims 5 to 8.

10. Bacterial strains according to claim 9, in that they are ecotypes of A chroococcum Berjerinkcii.

11. Application of the bacterial biomasses according to any one of claims 5 to 9 and of the bacterial strains according to claim 10, to the microbiological bacterization of soils and crop residues, in particular residues of cereal crops, including those of maize -grain.

12. Application according to claim 11, characterized by inoculation of said bacterial biomasses and strains by spraying formulations containing them on the culture residues.