FR2833016A1 - NEW BACTERIAL BIOMASSES, THEIR OBTAINING PROTOCOL, AND THEIR USE FOR BACTERIZATION OF SOILS AND CROP RESIDUES - Google Patents

Abstract

The invention relates to a method for obtaining in vitro bacterial biomasses of edaphic origin comprising the steps of: bringing an aqueous suspension of soil into contact with a matrix substrate, so as to form a hydrated zone, or biofilm, on the surface of the substrate, similar to that added to the ACH of the soil, essentially housing the indigenous bacterial component of the soil, in order to retain on the surface of the matrix substrate, most of the bacterial microflora endogenous to the edaphic media of origin.- 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 to the liquid phase supernatant the matrix substrate, and gradually to dominate it, - recovery and culture of the strains most prolific bacteria in rich liquid culture media, with an autochthonous character, hardy and persistent in situ, cultivable in liquid media uids, and therefore suitable for industrial production. Application of bacterial biomasses and strains obtained for the microbiological bacterization of soils and crop residues, in particular cereal crop residues, including those of grain corn.

Description

<Desc / Clms Page number 1>

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

Bacterization of arable soils consists of providing, by (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, bacterization has mainly been synonymous with the use of inocula from Rhizobium strains for the coating of seeds of Leguminosea species such as soybean, alfalfa and red clover.

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

The use of bacteria in agronomy, and more particularly in cereal production, however, came up against the problem of their non-persistence, once reintroduced into the soil. As a result, 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 input levels, in particular the mineral nitrogen.

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

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 occurrence of three phenomena, sometimes harmful,

<Desc / Clms Page number 2>

well known ; (1) the continual presence of agronomic quantities of straw residues on the soil surface during tillage work, (2) immobilization of nitrogen by non-diazotrophic microorganisms in the soil causing nitrogen hunger in the crop. place, and, paradoxically, (3) an appreciable drop in carbon contents (soluble, labile and total) and microbiotic biomasses.

Another problem encountered with bacteria is related to their isolation and mass production traditionally carried out by serial dilution of a soil suspension and culturing an aliquot of an nth dilution of this aliquot on an agar medium. rich. The strains thus isolated, once sufficiently characterized, are subsequently used to inoculate conventional bioindustrial fermenters. The capacity of the inocula produced in this way to sustainably increase the in situ activity of the organisms concerned, diazotrophy in topsoil amended with crop residues, for example, is therefore very low and / or uncertain today. The recourse to 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 make a greater effectiveness of this type of inoculation.

The study of these problems by the inventors has 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 bacterization of soils once (re) introduced, thanks to their persistence properties in situ.

Historically, we can identify three approaches to obtaining bacterial strains of edaphic origin (that is to say from arable soils) in principle the best suited to active persistence in situ once reintroduced: a. isolation on abiotic matrices: with the use of media with edaphic characteristics (i.e. silicic gels with or without earth extracts), without however integrating the main components (silicic, humic, carbonaceous, and hydrological) of these media; 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 cultured conventionally;

<Desc / Clms Page number 3>

vs. culture in depleted media: a voluntary depletion, in particular in carbon, of the culture media of conventionally isolated bacterial cells is carried out.

The solution provided by the invention integrates these 3 approaches, and more particularly approach a), within the production protocol defined below, which leads to the obtaining of the constitutive 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 the so-called more conventional approaches a), b) and / or c).

In addition, the invention takes into account the process of formation of biofilms in an aqueous medium. In this regard, the inventors 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 (planktonic) 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 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.

<Desc / Clms Page number 4>

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 (silicic gels or agar for example)
2. The basic biofilm containing most of the bacterial cells
3. The surface biofilm mainly containing the complementary cells
4. The supernatant liquid phase
5. The ultimate aqueous phase Now, it turns out that the biofilm as such consists mainly of 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 indigenous component. , the rather copiotrophic zymogenic component associated with this autochthonous component, and finally the component, very marginal but easily isolable, essentially zymogenic and very 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 ACHs of the soils.

It therefore aims to provide a process for obtaining bacterial biomasses essentially formed of 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 possessing the commonly sought-after metabolo-physiological characteristics, such as diazotrophy, fungal-static and / or phytosanitary activity, the production and secretion of growth regulators, the catabolism of xenobiotics, etc.).

The invention also relates to the use of these bacterial biomasses and strains for the bacterization of soils and crop residues, in particular of cereal crops.

<Desc / Clms Page number 5>

The process for obtaining in vitro bacterial biomasses of edaphic origin is characterized in that it comprises the steps of: bringing an aqueous suspension of soil into contact with a matrix substrate, so as to form a hydrated zone, or biofilm, on the surface of the substrate, similar to that added to the ACH of the soil, essentially housing the indigenous bacterial component of the soil, in order to retain, on the surface of the matrix substrate, most of the bacterial microflora endogenous to the soil. original edaphic environments.

- maturation of the biofilm with establishment of oligotrophic conditions allowing the cells most constitutive 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 bacterial strains prolific in medium of rich liquid cultures, with an autochthonous character, hardy and persistent in situ, cultivable in liquid medium and therefore suitable for being produced industrially.

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

Suitable materials as substrates include silica gel, agar, polystyrene, clay, porous glass, or 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, carbon compounds and in particular mono-, oligo- and polysaccharides, of plant origin, animal, and bacteriofungal, nutrients.

Maturation of the biofilm is carried out by alternating a phase in which the biofilm formed on the abiotic surface is not in the presence of a supernatant liquid phase, with a liquid phase comprising all the bacterial cells supernatant the solid phase.

<Desc / Clms Page number 6>

This maturation results in principle from the migration of native bacteria to the liquid phase (Pochon and Tchan, above) of the biofilm formed on the matrix substrate. Volumetric displacement only takes place during and after aging (maturation) of the biofilm (i.e. biofilm on the surface of the matrix substrate). This maturation allows the release, in the aqueous phase supernatant of the matrix substrate, of the bacterial cells which constitute the most constitutive 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 (especially bacterial) diversity of the soils with the microbiological purity of the biomasses to be produced in conventional bioprocesses, which has never really been attempted until now. The isolation of bacterial strains is in fact carried out above all 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 genomic (eg. using oligonucleotide probes specific for certain operons of agronomic or industrial interest) easily identifiable, but which basically have little or nothing to do with the adaptation and active persistence of these cells indigenous bacteria rather than zymogens once (re) introduced into various soil environments.

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

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

<Desc / Clms Page number 7>

bacterial biomasses 2) obtained from abiotic surfaces according to the protocol in question, but a) with a humic supplement not modeled on that of the target soil and / or b) with a carbonaceous substrate supplied in a quantity greater than the concentrations normally present in the soil , and / or bacterial biomasses 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 considered above was produced, namely concept 1: adhesion to inorganic and abiotic surfaces; concept 2: oligotrophic growth from carbohydrate and humic supplements; concept 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 with the fact that the integration of the 3 concepts (1, 2, 3) mentioned above is possible to obtain the desired results and constitute a means of identification of the biomasses of the invention as described in the examples.

According to a preferred embodiment of the process of the invention for the gradual isolation of hardy and persistent bacterial biomasses in situ, the following steps are advantageously used: a) immediate and temporary retention of most of the endogenous bacterial microflora to the bacteria. original edaphic media: In order to make this retention step less drastic in terms of elimination and loss of less copiotrophic strains, the 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, agar. The surface of the substrate is conditioned by contacting with a retention medium essentially modeled on the characteristics of the original target soil. These characteristics include, not exclusively: (1) the contents of humic and fulvic substances and their constitutions in terms of the various phenolic and benzoic compounds, (2) the contents of carbon compounds, and in particular the mono-, oligo-and polysaccharides of plant, animal and / or bacteriofungal origin, (3) the

<Desc / Clms Page number 8>

granulometry 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 contents of nutrients (nitrogen, phosphorus, sulfur, etc.) capable to influence 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 the 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 apt to persist actively once reintroduced into these APCs. b) -progressive dominance of the desired bacterial strains, diazotrophic and / or fungistatic for example, on these retention media specific and characteristic of the original edaphic media: The culture media, or retention media, become de facto the source of any biomasses candidate bacteria. Bacterial cells being good producers of exo-polysaccharides (EPS) serving for the formation of biofilms on the surface of the substrate, they can also be made to dominate the liquid phase restored on its surface. In fact, the biofilms thus formed can mature in this sense. It is during this maturation process that the multiplication of cells in the biofilm gradually moves the bacterial cells to the surface of the biofilm, through 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 at the surface of the biofilm. Insofar as this liquid phase and the biofilm substrate are virtually devoid of assimilable mineral nitrogen, the cells concerned will for the most part be azotobacterial. c) isolation, by micro-biological dominance, of the most prolific desired bacterial strains 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 media, such as they are used during industrial bio-processes. However, and since the aliquots from the liquid phases supernatant the media for obtaining these

<Desc / Clms Page number 9>

strains are not necessarily of absolute microbiological purity, there is a risk that the desired candidate strains which they contain will be displaced (ie. dominated in number) by contaminations that are more copiotrophic and contrary to the desired aim. To avoid such contaminations as much as possible, the culture medium is restored with an aliquot containing approximately 90% of the cells originating from the supernatant phase of the media obtained and only approximately 10% of cells actually pre-incubated in order to promote, possibly, the dominance of rather oligotrophic cells. Following the third, or even fourth, pre-incubation cycle, an aliquot of approximately 1 in 1000 of this last pre-culture 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 follow-up and control of the purity of these biomasses and strains in addition to a series of non-functional RAPD-type genomic profiles, and more functional according to the particular expression of certain most important mechanisms in term. of edaphic adequacy (eg. alg, moult, eps, pob, rhl, etc.).

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

conservation satisfaisant d'un minimum de 6 mois, soit à 4 C, soit à température ambiante (i. e. de 18 à 27 C), mais aussi d'assurer encore plus une pré-adaptation des cellules bactériennes déjà sélectionnées selon la présente invention. Liquid and solid formulations not only make it possible to ensure a

satisfactory storage for a minimum of 6 months, either at 4 ° C. or at room temperature (ie from 18 to 27 ° C.), but also to ensure even more pre-adaptation of the bacterial cells already selected according to the present invention.

The invention is also aimed at isolated bacterial biomasses, characterized in that they comprise, as dominant strains, GRAM negative bacterial strains, of an edaphic character, indigenous, oligotrophic, mainly aerobic, exhibiting metabolo-physiological characteristics of diazotrophy, font-static and / or phytosanitary activity.

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

<Desc / Clms Page number 10>

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

Compared to said controls, they are capable of maintaining the mineral nitrogen content of soils reconstituted in microcosms with 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 the characteristic fragments of aIgD and acuteU responsible for the production of EPS, as well as the 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 cereal crop residues, including those of grain corn.

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

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

To this end, inocula prepared from bacterial biomasses and isolated strains are advantageously sprayed onto the crop residues, in particular at the time of their

<Desc / Clms Page number 11>

burial in soil. For example, inoculation rates of 100,000 to 1 million viable cells / g of residues, or alternatively 1 g / t of residues, are used.

At the edapho-agronomic level, we observe in situ or in microcosms, a greater persistence in situ and over time of nitrogenase activity, in the case of azotabacterial biomasses capable of diazotrophy, an increase in the accumulation of l 'mineral nitrogen, especially in ammoniacal form, an increase in plant biomass, the ratio between vegetative and root parts, an initial delay in the degradation of crop residues attributable to the fungistatic activity of the (re) introduced bacteria.

- les figures 1A et 1B, les bandes PCR produites par des amorces AIgU y selon le type de matrices utilisées ; - la figure 2, les bandes produites par RAPD, selon une série de six amorces (nlO) caractéristiques des souches de type Azotobacteraceae candidates, comparées à une souche d'A. chroococcum 76.112 témoin, - la figure 3, les teneurs en azote minéral mesurées, sur des microcosmes et des microparcelles inoculés avec divers témoins et des souches candidates, - la figure 4, le rapport, en fonction du temps d'incubation entre l'activité de la réduction de l'acétylène en présence de résidus de culture inoculés selon l'invention ou avec des témoins, et non inoculés, - la figure 5, les teneurs en azote minéral de 2 séries de microcosmes traités selon l'invention ou par des témoins, avec addition de composés para-humiques ; - la figure 6, l'effet d'un antibiotique sur le traitement de résidus de culture selon l'invention et par des témoins, - les figures 7A et 7B, la décomposition de résidus de culture en fonction du temps d'incubation (laboratoire et au champ) ; - les figures 8A et 8B, l'effet de la granulométrie de sols reconstitués en microcosmes sur les propriétés des bactéries, et - les figures 9A à 9E, les effets de biomasses de l'invention par rapport à des témoins. 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 1A and 1B, the PCR bands produced by primers AIgU y according to the type of matrices used; - Figure 2, the bands produced by RAPD, according to a series of six primers (n10) characteristic of candidate Azotobacteraceae type strains, compared to a strain of A. chroococcum 76.112 control, - figure 3, the measured mineral nitrogen contents, on microcosms and microplots inoculated with various controls and candidate strains, - figure 4, the ratio, as a function of the incubation time between the activity reduction of acetylene 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 with controls, with the addition of para-humic compounds; - Figure 6, the effect of an antibiotic on the treatment of culture residues according to the invention and by controls, - Figures 7A and 7B, the decomposition of culture residues as a function of incubation time (laboratory and in the field); - Figures 8A and 8B, the effect of the particle size distribution of soils reconstituted in microcosms on the properties of bacteria, and - Figures 9A to 9E, the effects of biomasses of the invention compared to controls.

<Desc / Clms Page number 12>

Example 1: Isolation of candidate strains of the Azotobacteraceae type.

. soils used for sampling The results obtained on 2 sites (Terrefort and Groies) are reported, the APC characteristics of which are as follows:

<tb>
<tb> Table <SEP> the <SEP>: <SEP> Physico-chemical <SEP> analyzes <SEP> of <SEP> soils <SEP> F4 <SEP> and <SEP> 19 <SEP>(<SEP> Terrefort <SEP>) <SEP> on <SEP> the <SEP> domain
<tb> experimental <SEP> of <SEP> Inra-Toulouse
<tb> Plot
<tb> F4 <SEP> I9
<tb> Granulometry
<tb> Clay <SEP> 230 <SEP> 260
<tb> Limon <SEP> end <SEP> 242 <SEP> 227
<tb> Silt <SEP> coarse <SEP> 139 <SEP> 165
<tb> Sand <SEP> fine <SEP> 167 <SEP> 207
<tb> Sand <SEP> coarse <SEP> 222 <SEP> 141
<tb> Carbon <SEP> organic <SEP> 8. <SEP> 5 <SEP> 9.2
<tb> Materials <SEP> organic <SEP> 14.6 <SEP> 15.8
<tb> Organic <SEP> nitrogen <SEP> 1.07 <SEP> 1.13
<tb> pH <SEP> water <SEP> 5.9 <SEP> 6.6
<tb> CaCO <SEP> 0 <SEP> 0
<tb> P20s <SEP> 0.11 <SEP> 0.06
<tb> Exchangeable <SEP><SEP> 1.91 <SEP> 2.75
<tb> Na <SEP> exchangeable <SEP> 0.014 <SEP> 0.015
<tb> Mg <SEP> exchangeable <SEP> 0.173 <SEP> 0.187
<tb> K <SEP> exchangeable <SEP> 0.183 <SEP> 0.134
<tb> Humidity <SEP> to <SEP> 105 <SEP> C <SEP> 24 <SEP> 28
<tb>

<Desc / Clms Page number 13>

Table l. b: Edaphic characteristics of Groies soils near Poitiers (86) TERRES ... e Séchantes. Mainly in the north of the department CHARACTERS Brown color Silty clay Substrate: limestone PROPERTIES 'Limestone soil, shallow, stony (limestone) * Healthy soil Low water reserve (suitable for irrigation) * Good to medium fertility VARIATION: Large groie or clay -calcaire * Deep soil, more clayey, good water reserve. isolation protocol A sol / water suspension is brought into contact with a matrix substrate of agar or silicic gel. The biofilm which forms on contact with the matrix substrate meets the characteristics of the original target soil, in particular with regard to (1) the contents of humic and fulvic substances and their constitution in terms of the various phenolic and benzoic compounds, (2) the content of carbon compounds, and in particular the mono-, oligo- and polysaccharides of plant, animal and / or bacteriofungal origin, (3) the particle size of the mineral part of the soil, and especially the finest, or at least to the exclusion of aggregates greater than 5 mm in diameter, (4) the immediate contents of nutrients (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, for example once a week, in order to avoid drying out of the biofilm and to wait long enough between two restorations (eg about seven days), so in allowing time for the most constitutive azotobacterial cells of the biofilm thus formed to migrate by volumetric displacement towards the surface of the biofilm. In order to avoid the dominance of copiotrophic azotobacterial cells at the expense of azotobacterial cells

<Desc / Clms Page number 14>

sought after, which are rather oligo-or mesotrophic, more autochthonous and persistent once (re) introduced, but also therefore easily dominated in rich in vitro media, it is necessary to avoid loading the gels and the liquid phases which supernate them with carbonaceous substrates, in particular simple sugars such as glucose, and / or mineral nutrients. Aseptic distilled water (in particular by micro-filtration) is preferably used.

It is also necessary to avoid disrupting or excessively disturbing the biofilm at the time of restoration by using a jet of water that is too abrupt or directed at the time of restoration of the liquid phase. In general, it is sufficient to use 1 ml of water on gels contained in Petri dishes of 10 mm in diameter and 5 ml for dishes of 33 mm in diameter. After approximately seven days of incubation at 22-27 C, the last restoration, usually the third, aliquots of these liquid phases will be 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 glucose liquid medium (with Winogradsky salts comprising those originating from an extract of earth in an amount of 10% w / v) without mineral and / or assimilable nitrogen, and it is left to preincubate for 1 to 2 days at 22-27 C. This transfer is carried out from the culture thus obtained two to three more times, but using only 100 micro-liters (1/10 of mL) of this Rank 1 culture 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 that is rather less oligotrophic take the upper hand, in terms of number of cells per mL of culture medium, over the candidate biomasses that are even more oligotrophic, persistent and active in situ. .

- Identity of the organisms 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 selected are Gram negative, aerobic, diazotrophic and in the form of rods of about 1 to 3 microns.

We know that A. chroococcum is recognized as being the most adapted to edaphic living conditions. 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

<Desc / Clms Page number 15>

with the descriptions of Azotobacter chroococcum Beijerinckii 1901 from the ATCC, the DMS and the Institut Pasteur. A genomic and molecular characterization of these strains has therefore been established using mechanisms known in Azotobacter.

- biological properties of the organisms Bacteria of the Azotobacteracea 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 decomposition of grain and corn straw. In principle, some azotobacterial ecotypes are also able to establish rhizospheric associations and thus promote plant growth, but are not necessarily virulent. They are not either, and this unlike some Acetobacter, endophytes.

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

These bacteria are responsible for fixing diatomic nitrogen in soils by a non-symbiotic route. They are aerobic, unlike Clostridium, and are generally recognized as being able, depending on the circumstances, to fix by diazotrophy only a few kg, or even a few tens of kg of nitrogen per hectare and per year. However, these biological fixation rates are above all limited by the (non) availability of carbonaceous 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 in fact been demonstrated with the aid of experimental data obtained in the laboratory, in a greenhouse, in a culture chamber and in experimental plots in the field that the invention effectively allows greater relative efficiency of the strains.

<Desc / Clms Page number 16>

azotobacterials isolated, once they (re) introduced into their agro-pedo-climate (APC) of origin.

- methods of analysis. establishment of the identity of organizations
The identity of the organisms was established on the basis of their adaptation to CPAs. The results reported below concern the 2 APCs from which the strains were isolated, and in which they will be re-introduced in the form of bacterial inocula: one in the Toulouse region (31) (Terrefbrt or TF31, see Table l. a) and the other in the region of Poitiers (86) (Groies or GR86, see Table l. b).

For characterization purposes, we used oligonucleotides 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.

Pour AI t, gD

> Amorce AlgDl : TGG GCT ATG TMG GTG CAG T Amorce AlgD2 : ACSACCACGGTGTGGCGAA > Amorce AlgD3 : GGT GTA CTT GAT CAT CTC GGC We chose the two regions considered to be the most conserved of the genomic segments aIgU and algD already reported as belonging to the genera Pseudomonas and Azotobacter (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 easily identifiable by electrophoresis:

For AI t, gD

> Primer AlgDl: TGG GCT ATG TMG GTG CAG T Primer AlgD2: ACSACCACGGTGTGGCGAA> Primer AlgD3: GGT GTA CTT GAT CAT CTC GGC

<Desc / Clms Page number 17>

Pour AIgU

> Amorce AlgUI : TGG TYG QRC GRG TRC AGC G > Amorce AlgU2 : ACG RTA KGC TYY GAT GAA > Amorce AlgU3 : TTY TAT ACM TGG CTG TAT C > Amorce AlgU4 : GAC CCY ACS GTY CCM AC Les résultats obtenus sont donnés sur les figures 1A et 1B qui représentent : A. Les bandes PCR produites par les amorces AIgU 1/2 selon le type de matrices utilisées. L'expression des bandes PCR-AlgU dénote selon toute vraisemblance des segments de masses différentes et donc caractéristiques des souches candidates TF31 et R86, respectivement ; B. La mise en évidence de la différence entre les bandes PCR AIgU 1/2 pour les souches candidates TF31 et GR86.

For AIgU

> Primer AlgUI: TGG TYG QRC GRG TRC AGC G> Primer AlgU2: ACG RTA KGC TYY GAT GAA> Primer AlgU3: TTY TAT ACM TGG CTG TAT C> Primer AlgU4: GAC CCY ACS GTY CCM AC The results obtained are given in the figures 1A and 1B which represent: A. The PCR bands produced by the primers AIgU 1/2 according to the type of templates used. The expression of the PCR-AlgU bands denotes in all likelihood segments of different masses and therefore characteristic of the candidate strains TF31 and R86, respectively; B. Demonstration of the difference between the PCR bands AIgU 1/2 for the candidate strains TF31 and GR86.

The primers Algal and U2 prove to be the most discriminating, making it possible to reproducibly obtain PCR products characteristic of the two candidate strains selected, and this with respect to the strain used as a control, Azotobacter chroococcum 76.116 from the CNCM of the 'Institut Pasteur (see figure 1).

. control of the microbiological purity of the candidate strain and of the preparation The technique known as RAPD (Random Amplified Polymorphism DNA) is most generally used for rapidly obtaining a genetic profile of a bacterial organism.

A choice of oligonucleotide primers used to amplify DNA segments by PCR, a priori arranged randomly within the genome of the organism, can therefore serve as a QQA (Quality Assurance, Quality Control) tool during the production and experimental deployment of bacterial organisms. This choice is dictated by (i) the appearance of a PCR band, initially, (ii) the systematic reappearance of this band during successive PCRs, and (iii) the relationship that this community will establish. bands between the various strains that we try to distinguish from one another.

The candidate strains having been characterized as being of the Azotobacteraceae type and probably ecotypes of A. chroococcum Berjerinckii, Gram negative, aerobic,

<Desc / Clms Page number 18>

diazotrophs, in the form of rods, sometimes ovoid, and of size of 1 to 3 microns depending on the stage of development of the culture and the composition of the medium, it was mainly a question of distinguishing the various candidate strains TF31 and GR86, of 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 Burucoa et al. 1999).

Table 2: Synopsis of all the primers (decamers) tested in the context of the RAPD analysis of the three azotobacterial strains and biomasses, including the reference strain Azotobacter chroococcum 76.116 from the CNCM of the Institut Pasteur.

<tb>
<tb>

Production <SEP> of <SEP> Tapes <SEP> PCR-RAPD
<tb> Sequence <SEP> Azotobacter <SEP> strains <SEP> Strains
<tb> Decamer <SEP> Oligonucleotides <SEP> chroococcum <SEP> candidates <SEP> candidates
<tb> TF31 <SEP> GR86
<tb> Vtl-3 <SEP> TCGTAGCCAA <SEP>? - <SEP>? -Vt2-4a <SEP> TCACGATGGA <SEP>? - **
<tb> Vt3-4c <SEP> TCT <SEP> CGA <SEP> TGC <SEP> A <SEP>? --- Vt4-5a <SEP> CTG <SEP> TTG <SEP> CTA <SEP> C <SEP >? ---- <SEP>? --- Vt5-8 <SEP> TGG <SEP> TCA <SEP> CTG <SEP> A <SEP>? --- Vt6-10b <SEP> GGA <SEP> AGT <SEP> AGT <SEP> G <SEP>? ---- <SEP>? --- Vt7-lla <SEP> ACCTCGAGCA <SEP>? - <SEP>? -Vt8-12b <SEP> CGG <SEP > CCC <SEP> CTG <SEP> T <SEP>? <SEP> **** <SEP> ****
<tb> Vt9-13 <SEP> ATTGCGTCCA <SEP>? - <SEP>? -Azrl <SEP> GGC <SEP> CCGATC <SEP> G <SEP>? - <SEP>? -Azr2 <SEP> TACCCCGAGG <SEP>? - <SEP>? Azr3 <SEP> TGC <SEP> GGC <SEP> TCA <SEP> G <SEP>? <SEP> *** <SEP> ***
<tb> Azr4 <SEP> TCCCAGCGCG <SEP>? - <SEP>? Azr5 <SEP> GCA <SEP> TGC <SEP> ACG <SEP> G <SEP> * <SEP> ** <SEP> **
<tb> Azr6 <SEP> GCC <SEP> AGTCCCG <SEP>? <SEP> ** <SEP>? Azr7 <SEP> GCTGCCCGAG <SEP>? <SEP>? - <SEP>? Azr8 <SEP> GTG <SEP> CGA <SEP> CCA <SEP> C <SEP>? <SEP> ** <SEP> **
<tb> Azr9 <SEP> TTC <SEP> GGC <SEP> GCG <SEP> A <SEP> * <SEP> ** <SEP> **
<tb>

Notes? : primer used but no PCR band -: primer abandoned due to lack of PCR band.

*: PCR band produced **: PCR band not produced The results obtained are reported in FIG. 2, which represents the bands produced by RAPD.

<Desc / Clms Page number 19>

The six primers, which are decamers, made it possible to ensure that the candidate strains were indeed related to A. chroococcum 1901 / 76.116 from the Institut Pasteur (see bands a2 and a5 in Figure 2), while being distinguishing from these (see bands a1, a3, a4 and a6 in Figure 2). This PCR-RAPD profile also makes it possible to distinguish between candidate strains TF31 and GR86, which corroborates the more functional analysis of these two strains as a function of the segments of the alg genes (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 (ni 0) characteristic of the candidate Azotobacteraceae type strains, compared to a strain of A. chroococcum 76,112 control.

Identification of biomasses of the invention relative to controls It is recalled that, in general, the in vitro retention protocol of bacterial biomasses with edaphic characteristics best suited to survival, persistence and activity during the period of cultivation of the agronomic crops concerned integrates the following three (3) 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 planktonic characters These biomasses can in fact be identified and distinguished from 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 one. alone, of the 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 said protocol;

<tb>
<tb> Integration <SEP> of the <SEP> Notions <SEP> Biomasses <SEP> candidates <SEP> Number <SEP> of the <SEP> control <SEP> in <SEP> the
<tb> Bacterial <SEP> Retainers <SEP> examples
<tb> 1,2, <SEP> 3 <SEP> AZB <SEP> Na
<tb> 2, <SEP> 3 <SEP> Witness <SEP> Azb <SEP> Type <SEP> 1 <SEP> TRI
<tb> 1, <SEP> 3 <SEP> Witness <SEP> Azb <SEP> Type <SEP> IITM2
<tb> 1, <SEP> 2 <SEP> Witness <SEP> Azb <SEP> Type <SEP> lll <SEP> TM3
<tb> 1 <SEP> Witness <SEP> azb <SEP> 1 <SEP> TM4
<tb> 2 <SEP> Witness <SEP> azb <SEP> 2 <SEP> TM5
<tb> 3 <SEP> Witness <SEP> azb <SEP> 3 <SEP> TM6
<tb>

<Desc / Clms Page number 20>

* TM l - Azb Type 1 witness: 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. By virtue of the presence of a relatively rich matrix substrate, they are therefore in principle above all of an oligotrophic nature and contrary to the principle of active persistence as referred to by the invention; . TM2 - Azb Type II control: These biomasses are isolated in the presence of silica gels by alternating the liquid and solid phases by gradual drying of the supply of distilled water on the surface of the gels which have not received any earth extract, or else having received a soil extract (ie glucido-humic supplement modeled on the target soil) from a soil of origin different from that of the target soil.

* TM3-Azb Type III witness: As with the Type 1 Azb witnesses, these biomasses are isolated from gels, but silicic and / or inorganic and abiotic, on which an earth extract is modeled on the edaphic situation of the original soil /target. An earth extract is brought to the surface of the silicic gel (i.e. installation of a first and last liquid phase), but this is not restored once it has dried.

. TM4 - Control azb 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 the silica gels, and / or other inorganic and abiotic matrix surfaces, with any earth extract.

* TM5 - Azb 2 control: These biomasses are obtained by obtaining protocols with edaphic characteristics but strictly with regard to the carbohydrate and humic complement of the original / target soil; this complement (earth 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.

* 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 special consideration seeking to reproduce in vitro, 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 Witnesses; (a) on the one hand the endogenous azb 3 Witnesses (azb3a), i.e. these biomasses

<Desc / Clms Page number 21>

bacteria obtained in a conventional manner, but directly from a sample of the original / target soil, and (b) on the other hand the exogenous azb 3 Witnesses (azb3b), i.e. 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 graphically represented by ordering the reports of the agronomic results obtained either in microcosms, in greenhouse, or in micro-plots in the following fields (see Figure 9A and B): 'AZB / Witness Azb ratios (I, II and III) versus AZB / Witness Azb ratio (1,2 and 3) * AZB / Witness Azb ratios (1, II and III) versus AZB ratio in favorable conditions / unfavorable to the expression of the AZB effect resulting from the implementation of the invention.

This presentation makes it possible to demonstrate the effectiveness of the bacterial biomasses obtained according to the invention compared with 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 commercialization.

By way of example, 3 sets of experimental data have been reported and are illustrated respectively by Figures 9C, 9D and 9E: * Figure 9C: (a) Relative efficiency of various inoculant preparations from azotobacterial biomasses isolated from two types of soils (F4 and 19); Figure 9D: (b) Relative efficacy 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 aggregates of intact soil; * 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).

<Desc / Clms Page number 22>

Relative AZB / control efficiencies greater than 1.00 denote inoculating preparations that are more effective than conventional preparations. This improvement in efficiency is necessarily attributable to the practice of the invention.

Experimental data comprising conventional Azb controls and Type I Azb controls :. Nitrogen contents / kg of soil Table 3 below shows the mineral nitrogen contents (Nm) of the soils in microcosms incubated in the presence of 1% w / w of corn crop residues inoculated with bacterial preparations according to the invention (AZB) and their witnesses TM6 and TMI (ie internal witnesses).

Table 3

<tb>
<tb> ----------- <SEP> mg <SEP> Nm / kg <SEP> soil --- Tests <SEP> 1 <SEP> Witness <SEP> TM6 <SEP> Witness <SEP > TM1 <SEP> AZB <SEP> AZB / TMI <SEP> AZB / TI \ t6
<tb> Val <SEP> 2 <SEP> 54 <SEP> 57 <SEP> 71 <SEP> 1.25 <SEP> 1.31
<tb> Val <SEP> 3 <SEP> 58 <SEP> 62 <SEP> 64 <SEP> 1.03 <SEP> 1.10
<tb> Val <SEP> 4 <SEP> 57 <SEP> 55 <SEP> 71 <SEP> 1.29 <SEP> 1.25
<tb> Val <SEP> 5 <SEP> 62 <SEP> 62 <SEP> 62 <SEP> 1.00 <SEP> 1.00
<tb> Val <SEP> 6 <SEP> 60 <SEP> 62 <SEP> 63 <SEP> 1.02 <SEP> 1.05
<tb> Average <SEP> 59 <SEP> 62 <SEP> 68 <SEP> 1.10 <SEP> 1. <SEP> 16
<tb>
1 Phase II: from January 98 to December 1999 2 Witness Azb Type I. accumulation of mineral nitrogen (Nm) per kg of soil according to the retention conditions on media such as used in the invention.

In general, the isolation protocol of the invention promotes the retention in vitro at the surface of abiotic materials conditioned so as to harbor 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 demonstrate this rustic oligotrophy, sometimes called zymogeny, the experiment summarized in Table 4 below was carried out:

<Desc / Clms Page number 23>

Table 4 Accumulation of mineral nitrogen (Nm) per kg of soil according to the retention conditions on the media of the invention. The conditions for obtaining bacterial biomasses (azoto) in italics cancel out the effectiveness of the protocol of the invention, by establishing retention conditions that are too rich in carbonaceous substrates.

<tb>
<tb>

Pre-incubation <SEP> Presence <SEP> of <SEP> glucose <SEP> By <SEP> report
<tb><SEP> soils <SEP> on <SEP> the <SEP> environments <SEP> of <SEP> azb, <SEP> witness
<tb> With <SEP> cellulose <SEP> retention <SEP> conventional <SEP> mg <SEP> Nm / kg <SEP> sol3
<tb> yes <SEP> yes <SEP> 1. <SEP> 02 <SEP> 26 <SEP> (3)
<tb> no <SEP> yes <SEP> 0. <SEP> 99 <SEP> 26 <SEP> (l)
<tb> yes <SEP> no <SEP> 1. <SEP> 14 <SEP> 29 <SEP> (2)
<tb> no <SEP> no <SEP> 1.38 <SEP> 36 <SEP> (2)
<tb> 3 <SEP> Microcosms <SEP> of <SEP> 25 <SEP> g <SEP> of <SEP> soil <SEP> Terrefort <SEP> reconstituted <SEP> on <SEP> 28 <SEP> days <SEP > incubation <SEP> to <SEP> 22 C
<tb>
Control experimental data Azb Type II (TM2):. Effectiveness of the protocol of the invention for promoting the production of bacterial biomasses (azoto) according to the edaphic nature of the original and target soil.

The results obtained are summarized in Table 5 below: The contents of mineral nitrogen (Nm) / kg of soil are given in FIG. 3 by inoculating various controls and candidate strains, in micromes and on microplots.

<Desc / Clms Page number 24>

Table 5

<tb>
<tb> Biomass <SEP> Nature <SEP> edaphic <SEP> Nature <SEP> edaphic <SEP> Report <SEP> AZB / Azb <SEP> Tvpe <SEP> II <SEP> (TM2)
<tb> candidates <SEP> of the <SEP> soil <SEP> of origin <SEP> of the <SEP> soil <SEP><SEP> target <SEP><SEP> First <SEP> series <SEP> Second <SEP > series
<tb> Witness <SEP> T <SEP>: 'I12 <SEP> Argilo-silt <SEP> Arglle <SEP> 1. <SEP> 52 <SEP> 1. <SEP> 07
<tb> TMHMKn <SEP> TAf2 <SEP> Silt <SEP> Clay <SEP> 0. <SEP> 75 <SEP> 0.89
<tb> Witness <SEP> trin <SEP> Sandy <SEP> Clay <SEP> 0.63 <SEP> 1. <SEP> 31
<tb> AZB <SEP> Clay <SEP> Clay <SEP> 2.02 <SEP> 2.81
<tb>
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% W / V of cellulose.

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

Under such conditions, the strains obtained by means of the protocol of the invention (AZB) should be little or no more efficient than the conventional azobacterial biomasses (Azb). In addition, a 1% glucose solution sufficient to overcome 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 hardy bacterial biomasses (azoto) (see table).

It should be noted that only the complete absence of the carbonaceous substrates provided makes it possible to obtain, according to the protocol described above, AZB bacterial biomasses (azoto) which are truly more efficient than the azotobacterial azb biomasses isolated in a more conventional manner.

This experiment perfectly demonstrates the roles of oligotrophy during the retention of the most hardy and effective bacterial biomasses (azoto) once reintroduced into edaphic zones as inocula. It should be remembered that this condition for obtaining, which is oligotrophy, is necessary but not sufficient to ensure the isolation of the most edaphic bacterial strains. In fact, hardiness and resistance to nutritional stress do not correspond exclusively to soil bacteria, but remain

<Desc / Clms Page number 25>

nevertheless here essential given the recrudescence of 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 nitrogenase activity over time measured according to the ratio of the activity of the reduction acetylene (ARA) at time t2 and at time t1.

The results obtained are summarized in Table 6 below:
Table 6

<tb>
<tb> Test <SEP> 1 <SEP> Test <SEP> 2 <SEP> Test <SEP> 3
<tb> Microcosm <SEP> Nm <SEP> ARA3 <SEP> NmARA * <SEP> NmARA
<tb> Residues <SEP> from <SEP> culture <SEP> 28 <SEP> 3.87 <SEP> 10.8 <SEP> 22 <SEP> 60 <SEP> 1.6
<tb> Residues <SEP> from <SEP> culture <SEP> with <SEP> control <SEP> TM5 <SEP> 29 <SEP> 4.42 <SEP> 14.8 <SEP> 45 <SEP> 66 <SEP> 2.1
<tb> Residues <SEP> of <SEP> culture <SEP> with <SEP> AZB <SEP> 40 <SEP> 6.67 <SEP> 15.3 <SEP> 112 <SEP> 72 <SEP> 2.7
<tb>
. Mineral nitrogen content (Nm) of soils in microcosms 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) 3 t2 = 26 h after start incubation of microcosms; t1 = 22 h 4 t2 = 72 h after the start of incubation of the microcosms; t1 = 24 h 5 t2 = 66 h after the start of incubation of the microcosms; tl = 4 p.m.

<Desc / Clms Page number 26>

The results are summarized in Table 7 below
Table 7

<tb>
<tb> ---- <SEP> mg <SEP> Nm / kg <SEP> soil --- Tests <SEP> Phase <SEP> 16 <SEP> indicator <SEP> TM6 <SEP> indicator <SEP> TMI <SEP> AZB <SEP> AZB / TM] <SEP> AZB <SEP> TM6
<tb> II9 <SEP> 51 <SEP> Na <SEP> 56 <SEP> Na <SEP> 1.10
<tb> III <SEP> 46 <SEP> Na <SEP> 61 <SEP> Na <SEP> 1. <SEP> 33
<tb> IV <SEP> 53 <SEP> na <SEP> 54 <SEP> Na <SEP> 1.02
<tb> Eal <SEP> 53 <SEP> na <SEP> 54 <SEP> Na <SEP> 1.02
<tb> Ea2 <SEP> 51 <SEP> na <SEP> 56 <SEP> Na <SEP> 1. <SEP> 10
<tb> Ea3F4 <SEP> 51 <SEP> na <SEP> 56 <SEP> Na <SEP> 1.10
<tb> Ea319 <SEP> 52 <SEP> na <SEP> 55 <SEP> Na <SEP> 1.06
<tb> Ea4 <SEP> 49 <SEP> na <SEP> 58 <SEP> Na <SEP> 1. <SEP> 18
<tb> ea4bis <SEP> 52 <SEP> na <SEP> 55 <SEP> Na <SEP> 1.06
<tb> Ea4tris <SEP> 53 <SEP> na <SEP> 54 <SEP> Na <SEP> 1.02
<tb> Average <SEP> 51 <SEP> na <SEP> 56 <SEP> Na <SEP> 1.10
<tb> Tests <SEP> Phase <SEP> II <SEP> Indicator <SEP> TM6 <SEP> indicator <SEP> TM1 <SEP> AZB <SEP> AZB / TM <SEP>! <SEP> AZB-TM6
<tb> Val <SEP> 2 <SEP> 54 <SEP> 57 <SEP> 71 <SEP> 1.25 <SEP> 1.31
<tb> Val <SEP> 3 <SEP> 58 <SEP> 62 <SEP> 64 <SEP> 1.03 <SEP> 1.10
<tb> Val <SEP> 4 <SEP> 57 <SEP> 55 <SEP> 71 <SEP> 1.29 <SEP> 1.25
<tb> Val <SEP> 5 <SEP> 62 <SEP> 62 <SEP> 62 <SEP> 1.00 <SEP> 1.00
<tb> Val <SEP> 6 <SEP> 60 <SEP> 62 <SEP> 63 <SEP> 1.02 <SEP> 1. <SEP> 05
<tb> Average <SEP> 59 <SEP> 62 <SEP> 68 <SEP> 1. <SEP> 10 <SEP> 1.16
<tb> 6 <SEP> Phase <SEP> 1 <SEP>: <SEP> from <SEP> September <SEP> 96 <SEP> to <SEP> December <SEP> 97
<tb> 7 <SEP> Phase <SEP> Il <SEP>: <SEP> from <SEP> January <SEP> 98 <SEP> to <SEP> December <SEP> 99
<tb>
Figure 5 gives the mineral nitrogen contents (mg Nm / kg soil) 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 (conventional control azb-inocula consisting of conventionally isolated azotobacterial strains; silicic gels (GS) packaged according to the invention plus various para-humic substances (SpH 1, 2 and 3), a polycondensate of catechol (PC) and a SpH).

. Kinetics of degradation of crop residues in the field over a period of 5 months (from October 2000 to March 2001) on red chestnut soil type, Inra experimental area, Lusignan (86).

<Desc / Clms Page number 27>

The results obtained are reported in Table 8 below:
Table 8

<tb>
<tb> Inocula <SEP> according to <SEP> Coefficient <SEP> of <SEP> g <SEP> residues <SEP> remaining <SEP> by <SEP> report
<tb> the invention <SEP> and <SEP> degradation8 <SEP> Coefficient <SEP> R2 <SEP> to the <SEP> witnesses <SEP> non-inoculated <SEP> (t
<tb> cookies-ka <SEP> x <SEP> 10-2 <SEP> = <SEP> 5 <SEP> months)
<tb> Witness <SEP> TM6 <SEP> 37 <SEP> 0. <SEP> 93 <SEP> 1. <SEP> 47
<tb> Witness <SEP> TM2 <SEP> 41 <SEP> 0.97 <SEP> 1. <SEP> 11
<tb> AZB <SEP> 47 <SEP> 0. <SEP> 91 <SEP> 0. <SEP> 91
<tb>

8 Selon la cinétique de dégradation dRC = A x e-kt, où dRC représente la quantité de résidus de culture dégradés au temps t, et A la quantité de résidus au temps 0, soit 4. 00 g de matière sèche.

8 According to the degradation kinetics dRC = A x e-kt, 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 (RC) -effect of a finer grinding on the use of substrates resulting from their degradation and in principle usable by the bacterial biomasses (nitrogen) thus brought to the surface of these residues.

The results obtained are summarized in Table 9 below Table 9

<tb>
<tb> -------------------------- <SEP> mg <SEP> Nm / kg <SEP> Soil ------- -Inocula <SEP> Grind <SEP> Fine <SEP> of <SEP> RC <SEP> Grind <SEP> Coarse <SEP> of <SEP> RC
<tb> Witness <SEP> TM6 <SEP> 58 <SEP> 52
<tb> Witness <SEP> TM1 <SEP> 52 <SEP> 48
<tb> AZB <SEP> 82 <SEP> 55
<tb>
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 content (Nm) of the microcosms compared to the inoculated TM6 controls (ie isolated nitrogenobacterial biomass conventionally) and TM1 (ie azotobacterial biomass isolated according to an inefficient variant of the invention.

<Desc / Clms Page number 28>

. 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 multiflorum and Triticum aestivum according to the method of cultivation in pots ( greenhouse) during the incubation of cereal crop residues (wheat straw) over a period of 28 to 56 days (Test 5) with and without the various inocula of the invention and their controls TM6 and TM1.

Table 10

<tb>
<tb> mq <SEP> of <SEP> biomass
<tb> Mode <SEP> of <SEP> culture <SEP> in <SEP> pot <SEP> Test1a <SEP> Test2a <SEP> Test3b <SEP> Test4b <SEP> Test5b
<tb> Residues <SEP> from <SEP> culture <SEP> 9.1 <SEP> 7.1 <SEP> 33 <SEP> 37 <SEP> 35
<tb> Residues <SEP> of <SEP> culture <SEP> with <SEP> witness <SEP> TM6 <SEP> 8.7 <SEP> 5.9 <SEP> 39 <SEP> 32 <SEP> 37
<tb> Residues <SEP> of <SEP> culture <SEP> with <SEP> witness <SEP> TM1 <SEP> na <SEP> 8.1 <SEP> 39 <SEP> 32 <SEP> 37
<tb> Residues <SEP> of <SEP> culture <SEP> with <SEP> witness <SEP> TM2 <SEP> na <SEP> na <SEP> 39 <SEP> 32 <SEP> 37
<tb> Residues <SEP> of <SEP> culture <SEP> with <SEP> AZB <SEP> 10.0 <SEP> 8.7 <SEP> 40 <SEP> 41 <SEP> 39
<tb> Note <SEP>: <SEP> a <SEP>;<SEP> g <SEP> of <SEP> biomass <SEP> Lohum <SEP> multiflorum <SEP> by <SEP> pot
<tb> b <SEP>;<SEP> mg <SEP> of <SEP> biomass <SEP> Triticum <SEP> aestivum <SEP> per <SEP> seedling
<tb>
B) Percentage (%) increase in grain yields (yield at 15% humidity) and PMG (Thousand grain weight) during agronomic field trials (1999-2000 campaign in Toulouse 31 on Terrefort soil, and 2000-2001 campaign in Lusignan on red chestnut soil) compared to non-inoculated controls paired with TM6, TM1 and AZB.

Table 11

<tb>
<tb> Rdt <SEP> 15% <SEP> hum <SEP> PMG
<tb> Mode <SEP> from <SEP> culture <SEP> to <SEP> field <SEP> Toulouse <SEP> Lusignan <SEP> Toulouse <SEP> Lusignan
<tb> Residues <SEP> of <SEP> culture <SEP> with <SEP> control <SEP> TM6 <SEP> 4% <SEP> 0. <SEP> 73% <SEP> 5% <SEP> 0. <MS> 08%
<tb> Residues <SEP> of <SEP> culture <SEP> with <SEP> control <SEP> TM1 / 2a <SEP> 13 <SEP>% <SEP> 0. <SEP> 86% <SEP> 4% <SEP> 0. <SEP> 02%
<tb> Residues <SEP> of <SEP> culture <SEP> with <SEP> AZB <SEP> 20% <SEP> 1. <SEP> 47% <SEP> 6% <SEP> 0. <SEP> 94%
<tb> Note <SEP>: <SEP> a <SEP>;<SEP> Witnesses <SEP> TMI <SEP> for <SEP> Toulouse, <SEP> TM2 <SEP> for <SEP> Lusignan
<tb>
C) Coincidence of increases in yields and PMG during agronomic trials in the field of the 2001-2001 campaign in Lusignan on red chestnut soil soil and the various indices of valuation of crop residues obtained in parallel on these same plots.

<Desc / Clms Page number 29>

Table 12

<tb>
<tb> Mode <SEP> from <SEP> culture <SEP> to <SEP> field <SEP> Rdta <SEP> PMGb <SEP> -ktC <SEP> dRCd <SEP> dRCe
<tb> Residues <SEP> of <SEP> culture <SEP> with <SEP> control <SEP> TM6 <SEP> 63 <SEP> 0. <SEP> 08% <SEP> 41 <SEP> 2.10 <SEP> 0.70
<tb> Residues <SEP> of <SEP> culture <SEP> with <SEP> control <SEP> TM2 <SEP> 71 <SEP> 0. <SEP> 02% <SEP> 37 <SEP> 2.02 <SEP> 0.81
<tb> Residues <SEP> from <SEP> culture <SEP> with <SEP> AZB <SEP> 126 <SEP> 0.94% <SEP> 47 <SEP> 2.42 <SEP> 1.19
<tb> Note <SEP>: <SEP> a <SEP>;<SEP> increase <SEP> in <SEP> yields <SEP> in <SEP> kg <SEP> grain / ha <SEP> at <SEP> 15% <SEP> humidity
<tb> b <SEP>;<SEP> weight <SEP> thousand <SEP> grains <SEP> of <SEP> wheat <SEP> by <SEP> ratio <SEP> to the <SEP> controls <SEP> non-inoculated <SEP> paired <SEP> to <SEP> indicators <SEP> TM2 / 6 <SEP> and <SEP> AZB
<tb> c <SEP>;<SEP> coefficient <SEP> of <SEP> degradation <SEP> x <SEP> 104 <SEP> according to <SEP> dRC <SEP> = <SEP> A * ed <SEP>;<SEP> degradation <SEP> of <SEP> residues <SEP> of <SEP> culture <SEP> (4.00 <SEP> g <SEP> to <SEP> tO) <SEP> on <SEP> 164 <SEP > days
<tb> e <SEP>;<SEP> degradation <SEP> of <SEP> residues <SEP> of <SEP> culture <SEP> by <SEP> report <SEP> to <SEP> controls <SEP> non-inoculated <SEP> paired <SEP> to <SEP> testimonials <SEP> TM2 / 6 <SEP> and <SEP> AZB
<tb>
. 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 grown on soils incubated in the presence of treated cereal crop residues (wheat straw). and not treated with the inocula according to the invention and their controls TM6 and TM2.

<tb>
<tb>

Treatment <SEP> of <SEP> residues <SEP> of <SEP> crop --- report <SEP> vegetation / roots <SEP> (Index <SEP> Vegetative) ----------- Test <SEP> 1 <SEP> Test <SEP> 2 <SEP> Test <SEP> 3 <SEP> Average
<tb> Witnesses <SEP> TM6 <SEP> 0.85 <SEP> na <SEP> na <SEP> 0.85
<tb> Witness <SEP> TM2 <SEP> na <SEP> na <SEP> na
<tb> TF31 <SEP> (AZB <SEP> TF31) <SEP> 0.90 <SEP> na <SEP> na <SEP> 0.90
<tb> Witness <SEP> TM6 <SEP> 1.02 <SEP> 0.90 <SEP> 1.03 <SEP> 0.98 <SEP> (0.03) <SEP> 9
<tb> Witness <SEP> TM2 <SEP> na <SEP> 1.03 <SEP> 1.09 <SEP> 1.06 <SEP> (0.04)
<tb> GR86 <SEP> (AZB <SEP> GF86) <SEP> 1.05 <SEP> 0.92 <SEP> 1.36 <SEP> 1. <SEP> 11 <SEP> (0. <SEP> 09)
<tb> 9 <SEP> (standard deviation)
<tb>
. Delay of the effect of nitrogen immobilization compared to that obtained on control crop residues treated with an antibiotic.

We are studying the effect of treating cereal crop residues (wheat straw) with an antibiotic (ampicillin) at an agronomic rate (1000 kg / 10 Mg of residues / hectare (or about 1000 Mg of soil)).

<Desc / Clms Page number 30>

The results obtained are summarized in Table 14 below.

Table 14

<tb>
<tb> Mode <SEP> of <SEP> culture <SEP> in <SEP> pot <SEP> Test <SEP> 1Test <SEP> 2Test <SEP> 3Average
<tb> Residues <SEP> from <SEP> culture <SEP> 33 <SEP> 37 <SEP> 35 <SEP> 35 <SEP> (2) <SEP> 10
<tb> Residues <SEP> from <SEP> culture <SEP> with <SEP> antibiotic "37 <SEP> 39 <SEP> 38 <SEP> 38 <SEP> (1)
<tb> Residues <SEP> of <SEP> culture <SEP> with <SEP> AZB <SEP> 40 <SEP> 41 <SEP> 39 <SEP> 40 <SEP> (1)
<tb>'"<SEP> (deviation <SEP> standard)
<tb> Ampicillin
<tb>
The effect observed 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 attenuation of the immobilization of assimilable nitrogen allows greater plant growth of the tested seedlings in place; this increase in vegetative growth is similar 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.

FIG. 6 gives a graphic representation of three VB9 experiments with the use of ampicillin as an antibiotic control. It will be observed that ampicillin is more favorable to vegetative growth than the controls inoculated 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 relative to their uninoculated twin controls is studied.

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

. effect of the granulometry of soils The results relating to the study of the relative efficiency of the bacterial biomasses (nitrogen) of the invention for maintaining the mineral nitrogen content of soils are reported in FIGS. 8A and SB.

<Desc / Clms Page number 31>

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 AZB biomasses are effective and that this efficiency compared to inoculated (azb and Azb) and uninoculated (control) controls is no longer observed when the biomasses evolve within the 1 mm fraction.

<Desc / Clms Page number 32>

Bibliographical references Burucoa, C.; V. Lhomme and J. L. Fauchere. 1999. Performance criteria of DNA fingerprinting methods for typing of Heliobacter pylori isolates: experimental results and meta-analysis. J. 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.

:"Bioremediation : principals and practice, Vol. I"-Fundamentals and Applications (S. K. Davies, L .; HC. Flemming and PA Wilderer. 1998. Microorganisms and their roel in soil. In

: "Bioremediation: principals and practice, Vol. I" -Fundamentals and Applications (SK

Sikdar and R. L. Irvine, eds.). Technomic Publ. Inc., Lancaster (UK), Basel (CH).

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

Pochon, J. and Y-T. Tchan. 1948. Precise 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 (12)

1. Process for obtaining in vitro bacterial biomasses of edaphic origin, characterized in that it comprises the steps of: bringing an aqueous suspension of soil into contact with a matrix substrate, so as to form a hydrated zone , or biofilm, on the surface of the substrate, similar to that added to the ACH of the soil, essentially housing the indigenous bacterial component of the soil, in order to retain on the surface of the matrix substrate, most of the bacterial microflora endogenous to the soil. original edaphic environments. - maturation of the biofilm with establishment of oligotrophic conditions allowing the cells most constitutive of the edaphic biofilm which have colonized the matrix substrate, to migrate to the liquid phase supernatant the matrix substrate, and gradually to dominate it, - recovery and culture of the bacterial strains. more prolific in medium of rich liquid cultures, with an autochthonous character, hardy and persistent in situ, cultivable in liquid medium, and therefore suitable for being produced industrially. 2. Method according to claim 1, characterized in that the matrix substrate is an inorganic and abiotic matrix substrate, in aqueous medium. 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, carbon compounds and in particular mono -, oligo-and polysaccharides, of plant, animal and bacteriofungal origin, nutrients. 4. Method according to any one of claims 1 to 3, characterized in that the maturation of the biofilm is carried out by alternating a phase in which the biofilm formed on the abiotic surface is not in the presence of a supernatant liquid phase, with a liquid phase comprising all the bacterial cells supernatant the solid phase. 5. Isolated bacterial biomasses, characterized in that they comprise, such as <Desc / Clms Page number 34> dominant strains, Gram negative bacterial strains with edaphic character, autochthonous, oligotrophic, mostly aerobic. 6. Biomasses according to claim 5, characterized by the following phenotypes expressed in vitro and in situ relative to control bacterial biomasses obtained from biotic surfaces, but without the presence of a biofilm or a humic complement 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 supplied in a quantity greater than the concentrations normally present in the soil, and / or obtained from unconditioned abiotic surfaces, the aforesaid Bacterial biomasses are more mucoid than these controls, more encysted and more durable, less copiotrophic than said controls, and produce self-induction factors at higher levels than the controls, for example octanoyl homoserine lactone. 7. Bacterial biomasses according to claim 6, characterized in that, compared 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, Pseudomonaceae. , or Rhizobiaceae. 8. Bacterial biomasses according to any one of claims 5 to 7, characterized in that it is Azobacteriaceae, Pseudomonaceae, or Rhizobiaceae. 9. Bacterial strains isolated from bacterial biomasses 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 8 and of the bacterial strains according to claim 9 or 10, to the microbiological bacterization of soils and crop residues, in particular cereal crop residues, including those. corn for grain. 12. Use according to claim 11, characterized by inoculating said <Desc / Clms Page number 35> bacterial biomasses and strains by spraying formulations containing them on crop residues.