CA2467441A1 - 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 protocol and their use for the bacterization of soils and crop residues.
The bacterization of arable soils consists in bringing, by means (mi cro) biological to soil and to agronomic crops, part of the nutrients, in particular diatomic nitrogen from the atmosphere, necessary for obtaining yields economic and sustainable.
Traditionally, bacteria have mainly been used strain inocula Rh: ~~ obizrm for the coating of seeds of Legranirzosecc species such as soy, alfalfa and red clover.
It has also been proposed to use endomycorrhizae, acetobacters diazotrophs endophytes, PGPB (Plant Growth Promoting Bacteria) rhizobacteria micro algae and cyanobacteria, where there are non-symbiotic diazotrophic bacteria in association with crop residues.
The use of bacteria in agronomy, and more particularly in cereal production, has encountered, however, the problem of their non-persistence, once reintroduced in soils. Of this fact, they cannot ensure the achievement of the agronomic effect sought, namely the increase and stability of grain yields, their proteins, and / or the reduction of input levels, especially mineral nitrogen.
It follows that the bacterial biomasses proposed to date do not allow not one satisfactory use of compounds from degradation iti sitar (ie in the field) agricultural crop residues, especially crop residues intensive cereal, including including grain corn.
The soluble and assimilable compounds resulting from the ih situ degradation of crop residues cereals are poorly valued by the whole soil microflora, and more particularly its diazotrophic and non-symbiotic component called a ~ otobacterial. This lack of enhancement explains er ~ good part the realization of phenomena, sometimes harmful, WO 03/046156 ~ PCT / FR02 / 04119 well known ; (1) the continual presence of agronomic quantities of residues strawy soil surface at the time of agricultural work, (2) unc; immobilization of nitrogen through them non - diazotrophic microorganisms in .sol causing "nitrogen hunger"
in culture in place, and, paradoxically, (3) an appreciable drop in carbon content (soluble, labile and total) and microbiotic biomass.
Another problem encountered with bacteria is related to the isolation and their production in mass traditionally carried out by serial dilution of a suspension of soil and setting cultivation of an aliquot of a further dilution of this aliquot on a medium rich agar. The scuchN: thus isolated, once sufficiently characterized, serve by the following sowing conventional bioindustrial fermenters. The ability of inocula as well products to sustainably increase the activity of the organizations involved, the diazotrophy in soil arable amended with crop residues for example, is therefore today very weak and / or random. The use of various porous supports, solid or organic, to protect these bacteria once reintroduced) into the soil, does not seem to have been able to allow a more great ei ~ icacity of this type of inoculation.
The study of these problems by the inventors led them to develop conditions isolation of bacteria from the soil using protocols and: media isolation, or detention, 1fl hltl'O, capable of ensuring the maintenance and survival of the sou ~~ hes most capable bacteria active persistence in edaphic media, and bacteria satisfactory soil once (re) introduced, thanks to their properties of persistence in situ.
Historically, there are three approaches to obtaining strains bacterial of edaphic origin (i.e. arable soils) in principle the best adapted to the active persistence in sittc once reintroduced at. isolation on abiotic matrices: with the use of characters edaphic (ie silica gels with or without earth extracts), without as much to do integration of the main components (silicic, humic, carbonaceous, and hydrological) of these environments;
b. pull-incubation on abiotic supports: this is a pre-incubation in the middle edaphic, or within a more or less female fraction of it, of cell reindeer bacteria already isolated and conventionally cultivated;

J

vs. culture in depleted environments: we are impoverishing voluntary, especially carbon, culture media for isolated bacterial cells conventionally. ..
The solution provided by the invention integrates these 3 approaches, and more particularly approach a), within da obtaining protocol defined below, which leads to obtaining bacterial strains and biomass constituting, in terms of adaptation, persistence and of activity irz sitr. ~, and that beyond a simple conditioning c ~
posteriot ~ i bacteria already isolated and / or cultivated according to approaches a), b) and / or c) due to:
conventional.
In addition, the invention takes into account the process of formation of biofilms in middle aqueous. In this regard, the inventors considered that the. edaphic zone the more consistent l7pL! r soil bacteria corresponds to the adjacent hydrated zone (biofllm) to complexes clay-humic (CAH) of the soil, and that the formation and functioning of this is probably subject to the same mechanisms as those involved in training and the functioning of biofilms in the broad sense.
According to Characlclis (1990) and Characklis and Marshall (1990) the accumulation of biofilm at smrface cl '~ .zrle matrix, biotic or abiotic, is the result of eight physico- process chemical and biological dlStïIlCtS
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 G. Growth such as this, the bacteria constituting the biofilm at the expense of the sentence supernatant aqueous 7. Attachment of cells and particles in suspension in the phase aqueous 8. Release of cells making up the biofilm by displacement volumetric erosion, wear, etc. ...

WO 03/04615

6 4 PCT / FR02 / 04119 The realization of these processes allows in principle the formation of a biofilm to two phases (solid and liquid), and more precisely with five compartments 1. A matrix substrate (silica gels or agas for example) ?. The basic biofilm containing most of the bacterial cells 3. The surface biofilm containing essentially the cells Additional ~ 4. The supernatant liquid phase 5. The ultimate aqueous phase Gr, i1 turns out that biofilm as such consists mainly of compartments 2, 3 and 4.
According to the most recognized edaphic model (Davier et al. 1998; Gordienko 1990 ; Pouch and Tchan 1948; Pochon and De Barjac 1958), is the bacterial microflora also incorporated essentially three components, the most rustic Aboriginal component and oligotrophic, the rather copiotrophic zymogenic component associated with this component indigenous, and finally ia component, very marginal but easily isolable, essentially zymogen and t ~~ s copiotrophe.
The invention is based on an original conceptual rapprochement between the descriptions of bionlms in the broad sense and the hydrated zone adjoining the CAI3s of the soil.
i ~ lle then aims to provide a process for obtaining biomass bacterial essentially made up of persistent and active strains, once reintroduced into soil original target, characterized by the integration of APC (agro-pedo ), in particular by interacting with the CAHs and ISL (ground-lignocellulosic) of an identifiable edaphic zone.
It also targets bacterial biomass and the new strains of these biomass with the metabolabolophysiological characteristics commonly sought, such as diazotrophy, fungistatic and / or phytosanitary activity, production and the secretion of growth regulators, catabolism of xenobiotics, etc.).
The invention further relates to the use of these bacterial biomasses and strains for the bacteria from soils and crop residues, especially crops cereal.

i, e process for obtaining ifr panes of original bacterial biomass edaphic is characterized in that it includes the steps of - bringing an aqueous soil substrate into contact with a matrix substrate, so that ~
close a hydrated area, or biofilm, on the surface of the substrate, similar to the one attached to ~~ '.,. I ~ from the ground; mainly hosting the bacterial compo5at indigenous to the soil, to retain, on the surface of the matrix substrate, the essentials of the microflora endogenous bacterial to original edaphic environments e.
- maturation of the biofilm with establishment of oligotrophic conditions allowing most constitutive cells of the edaphic biofilm which OIIt colonized the matrix substrate, of migrate to the lic ~ iuide phase supernatant said substrate, and gradually to dominate her, recL: per ~~ tion and culture of the most bacterial strains. prolific in cultural backgrounds rich, indigenous liquids, rustic and persistent irz sit2r, cultivable in environments liyaicles and therefore suitable for being produced ~~: ites industrially.
~ sn uses in this process an inorganic and abioüc matrix substrate, in aqueous medium, faVCrïSaIlt 121 prOdl. ~ CtrC312 of exopolysaccharides (EP ~) and :. other components of the glycocalyx bacterial, as well as the networking of a sufficient number of capable cells to initiate the first steps of forming a biofilm at 1 <~ surface of substrate.
materials suitable for use as substrates include silica, polystyrene, clay, porous glass or vermiculüe.
The water in the aqueous suspension contains compounds present in the relative proportions characteristics of the original target soil, namely humic substances and fulvic, carbon compounds and in particular mono-, oligo- and polysaccharides, of origin plant, animal, and bacteriofungal, nutrients.
The biefilm maturation is carried out by alternating a phase where the biofilm formed on the surface abiotic is not in, .. presence of a supernatant liquid phase, with a liquid phase CU111prellaTlt all the bacterial cells supernat the phase solid.

This maturation results in principle from the migration of native bacteria to the phase biofilm liquid formed on the matrix substrate. Move volumetric only takes place course and after aging (maturation) of biofilm (i.e. biofilm on the surface of the matrix substrate). This maturation allows the release, in the phase aqueous supernatant matrix substrate, the most constitutive bacterial cells of this biofilm.
Maturation of biofiln: can be facilitated / increased if the liquid phase supernatant is periodically dried, or dt ~ less removed, fa ~~ on to favorïs the growth of bacterial biomasses making up the biofilm, which are mostly aerobic. She is then restored by adding aseptic water. This addition can be performed for example by microfiltratior ~.
The invention thus makes it possible to reconcile microbialogical diversity (especially bacterial) soils with microbiological purity of biomass dev, ~ .nt be produced in bioprocess conventional, which has never really been attempted so far.
Isolation of bacterial strains is in fact mainly carried out using relatively crops rich in metabolic characteristics (eg re:> resistance to certain antibiotics, in vitro diazotrophy ~~ o, specific activities of certain enzymes: ~ mes, etc.) and / or genomics (eg. to using specific oligo-nucleo probes from certa.ius of agronomic interest or industrial) easily identifiable, but which have basically little, or nothing to do with 'cda'j' ~ tatlUli and the active persistence of these bacterial cells natives rather than zymogens once (rë) introduced into various edaphic media.
The protocol for obtaining bacterial biomasses of the invention favors so dominance microbiological of bacterial strains with edaphic characters, and ensures nevertheless that these also remain sufficiently copiotrophic (i. ~~. capable of growing in circles rich) to be produced in industrial quantities by means of conventional bioprocesses.
On the contrary, the bacteria obtained according to processes which do not integrate dispositions that precede within the same protocol are ineffective. It's like that o bacterial biomass 1) obtained from biotic surfac ~ ds but without the presence a biofilm or humic supplement, and / or bacterial bibmasses 2) obtained from surface; abiotic according to protocol in WO 03/046156 ~ PCT / FR02 / 04119 question, but a) with a humic complement not modeled on that of the target soil and or b) with a carbon substrate provided in an amount greater than the concentrations normally present in the soil,. and / or ~ bacterial biomass 3) obtained from non-abiotic surfaces conditioned (ie without the development of a hydrated film or iofilm b).
will illustrate this derr ~ onstraticn, a series J'inocula witnesses ntégrant not at least one of the concepts envisaged above has been produced, namely notion l: adhesion to surfaces inorganic and abiotic; concept 2: oligotrophic growth from complements carbohydrates and humics; notion 3: character expression: benthic and planktonic, the direct comparison of the effects obtained with the biomasses of the invention and with these witnesses allows to interfere that the integration of the 3 notions (1,2,3) mentioned above is possible for obtain the desired results and constitute a means of identifying biomass of the invention as set out in the examples.
~ According to a preferred embodiment of the method of the invention of isolation progressive from rustic and persistent bacterial biomass zero situ, we have advantageously use ~
following steps a) - immediate and temporary retention of most of the microflora endogenous bacterial to the original edaphiclus mediums In order to make this retention step less drastic ~; n terms elimination and loss soucr ~ es less copiotrophic, we proceed to a pre-incubation of the substrate matrix with the soil suspension containing 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 setting contact with a retention medium essentially modeled on the soil characteristics original target. These characteristics include, but are not limited to: (1) humic substances and fivlviques and their constitutions in terms of the various phenolic compounds and benzoic, (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 part mineral soil, and s ~ u ~: out most nne, or at least excluding aggregates greater than 5 oh 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 from the retention media allows the formation of satisfactorily with a hydrated film or biofilm on the surface of matrix substrates solid.
Bacterial strains, which gradually come to dominate on the surface of these substrates and within these biofilins, are thus more specifically adapted to the APC on which the constitution of these biofilms has been modeled and are therefore also more able to persist actively once reintroduced in these APCs.
b) - progressive dominance of the desired bacterial strains, diazotrophs and / or fungus static for example, on these specific retention media and characteristics of environments original edaphic Culture or retention media become chu facto the source of potential Candidate bacterial biomass. Bacterial cells being good producing exo-polysaccharides (EPS) used to form biofilms on the surface of the substrate, they can also be brought to dominate the liquid phase r restored on its surface.
Indeed, the biofilms thus formed can mature in this sense. It's selling this process of maturation qz: e the multiplication of biofilm cells makes it possible to: move it gradually the cells bacterial to the surface: biofilm, by a process called displacement volumetric (Characl> iis 90 and Characklis and Marshall 90 above). These cells get possibly find free in the liquid phase supernatant at the surface of ~ biofilm. Insofar as this liquid phase and the biof ~ lm substrate are almost devoid assirr ~ ilable mineral nitrogen, most of the cells concerned will azotobactériennes.
c) - isolation, by microbiological dominance, of the bacterial strains searched for them more prolific in rich liquid culture environments and conventional The biomasses and strains of the invention, although relatively more oligotrophs as more conventional strains, must nevertheless remain cultivable in environments rich conventional, as used in industrial bio-processes.
However, and since the aliquots coming from the liquid phases suuissant the media obtaining these strains are not necessarily of absolute microbiological purity it there is a risk that the desired candidate strains they contain are moved (ie dominated in WO 03/046156 ~ PCT / FR02 / 04119 number) by more copiotrophic contaminations and contrary to purpose research. To avoid as much as possible of such contamination, the culture medium is restored with an aliquot containing approximately 90% cells. from the supernatant phase environments of production and only around 10% of cells actually pre-incubated in order to possibly favor the dominance of rather oligotrophic cells. After at the third, even fourth, pre-incubation cycle, an aliquot of 1 ~? 1000 approximately of the latter pre-treatment will be transferred to a conventional rich liquid medium, with nitrogen, cells which contain this aliquot can then be mass produced and way conventional.
It is recommended to follow up and control: the purity of these biomass and strains in addition to a series of non-i: emotional genomic profiles RAPD, and more functional according to the particular expression of some of the most important in term of edaphic adequacy (eg. czlg, nzurc, eps, pc ~ b, ~ lzl, etc.).
The candidate prototype inocula are formulated and packaged for their use at field.
Liquid and solid formulations not only ensure a timeframe.
satisfactory conservation of a minimum of 6 months, ie. 4 ° C, i.e.
ambient temperature (ie from 18 to 27 ° C), but also to ensure even more; a pre-cell adaptation bacteria already selected according to the present invention.
The invention also relates to bacterial biomasses, i: oleas, characterized in that they include, as dorr ~ inantes strains, GRAM negative bacterial strains, at edaphic, indigenous, eligotrophic, mainly aerobic, presenting metabolophysiological characteristics of diazotrophy, fungal activity static etlou plant.
Advantageously, these bacterial biomasses are capable of growing easily.
in circles liquids, which allows them to be produced in industrial quantities by bioprocess conventional.
As illustrated by the examples given below, these biomasses are characterized by following phenotypes expressed in vit ~~ o and / or in situ: compared to isolated witnesses by all WO 03/046156 1 ~ PCT / FR02 / 04119 or part of the conventional protocols 1), 21 and 3) recalled above, they are more mucoid than these controls, more encysted and more durably, less copiotrophs that said witnesses, and produce factors. self-induction (FAT) according to higher rates that witnesses, for example from! ' octanoyl homoserine lactone.
Compared to said witnesses, they are able to maintain the content of mineral nitrogen soils reconstituted in microcosms according to a particle size fraction> 3mm, on a period more than 28 days of incubation.
Preferred bacterial biomasses of the invention are Azobacteriaceae, of the 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 from algD and crlgU responsible for the production of EPS, as well as: fragments pobA features and pob ~ Z responsible for the production of PHBH, found in many of Azoto bacteriaceae, Pseudomonaceae and Rhizobiaceae.
Bacterial biomasses obtained are particularly suitable for the Bacterization microbiological of soils and crop residues, in particular residues of cultures grain, including this ~ .ix grain corn.
Thanks to the persistence of their activity in sztu once (re) introduced into the ground, during the period of cultivation of the agronomic crops concerned, they may use them residues in the process of decoinposition as energy and structural substrates.
The invention therefore relates to their use for such bacteria by introducing in sittc, to proximity of said residues, so as to exploit the soluble carbon released as a result of their degradation.
For this purpose, inoculas prepared at. from bacterial biomass and isolated strains are advantageously sprayed on the crop residues, especially when their burial in the soil. Gn uses for example inoculation rates of 100,000 to 1 million viable cells / g of residue, or lg / tonne of residue.

WO 03/046156 1 j PCT / FR02 / 04119 At the edapho-agronomic level, we observe will go sit2r or in microcosms, a more big persistence in sitar and over time of the activity of nitrogenase, in the case of biomass azotabacterial capable of diazotrophy, increased accumulation nitrogen mineral, in particular plowshares in the form of ammonia, an increase in biomass vegetable, relationship between the vegetative and root parts, an initial rfaard of the degradation of the cooking effect attributable to.1.4ct ~ Quick fungus of bacteria (Re) introduced.
Other characteristics and advantages of irwention are given in examples which follow, with reference to Figures I to ~, which represent, respectively FIGS. 1A and 1B, the PCR bands produced by AlgU'h primers according to the type of matrices used;
- Figure 2, the bands produced by RAPD, sel ~ an a series of six primers (N10) because acteristirjues strains type Nitrogen ~ bactet ° cccecce candidates, compared to a strain A.. chr ° ~ ococcum 76. i 12 witness, FIG. 3, the measured mineral nitrogen contents, on microcosms and of the microparcelles inoculated with various controls and candidate strains, - Figure 4, the ratio, depending on the incubation time between the activity of the reduction of (acetylene in the presence of inoculated culture residues according to the invention or with Ten2cins, not inoculated, - Figure 5, the mineral nitrogen contents of 2; series of microcosms treated according to the invention or by witnesses, with the addition of ara-humid compounds;
- Figure 6, the effect of an antibiotic on the treatment of residues of culture and by witnesses - Figures 7A and 7B, Ia decomposition of crop residues depending on the time incubation (laboratory and field);
- Figures ~ A ei ~ B, the effect of the particle size d ~ reconstituted soils microcosms on the properties of bacteria, and - Figures 9A to 9E, the effects of biomass of the invention compared to witnesses.

WO 03/046156 1 ~ PCT / FR02 / 04119 ~~ em ~ e 1: Isolation of candidate strains of the A.zotobacteraceae type.
soils used for sampling We reiterate the results obtained on 2 sites (Terrefort and Groies) whose ~ 1PC characters are 1G'S SLilVû.iit: i Table la: Physico-chemical analyzes of soil F4 and: I9 (“Terrefort”) on the domain experimental of INRA-Toulouse Parcel i ~ ranulor_rlétrie ~ r ~ ilP? 30 X60 Fine silt Coarse silt 139 165 able end 167 X07 ~ '. , L, gr ossier ~? ~ 141 It’s organic arbone 8.5 9.2 Organic matter 14.6 15.8 E ~ organic zote 1.07 1.13 water pH 5.9 6.6 CaCO 0 0 PaOs 0.11 0.06 Changeable 1.91 2.75 I'aa. changeable O.Olq ~ 0.015 Changeable mg 0.173 0.187 I ~ cha.ngea: ble 0.183 0.134 ~ Iusnidit 105 C 24 28 Table 1b: Edaphic characteristics of Groyne soils near Poitiers (86) LAND. . .
.. Dry .. Mainly north of the department C ~~~ ~ C'l'öI ~ ~ 'S
Color brrme o silty clay Substrate: limestone PROP: KIÉTöS
.- Limestone, shallow, stony soil (limestone).
~ Healthy soil o Low water reserve (opportunity for irrigation) Good to medium fertility V ~ FcI ~ TE: Large groie or clay-limestone o Deep, more clay soil water reserve r ~ r, ~ tooolû o ~ 'isclemcnt end n ~ ot ei ~ contact L! ne sl! sl ~ ensio2 ~ soil / water a-voc un s> matrix abstrate agar or gel silicic. The bioftlm rlui is formed in contact with the substrate rn.atriciel responds with characteristics of the original target soil, with particular regard to (1) The humic substances and fulvic and their constitution in terms of various phenolic compounds and benzoic, (2) the contents of carbonaceous compounds, and in particular the morio-, oligo- and polysaccharides of vegetable, animal and / or bacteriofungal origin, (3) the particle size of the part mineral 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.
to obtain the maturation of the oiofilm, the phase is periodically restored liquid, namely for example once - per week, in order to avoid the absorption of the biofilm and to wait , long enough between two restorations (eg about seven days), way to l ~~ isser temr5s to xc ell ~! the most constitutive azotobacterials of biofilm thus formed of WO 03/046156 l ~ PCT / FR02 / 04119 migrate by volumetric displacement towards the surface of the biofilm. To avoid dominance of copiotrophic azotobacterial cells at the expense of cells azotobactériennes sought after, which are rather oligo- or meso-trophes, more indigenous and persistent once (re) introduced, but also therefore easily dominated in iti vites environments rich, avoid load the gels and the liquid phases which float them with carbon substrates, in particular simple sugars such as glucose, and / or nutrients minerals. We ~~ tiïise ~ 1 ~: p refëreuce d, aseptic water dl5üllée {in particular by microwave tiltraüon).
Care should also be taken to avoid excessively breaking or disturbing the biofilm during time of the EStaLlratIOn e2111t1hv2 ~ nt jan water jet Too abrupt or directed at the time of the restoration of the liquid phase. In general, it is safflsant to use 1 ml of water on gels contained in 10 mm diameter petri dishes and 5 ml for 33 mm dishes diameter. After approximately seven ~ Otlrs of incubation at 22-27 ° C, the last restoration, either generally the third, aliquots of these liquid phases will be used to seed a series of backgrounds conventional liquid cultures.
To isolate the candidate candidate strains, we proceed advantageously like follows: orl place a 1 ml aliquot in 1000 ml of medium, (with salts <~ E
~: ~ Tinogradslcy including those coming from a soil extract up to 10% w / v) without mineral and / or assimilable nitrogen, and allowed to pre-incubate 1 to 2 days at 22-27 ° C. This transfer esi eff;: ctué ê. starting from the culture thus obtained still deu: ~ three times, but using only 100 micro-liters (1/10 tnL) of this “Rank I” culture and 9/10 ml of a new aliquot coming directly from the liquid phase supernatant of the medium obtaining original.
~ n reduces the probability, without eliminating it, of seeing a biomass candidate rather rnoir ~ s ohgotrophy take over, in terms of number of cells per mL of middle of CLiltürf :, on even more oligotrophic, persistent candidate biomasses and active: n sitar.
- gold identity. ~ anisms The bacteria were isolated on selective media lacking nitrogen mineral and / or organic thus ensuring the dominance of these diazotrophic organisms others' expense heterotrophic soil organizations. The organizations thus retained are Gram negative aerobic, diazotrophic and rod-shaped from about 1 to. 3 microns.
We know that A. chf ~ oococcLnn is recognized as being the most suitable for living conditions LAD: hic. This strain was therefore retained as a reference; e for the strains of the invention.
Indeed, macro- and microscopic observations of the strains of the invention they agree vs.
with descriptions of_Azotobczeter chrt? ocoec ~ tcm Bei ~ enir ~ crkü 1901 de ATCC, DMS and of the Pasteur Institute. A genomid and molecular characterization of these strains was therefore established from mechanisms known from Awtobcccter.
- biological properties of Gold ~ alllsmPS
L. Azetoberciercrcecc type bacteria obtained with the technology of the invention are able to fix diatomic nitrogen and use carbonaceous substrates from the decomposition of crop residues in the field, and in particular those from the ct ~ col ~~ l ~ csü; iol of cereal straws and: hatched. In principle, some ecotypes azotobacterials are also capable of establishing rhizospheric associations and of thus promote plant growth, but are not virulent. They don't are not either, and this unlike some Acetobcrctef ~, endophytes.
~ bone type dP bacteria! lzotobcrV ~ tetrcrcecce are generally found in the floors, they sqnt Ora? ~~? ~ .Egative, and have ~ _in mc: ~ e of growth and development conventional and Straight ~! .rohhe. In water stress conditions or other, they can be encysted in using exopolysacchar capsules ~~ ides (EPf) and more ~~ articuliérement alginate. In this meaning, this production of EPS is essential for survival; and the hardiness in an edaphic environment Ce5 bacteria; the more or less fine characterization of this mechanism will therefore be used to the identification of these organizations as described below.
These Boni series. responsible for the fixation of diatomic nitrogen in soils not Symbrotiqu ~. They are aerobic, unlike C'k ~ sty ~ idircm, and are usually recognized as unable; depending on the circumstances, fix by diazotrophy that a few lcg, .see a few tens of lcg of nitrogen per hectare per year. Now, these fixation rate bioiogics are mainly limited by the (non) availability of substrates carbonaceous following the (non) persistence of diazotrophic scuches (re) introduced into soils individuals.
Or ~ will note that, in accordance with the invention, these overall rates per hectare can be at less tripled; even fivefold. It was indeed dismantled using data experimental E

WO 03/046156 ~~ PCT / FR02 / 04119 obtained in laboratory, greenhouse, culture chamber and plots experimental at field that the invention actually allows greater relative efficiency strains isolated azotobacterials, once these (re) introduced into their agro-soil climate (APC) of origin, - methods of analysis eta ~ lisset. ~ cr ~ t de i'iG. ~ l'ï7lTlG keys OTg'C1177s77? i, '..
The identity of the organizations was established based on their adaptation to APC. The results reported below relate to the 2 APC from ë.esrluels the strains have been isolated, and in which they will be re-introduced under the forrzns J'inocula bacterial: one in the Toulousaine region (31) ('Terrefort or' TF31 ', see Tabl ~ saü la) and the other in the region of Poitiers (86) (Groies oti "GR ~ 6", see Table 1.b).
For characterization purposes, oligonucleotides capable of being used were used to initiate the iSClyri2éïlSatioil de S ~ grTiPrltS COriSerVBS Cl'iln genne reCOn? 1u As eSSentlel to synthesis exo - polysaccrarides (EPS), especially alginate, necessary for survival bacteria in 1 <= s soils.
Olig ~ ntrclér. ~ Tides t ~ tiiisé, s ~ orn ~ lcr rcrr ~ c ~ ctérisatirry ~ .f ~ fzction: ~ elle , ~ e segnver? tS AIgU and.
AigD clea ~ .sez! Ehes, ~ zatobc ~ cterireecre ecrrulic ~ '~~ ries.
C) did not choose the two regions considered to be the most conserved of the segments genonniques algU and algD already reported as belonging to the genera Pseurclomoncrs and A ~ vW bcccte; ° (information on Genebar ~ l ~ concerning the segment AVtJ11240. AIgD Azobacter vü: elandü ATCC 904 Campos et al. Tournai J. Bacteriol. 1713 (7), 1793-1799 (1996)). Seven series of oligonucleotide primers were developed from these regions to serve in amplify portions of a few hundred bases of these by PCR
Genoa easily identifiable by electrophoresis Poar3 ~ A lgD -Primer AIgD 1: TGG GCT ATG TIVTG GTG CAG T
9 Primer AlgD2: ACS ACC ACG GTG TGG CGA A
Primer AlgD3: GGT GTA CTT GAT CAT CTC GCiC

WO 03/046156 1 ~ PCT / FR02 / 04119 Po ~ arn AI. ~ U
~ AlgU primer ~ lTGG TYG QRC GRG TRC AGC
: G

d Primer AlgU2ACG RTA KGC TYY GAT GAA
:

Primer AigU3 TTY TAT AC1VI TGG CTG
: TAT C

Primer AlgU4 GAC CCY ACE GTY CCM AC
:

The results obtained are. given in FIGS. 1 A and 1B dui represent A. The PCR bands produced by the fllgU 1/2 primers according to the type of matrices used.
Expression of PCR-AIgU bands most likely denotes segments of different axasses and therefore characteristic of the candidate strains TF31 and R86, respectively;
B. L ~. highlighting of the difference between the AIgU 1/2 PCR bands for the strains CandiCl ~ tws TF31 and GR86.
The primers AIgTJl;, t U2 prove to be the most discriminating, allowing to get so reproducible PCR products characteristic of the two candidate strains retained, and that vis-à-vis the. strain used as térnoin, Azotobc ~ cteY c: lzroococczrzn 76.116 of the C1VCM
Binds the Pasteur Institute (see Figure 1).
eorat: vle de lcc parr ~ summer rzzierobzoiogiqare de lcz sozrehe ecendidcrte and de lcz p ~ épcroatzorz The technique called "R.APD" (Rcrrzdonz AJZZpI ~ ed Polymozphisnz DNA) is the more generally used to quickly obtain a genetic profile of a bacterial organism.
A choice of oligonucleotide primers used to amplify by PCR
DNA segments, a pl'dOTd arranged randomly within the genome of the organism, can therefore serve as a tool of A QCQ (Qz Assurance: bed rest, Quality Control) during production and deployment test of bacterial organisms. This choice is dictated: by (i) the appearance of a "band PC R ", firstly, {ü) the systematic reappearance of this strip during hCR successive, and (iii) the report that this community of bands will establish between the various strains that we are trying to distinguish from each other.
The Eandidate strains having been characterized as such;
A ~ otobcrcter ~ aceae and probably A. ecotypes. chroococczizzz Bezjerzrtclcü, Gram negatives, aerobic, WO 03/046156 1g PCT / FR02 / 04119 diazotrophs, in the form of rods, sometimes ovoid, and of size from 1 to 3 microns according the stage of development of the culture and the composition of the environment, was mainly about distinguish the various candidate strains TF31 and GR86, from a strain of standard culture, in particular A. chroococc ~~ n Be ~ jei ° ir3kciz 76.116 of the CNCM of the Pasteur Institute.
~ _ from two series "~. nine composite random primers: each of ten nucleic bases (see Tc ~ bleczzr 2), six primers were chosen according to the three criteria of choices mentioned above (See Bunicoa et al. 1 S99).
Table 2: Synopsis of all the primers (decamers) tested in the frame of RAPD analysis of three strains and biomass azotob; ~ ctériennes, y understood the strain of reference Azotobactef ~ chi ° oc: co ~ crrrr ~ 76.116 of the CNCM d ~ e the Pasteur Institute.
Produc; tior ~ of Bands PCR-RAPD

Azotobcrcte Seed ~~ Strains l ~ calnreC3ligonuclotidiqueschf ~ oococez ~ r3 ~~~ candidatescandidates ~ Tt l- ~ TCG T AG CCA A? ____? ____ Vt2-4a TCA CGA TGG A? ---- **

Vt3-arc TCT CGA TGC A? ----? ----Vt ~ -Sa CTG TTG CTA C? ----? ----Vt5-8 TGG TCA CTG A? ----? ----Vt 6-10 GGA AGT AGT G? ----? ----b ~; t7-1: 1ACC TC'r a ~ CA? ____? ____ ~ T.

~~ 't8-1? bCGG C.CC C TG T ~ *% jG *: ~ Jh' ~ '. ~ ::

Vt9-13 ATT GCG TCC A? ____? ____ A.zr l GGC CCG ATC G '7 -_? _-Azr2 TAC CCC GAG G? -? -Azr3 TGC GGC TCA G? ~ ***

Azr4 TCC CAG CGC G? -? -AzrS GCA TGC ACG G * ** **

Azr6 GCC AGT CCC G
**? _ ?

Azr7 GCT GCC CGA G '? ? -? -AzrB GTG CGA CCA C? ** **

A ~ r9 TTC GGC GCG A * * *

____- ~ ALL -? : primer used but no PCR band -: primer abandoned due to lack of tape PC: R ..
PCR strip performed PCPh embroidery not performed The results obtained are reported in Figure 2, which represents the tapes produced by RAPIN.

The six primers, which are decamers, ensured that the candidate strains were actually related to A. chr ~ oococcaun 1901 / 76.116 of 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 strains c ~. ~ i,! dic:? aaca TF ~ 1. and ~ P. ~ 6, which corroborates the analysis more functional of these two strains according to the alg gene segments (see Figure: 1).
The results obtained are reported in Figure 2, which represents the tapes produced by I ~ 4PD, according to a series of six primers (n10) characteristic of the strains of type : ~ ZOt !? JC! Nt2YC ~ G2Cl ~ Cand! DateS, compared to a strain of A. ehnoococcrr ~ z 76,112 witness.
Identification of biomasses of the invention in relation to controls It is recalled that, in general, the protocol of: retention will go ~~ o of biomasses bacteria with edaphic characters best suited to survival, persistence and activity during the period of cultivation of the agronomic crops concerned integrate the three (3) following concepts within the protocol, namely ~ Concept 1: Adherence to inorganic and abiotic surfaces .. - ~ ° ~ Ioüon '~: CïoisSa.r ~ Oligotrophic ge from carnplements carbohydrate and humic .. I ~~ ûtion 3: Expression of benthic and p characters: .a.nctonics These biomasses can indeed be identified and distinguished in relation to a range b! control eyelets produced more or less conventionally (traditionally) and / or ~. ~~ let,, 7ne variant dtz known as p! -otacole integrating only two :, even one only, of these notions.
More precisely, a series of tests in microcosms, even; in micro-plots incorporating a or more of the inoculated controls (azb etlou Azb) listed below, and the candidate biomass {~ 7 ~) p ~ ui highlight, a posteriori, the setting ~ u ~~ re said protocol;
rntga ~ ation Biomass candidates Witness number Retained Bactrian Notions for example 1.2 AZB Na _ Tmoin Azb T e I TMI
2.3 1.3 Tmoin Azb T e II TM2 1.2 Tmoin Azb T e III TM3 1 Witness azb 1 _ TM4 '- --2 Tmoin azb 2 _ ~ TMS

3 Tmoin azb 3 TM6 WO 03/046156 2 ~ PCT / FR02 / 04119 TMl - Azb Type I control: These biomasses are isolated from agar or other gels conventional organic and biotic matrix surfaces on which a extract of earth modeled on the edaphic situation of the original / target soil. By the presence of a relatively rich matrix substrate, so in principle they are mainly character oligotrophic and contrary to the principle of active persistence as targeted through the invention;
v TM2 - Azb Type II control: These biomasses are isolated in the presence of gels silicic by alternating the liquid and solid phases by gradual drying of water intake distilled on the surface of gels which have not received an earth extract, or Have received Lin e, would go from earth (ie supplement glucido - humique modeled on the ground target) of a soil of origin different from that of the target self.
TM3 - Witness Azb Type IiI: As with the Azb Type I teres, these biomasses are isolated from gels, but silicic and / or inorganic and abiotic, on which one soil extract is modeled on the soil condition of the original soil /
target. An extract c: ~ earth is at ~ departed = v the surface of the silica gel (ie installation of a first, and last liquid phase), but this is not restored once it has dried.
TM4 - Witness azb 1.: These baaterian biomasses; ~ candidates are obtained according to Pochon protocol (1950, Pochon and Tchan (19 ~ E8), Pochon et al. (1958) and Bassoon (1967). <~ epe ~ idant, and according to the present definition, these biomasses are obtained without impregnation of silicic gels, and / or other matrix surfaces inorganic and ; b ~ OtlC ~ L! 8â, a ~% ~ G l.Iil CILL ~ l ~ "i2C ~ tle eXtralt de terre.
Tï ~; ~ 'i5 - Azô witness ~: These biomasses are Obt2ilLl ~: ~ by procurement protocols with edaphic characters corn strictly as far as ~~ is concerned with the complement carbohydrate and humic soil origin / target; this supplement ("soil extract") is either brought on the surface of an organic / biotic matrix surface (eg. agar gel) or in one rich and more or less conventional liquid culture medium.
i1 '~ 6 - Witness azb 3: These biomasses are dci ~ to be listed in the various bacterial collections (ATCC, UTE ~, Institut Pasteur, D1 ~~ 1ZM, ete.) and have summer isolated conventionally, or at least without special consideration seeking to reproduce irz viL wo, during their isolation, the edaphic character, at sense of present invention. It is also possible to distinguish two types of Witnesses.
azb 3; (at) on the one hand the "endogenous" azb 3 Witnesses (azb3a), that is to say these biomasses WO 03/046156 ~ 1 PCT / FR02 / 04119 bacteria obtained conventionally. but directly from a sample of the original / target soil, and (b) on the other hand the Witnesses azb 3 "
exogenous "
(azb3b), i.e. these bacterial biomasses already listed in the collections.
Graphidue representation of the AZB effect compared to control inocula ~ 3r1 represented gra.phicluerr ~ ent lot réa.lisatiali of this effect <cAZB "in ordering reports say agr results or ~ amicluos obtel ~ read read e11 illCrOGOsmeS, ie eI1 Saarland, either in micro-plots to the. following fields (see Figure 9A and B) AZB / Azb Witnesses reports (I, II and III) - ~ e ~ s ~ xs AZB / Azb Witnesses reports (l, 2 and 3) n AZB reports / Azb witnesses (I, II and III; I ~ e ~ sLlS AZB report in condition favorable / unfavorable to the expression of the “A2B” effect arising from the setting in opens of the invention.
~~ wtte l: résentatian highlights the efficiency of biomass bacterial obtained according to the invention with respect to. their various witness inocula. This representation also allows easy identification of inoculating preparations relatively efflcacgs can be used as candidate strains for the manufacture '~ <~ tian of inocula intended for the cam_merr;. iaiisaüon.
As an example, 3 sets of experimental data have been reported and are illustrated respectively by FIGS. 9C, 9D and 9E
Figure 9C: (a) Relative efficacy of various inoculating preparations derived from azotobacterial biomasses isolated from two types of soil (F4 and I9);
o Figure 9D: (b) Relative efficacy of various inoculating preparations derived from lilal.W ~ 55C- ~ aZ: 3 ~ r0rJaCtérlenlle5 lSCléeS of two types of soil (F4 and I9) depending on the results obtained during incubation in microcosms with and without soil aggregates intact;
a Figure 9E: (c) Relative efficacy of various inoculating preparations obtained and tested with a series of experimental tests (Exp 17, 18, 19, 20 and 21).

AZB / control relative efficiencies greater than 1.00 indicate preparations inoculants more effective than conventional preparations. This improvement of the effectiveness is necessarily attributable to the implementation:
the invention.
T'o: ~ r ~ ûJs e2l, ériïneritales including witnesses! -1zb conventional and Azb witnesses from ~ r ~ e T
'~ ei ~ e ~! _rs en ~, ~ ote / ic ~ de sol Table 3 below indicates the mineral nitrogen contents (Nm) of the floors in microcosms incubated in the presence of 1% w / w of residues of; corn growing inoculated with preparaéio ~~ s oactériennes according to the invention (AZB) and leurwtoins Tl '~ I6 and TLVIl (ie térr ~ oins internas).
Table 3 ___________ mg Nm ll ~ g sol -_________ Témoin TIt ~ i6 Witness TI ~ / il AZB A.ZB / Tli'Il AZB / T1VI6 Vat 2 54 ~ 7 71 1.2 ~ 1.31 Val3 SR 62 64 1.03 1.10 vaI ~ t ~ ~ 1 7I 1.29 1.2 ~

Val ~ 62 62 62 1.00 1.U0 ~ / 1 c; GO 02 53 i.0 ?. 1.05 i ~~ o ~ T ~~ n ~~~ 'v2 & ls 1.10 ~ .3 ~ 5 '' Phase II: cle jru-wier 98 to December 1999 Témoir. Azb Tape I
. accumulation of mineral nitrogen (Nm) per Icg of soil according to the conditions of retention szir .mi1_ieux such elu'used in the invention.
L ~~ facv ~~ genc ~ rale, the isolation protocol of the inventïon promotes the irr vitro retention at surface of abiotic rnatières conditioned so, shelter biofilms conducive to the evolution of bacterial strains (azoto) with edaphic characters. Moon of the main characteristics of these edaphic bacterial biomasses thus retained is in principle their oligotrophy dui is associated with their greater hardiness and efficiency in site. To put in evidence of this rustic oligotrophy, sometimes called zymogeny, I'expërimentation ~~; .2: ~~ vr ~ ~? aizs lo ta '~ l ~ au._ ~ below has been made WO 03/046156 2 'PCT / FR02 / 04119 Table 4 Accumulation of mineral nitrogen (Nm) .par. lsg of soil according to the conditions of retention on backgrounds of the invention. '' The conditions for obtaining biomass (azoto) bacteria in itc ~ li ~ tre cancel the effectiveness of the protocol of the invention, by establishing conditions of retention too rich in carbon substrates.
Pre-incubation Presence of glucose By report t ~ ov soils on r ~ .zb media, witness With cellulose retention ~~ conventional mg Nm / kg soli oZCi o ~ ci L 0 ~ 26 (3) non ozri 0.99 26 (1) ~. ~ i no 1.1 ~ 29 (2) no no 1.3 8 3 6 (2) 3 Microcosms of 2 ~ g of SoI Terrefort reconstituted over 28 days of incubation at 22 ° C
Experimental Azb control data from T ~ tee II ~ TM21:
. effectiveness of the protocol of the invention for i: to favor obtaining biomasses ~ .- ~ z ~~ to ~ Llctériennes according ia edaphirlue nature of the soil of origin and "target ".
The results obtained are summarized in Table 5 below ~ n gives on the figure; i, the mineral nitrogen contents (rdm) / kg of soil in inoculating various t ~ üoins and candidate strains, in microcromes and on df; s mite oparcelles.

Table 5 . ~ iotzc! sseNature daphiqueNature daphitltteRapportA ~ BlAxb pe II iTM2) Ty ctat ~ rlidatesdtt original soil. tlu soil ~ c targeted, First series Second series T, aoitr ~ Ar ~ lo-silty Clay 1.52 1.U7 TNd2 ~ 's ~~ nit? Limon Argile U.75 U.89 a'l'vT2 ~~ 'G'7390? .L? ~ AbleLl \ Argile O.G3 1.31 t "s? ~ r ~

~ a ~ Ardu Arb on 2.02 2.81 Two series of the same soil were pre-incubated, from which we wish isolate according to the protocol of the invention the AZB bacterial bacteria with and without 2 ° Jo P / V of cellulose.
In principle, this pre-incubation should have favored the proliferation of bacteria rather zymogenic and less oligotrophic and thus make them easier, or at least more ;: ~ iJ ~ J! ~ i ~~ i ~ elrL, lsolaLIPs ~ nz their greatest number.
L. jaris of such conditions, the strains obtained by means of the protocol of the Inventi <on ( <<!'~'B;>) should be little or no more efficient than biomass azobactériennes conventional (A.zb). _ '. ~~ more, cn added a solution to 1 ° '° of glucose sufficient for alleviating the oligotroplucic conditions in principle present on the retention media according to i'111venti0il. Three negative witnesses: Fs canceling the action of the strains candidates could be established at the time of biomass retention (bacterial azoto'i more oligotrophs and rustic (see table).
Note that only the complete absence of carbon substrates provided allows to get according to the protocol described above for bacterial biomass (nitrogen) AZB
genuinely more e ~ caces that azb azotobacterial biomass isolated more Conventional.
~~ head experiment perfectly highlights the roles of oligotrophy during the retention of the most rustic and efficient bacterial (azoto) biomass a time reintroduced into the edaphic areas as an inocula. We must remember that this condition of obtaining qtl'e5t oligotrophy is necessary but not sufficient for ensure isolation the most edaphic b ~ .ctériennes strains. Indeed, the hardiness and the stress resistance r_utrüi.onnel does not correspond exclusively to only soil bacteria, but remain WO 03/046156 ~ 5 PCT / FR02 / 04119 nevertheless essential here in view of the recrudescence of conditions oligotrophs in edaphic areas.
Other experimental data . Study of the increase in the content of microcosms in mg of mineral nitrogen (Nm) per kg Cln sol re ~ onstitl ~ é., And the. persistence of nitrogenase activity in time measured according to the combination of the activity of acetylene reduction (AIaA) at time t2 and at time tl.
The results obtained are summarized in Table 6 below ;;
Table 6 Trial Trial 2 Trial MiCr "oCOSme Nm ARA3 Nm ARAS Nm ARA5 Crop residue 28 3.87 1U.8 22 60 1.6 Crop residue with control 29 4. ~~ 214.8 45 68 2.1 TWLS

Culture residue with AZB 40 6.67 15.3 112 72 2.7 . Mineral nitrogen content (Nm) of the salts in microcosms incubated in the presence of 1% w / w ~ -hay crop residues inoculated with A.ZB and their witnesses azb and Ab (ie "internal" witnesses) 't2 = 26 h after the start of illCllb ~ Lt1011 of the microcosms; tl = 10 p.m.
't2 = 72 i ~ after elébui of ~ ioa ion of microcosms; tl = 24 h 't2 = C6 ïi after start of incubation of microcosir ~ es; tl = 16h WO 03/046156 ~~ PCT / FR02 / 04119 i, the results are summarized in table 7 below . Table 7 _____.._____ mg Nm / Icg sol -_________ Tests Phase I6 witness TM6 witness TI ~ Il AZB AZB / TMl AZB / TM6 II9 51 Na 56 Na 1.10 III. ~ 5 Na 6i Na 1.33 IV 73 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 ~ 9 na 58 Na 1.18 ea ~ i ~ bis 52 na 55 Na 1.06 Ea4tris 53 na 54 .. Na 1.02 ~

lblonentze51 na 56 Na 1.10 Tmoil tests ~ tmoin TMl AZB AZB / TMl AZB / TM6 Pase Ih TIvI6 ZIal2 54 57 71 1.25 1.31 Val3 58 62 64 1.03 1.10 VaI4 57 55 71 1.29 1.25 Val s 62 62 62 1.00 1.00 -Vai 6 50 62 63 1.02 1.05 Vloyenne: 9 b2 68 1.10 1.16 f Phase l: from September 96 to December 97 Phase II: from January 98 to December 99 Figure 5 gives the mineral nitrogen content (mg Nm / kg sol) of two series of microcosms in parallel, with and without the contribution of the equivalent of a fifty units mineral nitrogen per hectare, depending on the type of bacterial inocula (nitrogen ~~) used (azb - inocula conventional control consisting of isolated azotobacterial strains conventionally;
süiciclues gels (~ W) co ~: summed according to the invention plus ~ Various substances para-humic (; ~ p ~ TI, 2 and 3), a polycondensate of catechol (PG) and a ~~ pH).
': inétielue ale clégr ~~ dation of crop residues <; stem over ur_ period 5 months (from October 2000 to March 2001) on soil of the “red chestnut ferres” type, Field experimental Inca, Lusignan (~ 6).

WO 03/046156 2 ~ PCT / FR02 / 04119 Or ~ reports in Table 8 below the results obtained Table 8 -..__-- ~ -_ Inocula according to Coefficient g rsidus remaining by report I Invewtior_dgradations Coefficient with non-inoculated witnesses and R '(t - '= 5 months I witness -lcl x 10 ") Trnoit ~ TA! Rb37 0.93 1 .. ~ 7 i'tr; ozo ~! 0.97 L 11 2 ~ 2 AZB ~ 7 0.91. 0.91 a According to the degradation kinetics dRC = A xe ~, where dRC represents ler quantity of degraded eultater residues ast time t, and At the qttat ~ tity of residues att time 0, ie. ~. 00 g of trtatière sèehe.
~ acted ~ -isation according to the invention of crop residues (RC) - effect of finer grind on the use of substrates resulting from their degradation and in principle usable by bielmasses; azoto) bacteria thus brought to the surface of these residues.
.eg obtainable results SOilt summarized in table 9 below:; under Table 9 __________________________ mg Nni / kg Sol -______________.._____.
inoo ~~ ia Fine Mouture of CR lVlouture Coarse of CR
Térr ~ oin TMG 58 52 Witness TMl 52 48 It will be observed that the coarser grind (3 mm) of these same residues does not allows likely not to AZB biomass to contribute to the increase relative of contents of mineral nitrogen (Nm) of microcosms compared to controls inoculated TM6 (ie azotobacterial biomass conventionally isolated) and TMl (ie biomass isolated azotobacterial ~ according to an ineffective variant of the invention.

WO 03/046156 ~ g PCT / FR02 / 04119 increase in biomass: the results obtained are reported in tables 10,11 and 12 below A) Increase in the production of plant (aerial) biomass by Lolia species ~ m fnttltif7.o ~ r.ti ~~ and T ~~ itiG ~ ann ctestiv2rtn according to the mode of culture in pot (greenhouse) during the incubation of cereal crop residues (wheat straw) on a 28 to 56 day period (Test 5) with and without the inocula cüvers of the invention and their TM6 controls and TMI ..
Table 10 __ my biomass ~ Ultut'e et1 pot Essail '; Lssai2a Test3 ~ 'Test4b Test5b Crop residue 9.1 7.1 33 37 35 Crop residue with control 8.7 5.9 39 32 37 Crop residue with control na 8.1 39 32 37 ; Zsidus ci ~ culture with trnoinna na 39 32 37 Culture RSidLIS with AZB 10.0 8.7 40 41 39 Note: a; g of biomassLolir.tm nrttltiflor7tm per pot b; ny of biomass Triticrnn aestivrtm by phntule B? Percentage (%) of increase in grain yields (Yield at 15 ° l ° humidity) and PI ~ Ifs (aids of a thousand grains) during agronomic field trials (1999-2000 campaign at Touïouse 31 on soil ierreféu, and 2000-2001 campaign at Lusignan on soil Red earth of chestnut) compared to the non-inoculated controls combined with TM6, TMl and AZB.
Table III
15% time savings PiVIG
hlEûCle ds. CUitut'e au champ ToulouseLusianan ToulouseLusianan Culture residue with TM6 control 4% 0.73% 5% 0.08 i ~~ sidus of copper with witness TM112a 13% 0.86 ° rô ~. % 0.02 f? ésidus cie culture with AZB 20% '1.47 ° ~ ô 6% 0.94 ~: ~ Tote.: ~ A; Witnesses TMl buddy Toulouse, TM2 for Lttsignan C) Coincidence of increases in yields and PMCr during trials in the 2001-2001 campaign field in Lusignan on red soil soil of chestnut and the various indices of recovery of crop residues obtained so parallel on these same plots.

? 9 Table 12 ~ the cultivation idea in the field Rdt ° PIvIGb -nt ° dRCd ctRCe Crop residues with TM6 G3 control 0.08% 41 2.10 0.70 culture residues with TM2 control ~ 71 0.02 ° rô 37 2.02 0.81 Crop residues with AZi3 12G 0.94 ° r ~ 47 2.42 1.19 2 ~ fote: a; increased yields in kg grain / ha to 15 %ununity h; weight thousand grlins of wheat compared to Y non-inoculated controls au ~ t witnesses TM2 / 6 and AzB
vs ; degradation coefficient; ~ l0a according to dRC = ATe- ~~
d; degradation of crop residues (4.00 g at t0) stu ~ 164 days e; degradation of culture residues compared to non-inoculated control Y
combined with Y witnesses 71V12 / 6 and AZB
. Increase in the plant biomass / macitary biomass ratio attributable to the action rhizosphere of (re) introduced bacteria.
Table 13 below gives the results obtained concerning the increase of the relationship between the vegetative (aerial) and root parts (Vegetative Index, IV) of seedlings grown on soils incubated in the presence of cereal crop residues t, wheat straw) treated and untreated with the inocula according to the invention and their controls TM6 and 7, M2.
Treatment of crop residues --------- root crops report (Index Vegetative ------------Trial 1 Trial 2 Trial 3 Average Witnesses TM6 0.85 na na 0.85 T1VI2 control na na na TF31 (AZB TF31) 0.90 na na 0.90 '-Witness TNI6 1.02 0.90 1.03 0.98 (0.03) 9 î en ~ oin fi ivi2 na 1.03 1.09 1.06 (0.04) GRBû (~ GB GF'8ô) i.05 0.92 1.36 i.ll (0.09) (STDEV) Delay in the effect of nitrogen immobilization compared to that obtained on residues of control culture treated with an antibiotic.
Gn is studying the effect of the treatment of cereal crop residues (straw of wheat) with a antI1710tiCllte (ampicillin) at an agronomic rate (from 1060 kg i 10 NIg of residues / hectare (about 1000 lVlg of soil)).

WO 03/046156 3o PCT / FR02 / 04119 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) '°
Crop residues with antibiotics 37 39 38 38 (1) Crop residues with AZB 40 41 39 40 (1) ° (standard deviation) ~ ~ Ampicillin The effect observed is attributable to the delay instilled in the bacteria involved in degradation crop residues, and therefore incidentally to the immobilization of nitrogen mineral assimilable from the soil. This mitigation of nitrogen immobilization assimilable allows a greater plant growth of seedlings tested in place; this increase in vegetative growth is similar to that observed in the presence of culture treated according to the invention. This treatment can therefore also be seen as a good alternative (micro) biological to possible treatment with antibiotics of residues of cultures.
Figure 6 gives a graphic representation of three VBS experiments with the use of ampicillin as an antibiotic control. We will observe 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 We study the decomposition into microcosms in the laboratory and in the field, in nylon sachets, cereal crop residues treated according to the invention with respect to their matched witnesses not inoculated.
The results obtained are given in FIGS. 7A AND 7B.
. effect of the particle size of soils The results relating to the study of the relative effectiveness of bacterial (nitrogen) biomass of (invention for maintaining nitrogen content soil mineral 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 biomasses A ~ B are effective and that this efficiency compared to the controls inoculated (azb and A: ~ b) and not inoculated (control), is more observed when the biomasses evolve within the 1 mm fraction.

References Bibüo ~ ra ~ phic ~ wes Bunicoa, C.; V. man and JL Fauchere. 1999. Performance criteria of DNA
ïin ~ er ~~ arintin ~ methods for typina ofl¿eliobacteo ~ ylooi isolates:
experimental results and meta-analysis. J. Clin. Microbiol. 37 (I2): 4071-4080.
Characlclis, WG 1990. Biofilm processes. In: "Biofilms" (Characklis and Marshall, eds.), Jchn Wiley and Sons, N '~', N'f.
Characlclis, WG and hC Marshall. 1990. Biofilm: a basic for an interdisciplinary approach.
In: "Biofihns" (Characklis and Marshall, eds.), John Wiley and Sons, NY, NY.
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Silcdar and RL Irvine, eds.). Technomic Publ. Inc., Lancaster. (UI ~), Bases (CH).
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Claims (12)

CLAIM 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, in a way to form a hydrated zone, or biofilm, on the surface of the substrate, analogous to that attached to CAH of the soil, essentially harboring the autochthonous bacterial component of the ground, in order to retain on the surface of the matrix substrate, most of the microflora bacterial endogenous to original soil environments.
- maturation of the biofilm with establishment of oligotrophic conditions allowing most constitutive cells of the edaphic biofilm which have colonized the matrix substrate, migrate to the liquid phase supernatant the matrix substrate, and gradually to dominate it, - recovery and culture of the most prolific bacterial strains in culture media rich liquids, autochthonous, hardy and persistent in situ, cultivable in medium liquids, and therefore suitable for industrial production. 2. Method according to claim 1, characterized in that the substrate matrix is a inorganic and abiotic matrix substrate, in aqueous medium, favoring the production exopolysaccharides (EPS) and other components of the bacterial glycocalyx, as well as the network organization of a sufficient number of cells capable of initiating the first stages of formation of a biofilm on the surface of the substrate. 3. Method according to claim 2, characterized in that the water from the suspension aqueous contains compounds present in the relative proportions soil characteristics original target, namely humic and fulvic substances, compounds carbon and in particular mono-, oligo- and polysaccharides, of vegetable, animal, and bacterio-fungal, nutrient. 4. Method according to any one of claims 1 to 3, characterized in that that the Biofilm maturation is achieved 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 the bacterial cells supernatant in the solid phase. 5. Isolated bacterial biomasses, characterized in that they comprise, as dominant strains of an edaphic, autochthonous, oligotrophic nature, in major part aerobic, presenting metabolo-physiological characteristics of diazotrophy, activity fonti-static and/or phytosanitary. 6. Biomass according to claim 5, characterized by the following phenotypes expressed in vitro and in situ compared to control bacterial biomasses obtained from biotic surfaces, but without the presence of biofilm or complement humic and/or obtained from abiotic surfaces, but a) with a non-patterned humic complement on the one target soil and/or b) with a carbonaceous substrate supplied in a greater quantity to concentrations normally present in soil, and/or obtained from surfaces non abiotic conditioned, said bacterial biomasses are more mucoid than these witnesses, more encysted and in a more durable way, less copiotrophic than the said controls, and produce auto-induction factors at higher rates than controls, e.g.
example of octanoyl homoserine lactone. 7. Bacterial biomass according to claim 6, characterized in that, by compared to said controls, they are able to maintain the nitrogen content soil mineral reconstituted in microcosms over a period of more than 28 days of incubation of Azobacteriaceae, Pseudomonaceae, or Rhizobiaceae. 8. Bacterial biomass according to any one of claims 5 to 7, characterized in that they are Azobacteriaceae, Pseudomonaceae, or Rhizobiaceae. 9. Bacterial strains isolated from bacterial biomasses according to moon any of claims 5 to 8. 10. Bacterial strains according to claim 9, in that it is of ecotypes of A.
chroococcum Berjerinkcii. 11. Application of the bacterial biomasses according to any of the claims to 9 and bacterial strains according to claim 10, to bacterization microbiological soil and crop residues, in particular crop residues cereals, including those of grain corn. 12. Application according to claim 11, characterized by inoculation of said bacterial biomasses and strains by spraying formulations containing on the crop residues.