Improving microbial activity

Abstract

The present invention relates to microorganisms, materials and methods for improving the activity of microorganisms. In particular, the present invention provides methods for preparing microorganisms to improve metabolic activity and / or synergy, metabolism-activated and / or synergistic microorganisms, and methods of using these metabolism-activated and / or synergistic microorganisms, particularly in industrial fermentation processes.

Description

[0001] The present invention relates to microorganisms, materials and methods for improving microbial activity. In particular, the present invention provides methods for preparing microorganisms to improve metabolic activity and / or alignment, metabolically activated and / or aligned microorganisms, and methods of using these metabolically activated and / or aligned microorganisms, particularly in industrial fermentation processes. BACKGROUND

[0002] The goal of an industrial fermentation process is typically to produce a desired product in the shortest possible time using the least expensive resources. In such a process, a microbial culture or cell-free extract converts one or typically multiple metabolizable nutrients into the desired product. A typical industrial fermentation process is a submerged fermentation. In such a process, a microbial culture is suspended in a fermentation medium, which typically provides all the nutrients needed to produce the desired product; gaseous nutrients are typically provided using a bubble column or other form of aeration.

[0003] Fermentation processes can be carried out as batch or continuous fermentations. In batch fermentation, a fermentation medium is inoculated with a production microbial seed culture. The inoculated medium is then treated in a fermenter under conditions that favor the production of the desired product for the actual fermentation. At the end of the fermentation, the fermentation medium containing the desired product is harvested. Typically, the fermenter is then cleaned and prepared for another fermentation batch. Fed-batch fermentation differs from batch fermentation in that additional material is added during the fermentation (feed). The feed can or can not have the same composition as the fermentation medium at the start of the fermentation. In continuous fermentation, all or a sufficient portion of the microbial culture is retained in the fermenter, where new feed is continuously or intermittently fed to the fermenter, and a portion of the fermenter contents containing the product is harvested (partial harvest or repeated batch process).

[0004] Growth of microorganisms during fermentation typically goes through several phases. During a lag phase after inoculation (see Monod, Ann. Rev. Microbiol 1949, 371-394), the doubling time of the microbial culture is high. It is thought that the microorganisms need time to germinate and can adapt their metabolism to the changes in the composition of the culture medium. The lag phase is followed by an exponential growth phase (“log phase”), in which the cells divide at approximately constant and high rates. For example, a doubling time of as little as 20 minutes has been observed for Salmonella enterica during the log phase. During the exponential growth, the rate of consumption of nutrients by the microorganisms is highest. It is thought that not all nutrients are consumed at the same speed at all times. Rather, it is thought that some complex nutrients require the synthesis of specialized enzymes to be consumed (“second growth”, see Salvy et al., Proc. Natl. Acad. Sci. USA 2021, doi: 10.1073 / pnas.2013836118). This can result in one or more periods of temporarily reduced growth rate during which the required enzyme concentrations change. With remaining, even less readily consumable nutrients, growth enters a stationary phase in which the doubling time increases significantly again. It is thought that microorganisms in the stationary phase enter a dormant phase with a strongly reduced metabolic activity and / or that microorganisms in the stationary phase survive on the remnants of dead cells, such that the doubling rate of the cells is roughly equal to the death rate. Once the stationary phase can no longer be maintained, the microbial culture enters a decline phase in which the death rate is higher than the doubling rate.

[0005] In industrial fermentations, it is desirable for the microorganism to enter the exponential phase as quickly as possible and for as long as possible to maximize product formation. Bertrand et al. (J. Bacteriol. 2019, https: / / doi.org / 10.1128 / JB.00697-18) have reviewed the factors influencing the duration of the lag phase. They found that the duration of the lag phase is not only influenced by the history of the culture used for inoculation, but also by the number of bacteria in the culture. As the number of cells increases, the duration of the lag phase decreases. Caipo et al. (J. App. Microbiology 2002, 879-884) tested the effect of different inoculum amounts (10E3 to 10E8 spores / ml) of a spore cell bank of Bacillus megaterium by microscopic evaluation. Their results showed that higher concentrations of B. megaterium spores promoted faster germination and promoted more spore germination. The authors pointed out that this is in agreement with the theory of Woese et al. (Proc. Natl. Acad Sci USA 1968, 869-875) according to which Bacillus spores can contain different amounts of “germinative molecules” such that at higher Bacillus spore densities, there will be a greater percentage of rapidly germinating spores. Therefore, higher inoculum amounts are positively correlated with faster germination. Since the completion of germination is a prerequisite for the entry of sporulating microorganisms into exponential growth, it is believed that large inoculum amounts are advantageous for industrial fermentations. Likewise, Webb et al. (Food Microbiol. 2012, 104-109) observed that the inoculum concentration of Clostridium botulinum spores significantly affected the germination time; decreasing the inoculum amount of C. botulinum spores increased the time of toxin formation in food products. The authors suggested that molecules from germinating spores can trigger the germination of dormant neighboring spores.

[0006] Inoculation of a fermenter is usually done by preparing a starter or seed culture of the production microorganism and adding a defined amount of this starter or seed culture to the fermentation medium in the fermenter or pre-culture vessel. The seed culture is prepared by inoculating a volume of a cell bank vial or a colony from an agar plate culture into a seed medium (which can or can not have the same composition as the fermentation medium) and then incubating the inoculated seed medium under conditions suitable for growth of the microorganism culture. Typically, the fermentation medium is inoculated with a seed culture volume of 2-10% v / v of the volume of the fermenter (see WO 2006096164 and WO2017068012).

[0007] Attempts have been made to remove the seed culture preparation step. For example, WO 2006096164 describes Clostridium botulinum fermentation for botulinum toxin production, wherein the step of seed culture preparation is omitted, so that the fermenter is inoculated directly with the contents of a cell bank vial. Such an inoculation scheme did not yield positive results, and the authors prefer to inoculate the fermentation medium with about 2-4% of a seed culture medium with Clostridium botulinum in the seed growth phase. Likewise, WO 2017068012 describes Trichoderma reesei fermentation for beta-xylosidase production, wherein the fermenter is inoculated directly from the contents of a cell bank vial or from the surface of a PDA agar plate. The authors speculate that such an inoculation scheme can be used not only for ascomycetes, but also for bacteria. However, there is no data to support this speculation, and in the preferred embodiment the microorganism is a filamentous fungus.

[0008] The quality of an industrial fermentation process depends not only on the duration of the lag phase, but also on the yield of the desired product and the extent of formation of undesired by-products, such as slime or foam. Furthermore, reproducibility of the fermentation results is required to avoid unexpectedly long durations of the lag phase, unexpectedly short durations of the log phase, or unforeseeable nutrient requirements.

[0009] It is therefore an object of the present invention to provide improved materials and methods for industrial fermentation, in particular with regard to microorganisms of the family Paenibacteriaceae. SUMMARY

[0010] The present invention provides a microbial culture, wherein the microorganism belongs to the family Paenibacteriaceae, preferably to the genus Paenibacillus, wherein the cells in the culture

[0011] - are metabolically cooperative and / or

[0012] - are in an activated metabolic state.

[0013] The present invention also provides a fermentation method, the fermentation method comprising

[0014] i) providing a fermentation broth comprising a microbial culture according to the present invention in a fermentation medium; and

[0015] ii) cultivating the fermentation broth under conditions suitable for producing a target substance; and

[0016] iii) preferably recovering the target substance.

[0017] Furthermore, the present invention provides a method for reducing the fermentation time required to produce a desired amount of a target substance, and / or for reducing the viscosity during fermentation, and / or for increasing the fermentation repeatability accuracy, the method comprising the steps of:

[0018] i) providing a culture according to the present application in a fermentation medium; and

[0019] ii) cultivating the culture in the fermentation medium.

[0020] And the present application provides the use of a culture according to the present application for reducing the fermentation time needed to produce a desired amount of a target substance, and / or for reducing the viscosity during fermentation, and / or for increasing the fermentation reproducibility.

[0021] The present application also provides a cultivation parameter data set for controlling a fermentation, the cultivation parameter data set comprising

[0022] - the name of a set of applicable fermentation media,

[0023] - the name of a set of applicable microbial samples,

[0024] - the name of fermentation conditions that facilitate obtaining a culture according to the present application by cultivating an applicable sample in an applicable fermentation medium, wherein the fermentation conditions preferably comprise at least one of the following: the type of medium, the amount of medium, the required cultivation temperature range, the required cultivation aeration parameter range, the required amount of produced cells, the required cultivation duration, the required pH during cultivation, the required yield or amount of target substance.

[0025] And the present application provides a computer-implemented fermentation method, the fermentation method comprising

[0026] - receiving, by a computing device, a cultivation parameter data set according to the present application,

[0027] - receiving a fermentation medium that complies with the cultivation parameter data set,

[0028] - receiving a sample of a volume of a microorganism that complies with the cultivation parameter data set, and

[0029] - controlling, by the computing device, a fermenter to cultivate the microorganism and / or its spores in the fermentation medium under the fermentation conditions of the cultivation parameter data set.

[0030] Further, the present application provides a method for producing one or more viscosity increasing substances, the method comprising

[0031] i) inoculating a fermentation medium with a culture of microorganisms according to the present application at a concentration that is at a predetermined concentration sufficient to obtain a desired viscosity during cultivation; and

[0032] ii) cultivating the culture obtained in step i); and

[0033] iii) preferably recovering the one or more viscosity increasing substances or tackifying substances. Attached Figure Description

[0034] Figure 1 The effect of inoculum volume on the growth of *Bacillus polymyxa* LU21132 in shake flasks is shown. Biomass formation is shown as OD600 [AU] after 24 h of culture in shake flasks containing PX-125 composite medium. A range of different volumes of working cell bank (WCB) 1041 were used for inoculation (0.1%–4% v / v of total culture volume, as listed in Table 1). As the amount of cells used for inoculation increased (i.e., the volume of the WCB spore composition increased), the optical density decreased at 24 h post-inoculation, indicating slower growth in densely inoculated cultures. The fastest growth was obtained at inoculation concentrations of 0.2%–0.4% (v / v), with growth rates decreasing again at lower concentrations.

[0035] Figure 2 The effect of inoculum volume on the growth of *Bacillus polymyxa* LU20754 in 2 ml deep-well plates is shown. Cultures were performed using deep-well plates with 48 sites at an oscillation frequency of 900 rpm in a BioLector apparatus (m2p labs, Germany). Biomass formation was periodically assessed by scattered light measurements at 620 nm ex / em. Inoculation was performed using different volumes of working cell banks of spores from strain LU20754 (0.6%–1.8% v / v of total culture volume). Higher inoculum concentrations resulted in a longer delay before biomass reached 20 AU.

[0036] Figure 3 Microscopic images (left) and derived particle outlines (right) of shake-flask cultures of *Bacillus polymyxa* LU21552 are shown. Images were taken after 28 h of culture. Spores WCB1059 (prepared as described in Table 5) were used with 0.2% v / v ( Figure 3 A) and 1% v / v ( Figure 3 B) Inoculate the shake flask with the inoculation volume.

[0037] Figure 4 Showing according to Figure 3 A and Figure 3 Cell corpuscular length is determined by the particle profile of B.

[0038] Figure 5The effect of shake flask inoculum volume on production of fusaricidin by Paenibacillus polymyxa LU21132 is shown. Fusaricidin titers are shown after 24 h cultivation in shake flasks containing PX-125 complex medium and using different working cell bank (WCB) inoculum volumes (0.1-4% v / v of total culture volume, Table 1). WCB1041 consists of 1.6E+08 (endogenous) spores / ml [cv% 9.5]. Cultivation conditions: 250 ml baffled shake flasks, 30 ml medium, 150 rpm, 2.5 cm throw, 33°C, pH 6.5, 24 h. Compared to the fusaricidin yield obtained at 0.2% (v / v) inoculum volume, the fusaricidin yield dropped sharply about 50-fold at inoculum volumes above 0.4% (v / v).

[0039] Figure 6 The effect of shake flask inoculum volume on growth of Paenibacillus polymyxa LU21132 using working cell banks (WCBs) from six different preparation procedures as described in Table 5 is shown. Biomass formation is shown as OD600 [A.U.] after 24 h cultivation in shake flasks containing PX-125 complex medium. Two different spore WCB inoculum volumes (0.3% and 0.6% v / v of total medium volume) were used to test the growth properties of each of the six working cell banks. Cultivation conditions: 250 ml baffled shake flasks, 30 ml medium, 150 rpm, 2.5 cm throw, 33°C, pH 6.5, 24 h. Independent of the working cell bank preparation, the growth rate indicated by the optical density at 600 nm after 24 h inoculation at 0.3% (v / v) was about twice as high as the growth rate of the corresponding culture inoculated at 0.6% (v / v).

[0040] Figure 7 The effect of shake flask inoculum volume on production of fusaricidin by Paenibacillus polymyxa LU21132 using six different working cell banks (WCBs) prepared under different conditions (described in Table 5) is shown. Fusaricidin titers are shown after 24 h cultivation in shake flasks containing PX-125 complex medium and using two WCB inoculum volumes (0.3% and 0.6% v / v of total culture volume). Cultivation conditions: 250 ml baffled shake flasks, 30 ml medium, 150 rpm, 2.5 cm throw, 33°C, pH 6.5, 24 h. Independent of the working cell bank preparation, the fusaricidin titers of cultures inoculated at 0.3% (v / v) were about 3-25-fold higher than cultures inoculated with the 0.6% (v / v) WCB spore composition.

[0041] Figure 8Example 6: Effect of initial preculture inoculum volume on growth synchronization in the main culture (two-stage process) It was shown that the initial preculture inoculum volume does affect fengicin production in the main culture in a two-stage process: fengicin A, B, and D levels in shake flask main cultures with PX-143 medium varied with preculture inoculum volume. Preculture flasks were inoculated with 0.3% and 0.6% (v / v) of different WCB types (see Table 5). After 24 h of cultivation, cultures from Example 3 were transferred to main culture flasks using a 2% (v / v) inoculum volume. The fengicin levels of the main cultures inoculated with preculture flasks with a 0.6% (v / v) preculture inoculum volume are shown relative to the fengicin levels detected in parallel main cultures from the 0.3% preculture inoculum variants. Cultivation conditions: 250 ml baffled shake flasks, 30 ml medium, 150 rpm, 2.5 cm throw, 33°C, pH 6.5, 24 h preculture, 48 h main culture. The fengicin content accumulated within 48 h of fermentation was roughly 10% less in fermentations inoculated with precultures inoculated with 0.6% (v / v) WCB spore composition at 2% (v / v) compared to the corresponding fermentations inoculated with precultures inoculated with 0.3% (v / v) WCB spore composition at 2% (v / v) (the absolute fengicin content was roughly the same in both cases, and in each case was higher than 2000 mg / l).

[0042] Figure 9 Effect of lower preculture inoculum volume on growth synchronization in the main culture (two-stage process) was shown. The effect of six different working cell banks (WCBs) of Paenibacillus polymyxa LU21132 (prepared according to Table 5) was demonstrated. Preculture flasks (PX-125 medium) were inoculated with 0.3% and 0.6% (v / v) of different WCB types of P. polymyxa LU21132 from Table 5. After 24 h of cultivation, the precultures were transferred to main culture flasks (PX-143 medium) using a 2% (v / v) inoculum volume. After 48 h of cultivation in the main medium, the OD600 (A.U.) of each culture from the six WCBs from Table 5 was measured. Cultivation conditions: 250 ml baffled shake flasks, 30 ml medium, 150 rpm, 2.5 cm throw, 33°C, pH 6.5, 24 h preculture, 48 h main culture.

[0043] Figure 10The main culture inoculum volume was shown to determine the broth viscosity in soy-based medium (EPS-03) in shake flask cultures (250 ml baffled shake flask, 30 ml culture medium, 150 rpm, 2.5 cm throw, 33 °C, pH 6.5). The viscosity was assessed after 16 h of cultivation. EPS formation was partially uncoupled from growth as indicated by OD600, especially at 2%, 4% and 6% inoculum volume. DETAILED DESCRIPTION

[0044] The technical teaching of the present application is expressed in this document using linguistic means, in particular by using scientific and technical terms. However, the skilled person understands that linguistic means, no matter how detailed and precise, can only give an approximate picture of the whole of the technical teaching, even if only because there are multiple ways of expressing the teaching, since each expression is necessarily finite and each is necessarily unable to express all the conceptual connections. With this in mind, the skilled person understands that the subject matter of the present application is the sum of the individual technical concepts expressed in this document or necessarily expressed in a way that parts replace the whole (pars pro toto) by the inherent constraints of the written description. In particular, the skilled person will understand that in this document the meaning of individual technical concepts is done in a way that is an abbreviation of every possible combination of concepts, which can be elucidated within the technically reasonable range, so that, for example, the disclosure of three concepts or embodiments A, B and C is an abbreviation for the concepts A+B, A+C, B+C, A+B+C. In particular, back-up solutions for features are described in this document by gathering lists of alternatives or examples. Unless otherwise stated, the present application described in this document includes any combination of such alternatives. The selection of more or less preferred elements from such lists is part of the present application and such selection is caused by the preference of the skilled person for the least extent of implementation of the conveyed advantage of the respective feature. Such multiple combination examples represent one or more fully preferred forms of the present application.

[0045] As far as database entries cited in this document (e.g. Uniprot entries) are concerned, these entries are the entries published on 2022-01-1010:00 CET. This also applies to sequences published under the respective database entry identifier.

[0046] As used herein, the singular and singular forms of terms, such as "a," "an," and "the," include plural referents unless the content clearly dictates otherwise. Thus, for example, the use of the term "a nucleic acid" optionally includes many copies of that nucleic acid molecule; similarly, the term "probe" optionally (and typically) encompasses many similar or identical probe molecules. Also as used herein, the word "comprising" is to be read to mean the inclusion of any recited elements, integers or steps, or groups of elements, integers or steps, but not the exclusion of other elements, integers or steps, or groups of elements, integers or steps. It is understood that the word "comprising" is to be interpreted as "including but not limited to."

[0047] As used herein, the term "and / or" refers to and encompasses any and all possible combinations of one or more of the associated listed items, as well as the lack of combinations when interpreted in the alternative ("or"). The term "comprising" also encompasses the terms "consisting of" and "consisting essentially of."

[0048] The term "about," when used in reference to a measurable value such as an amount of mass, dose, time, temperature, sequence identity, etc., is meant to encompass variations of ± 0.1%, 0.25%, 0.5%, 0.75%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, or even 20% of and from the specified value. Thus, if a given composition is described as containing "about 50% X," it is understood that in some embodiments the composition contains 50% X, while in other embodiments it can contain any value between 40% and 60% X (i.e., 50% ± 10%).

[0049] The term "microorganism" according to the present application denotes both an individual microbial cell and a plurality of such cells, the latter also being referred to as "population" or when combined with a culture medium as "culture". Preferably, the microorganism is a single taxonomic species, and even more preferably a single strain of a species. Thus, according to the present application, a pure culture of microorganisms is preferred. The population of microorganisms can also be a consortium of microbial cells of different strains of a common species and / or can be a consortium comprising different taxonomic species or genera. However, such mixed populations and cultures are less preferred. The microorganism can in principle be a cell of any species of the taxonomic class Bacilli. The microorganism can be a wild-type organism (i.e. a microorganism that has not been substantially altered with respect to the organism originally isolated from nature), or can be a modified microorganism that has been modified by one or more technical procedures (e.g. mutagenesis). The wild-type microorganism sample is preferably documented as a type strain in a culture collection. Genetic modification can be achieved by random mutagenesis (e.g. NTG chemical mutagenesis, UV irradiation or transposon mutagenesis), or by directed mutagenesis (e.g. incorporation of a heterologous plasmid or homologous recombination with a heterologous nucleic acid) and / or by site-directed mutagenesis (e.g. using meganucleases, TALENs or CRISPR-type mutagenesis). Preferred methods of mutagenesis of Bacillus and Paenibacillus are described, for example, in WO 2017117395, which is incorporated herein in its entirety.

[0050] The microorganisms used throughout the present invention belong to the taxonomic family Paenibacillaceae. Microorganisms of the taxonomic family Paenibacillaceae are defined by their 16S rDNA similarity (De Vos, P., W. Ludwig, K. H. Scheifer, and W. B. Whitman 2009. Family IV. [Family IV. "Paenibacillaceae". Bergey's Manual of Systematic Bacteriology. P. De Vos, G. M. Garrity, D. Jones, et al., New York, Springer. 3: 269). Their genome size is typically 3-9 Mb. They are usually small to medium-sized straight or curved rods, about 0.5-1.0 x 2-6 μιη in size. They usually have a gram-positive cell wall structure and are also usually capable of sporulation. Members of this family have been isolated from a variety of habitats ranging in distribution from Alaska to the Antarctic, encompassing deep-sea sediments, ice cores, desert brines, sandstone biofilms, prairie soils, forest soils, and agronomic plant roots. Due to the diversity of this family, taxonomic rearrangements are expected to occur in the future. Preferred genera and species according to the present invention are described in more detail below.

[0051] Preferably, the microorganism belongs to any one of the following genera: Ammoniibacillus, Ammoniphilus, Aneurinibacillus, Oxalophagus, Brevibacillus, Candidatus Reconcilbacillus, Chengkuizengella, Cohnella, Fontibacillus, Gorillibacterium, Longirhabdus, Marinicrinis, Paenibacillus, Paludirhabdus, Saccharibacillus, Thermobacillus, or Xylanibacillus, more preferably Paenibacillus.

[0052] Particularly preferred are microorganisms of one of the following species:

[0053] Paenibacillus sp.: P. abekawaensis, P. abyssi, P. aceris, P. aceti, P. aestuarii, P. agarexedens, P. agaridevorans, P. alba, P. albidus, P. albus, P. alginolyticus, P. algorifonticola, P. alkaliterrae, P. alvei, P. amylolyticus, P. anaericanus, P. antarcticus, P. antibioticophila, P. antri, P. apiaries, P. apiarius, P. apis, P. aquistagni, P. arachidis, P. arcticus, P. assamensis, P. aurantiacus, P. azoreducens, P. azotifigens, P. baekrokdamisoli, P. barcinonensis, P. barengoltzii, P. beijingensis, P. borealis, P. bouchesdurhonensis, P. bovis, P. brasilensis, P. brassicae, P. bryophyllum, P. caespitis, P. camelliae, P. camerounensis, P. campinasensis, P. campinensis, P. campinasensis, P. campinensis, P. campinensis, P. campinensis, P. campinensis, P. campinensis, P. campinensis, P. campinensis, P. campinensis, P. campinensis, P. campinensis, P. campinensis, P. campinensis, P. campinensis, P. campinensis, P. campinensis, P. campinensis, P. campinensis, P. campinensis, P. campinensis, P. campinensis, P. campinensis, P. campinensis, P. campinensis, P. campinensis, P. campinensis, P. campinensis, P. campinensis, P. campinensis, P. campinensis, P. campinensis, P. campinensis, P. campinensis, P. campinensis, P. campinensis, P. campinensis, P. campinensis, P. campinensis, P. campinensis, P. campinensis, P. campinensis, P. campinensis, P. campinensis, P. campinensis, P. campinensis, P. campinensis, P. campinensis, P. campinensis, P. campinensis, P. campinensis, P. campinensis, P. campinensis, P. campinensis, P. campinensis, P. campinensis, P. campinensis, P. campinensis, P. campinensis, P. campinensis, P. campinensis, P. campinensis, P. campinensis, P. campinensis, P. campinensis, P. campinensis, P. campinensis, P. campinensis, P. campinensis, P. campinensis, P. campinensis, P. campinensis, P. campinensis, P. campinensis, P. campinensis, P. campinensis, P. campinensis, P. campinensis, P. campinensis, P. campinensis, P. campinensis, P. campinensis, P. 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campinensis, P. campinensis, P. campinensis, P. campinensis, P. campinensis, P. campinensis, P. campinensis, P. campinensis, P. campinensis, P. campinensis, P. campinensis, P. campinensis, P. campinensis, P. campinensis, P. campinensis, P. campinensis, P. campinensis, P. campinensis, P. campinensis, P. campinensis, P. campinensis, P. campinensis, P. campinensis, P. campinensis, P. campinensis, P. campinensis, P. campinensis, P. campinensis, P. campinensis, P. campinensis, P. campinensis, P. campinensis, P. campinensis, P. campinensis, P. campinensis, P. campinensis, P. campinensis, P. campinensis, P. campinensis, P. campinensis, P. campinensis, P. campinensis, P. campinensis, P. campinensis, P. campinensis, P. campinensis, P. campinensis, P. campinensis, P. campinensis, P. campinensis, P. campinensis, P. campinensis, P. campinensis, P. campinensis, P. campinensis, P. campinensis, P. campinensis, P. campinensis, P. campinensis, P. campinensis, P. campinensis, P. campinensis, P. campinensis, P. campinensis, P. campinensis, P. campinensis, P. campinensis, P. campinensis, P. campinensis, P. campinensis, P. campinensis, P. campinensis, P. campinensis, P. campinensis, P. campinensis, P. campinensis, P. campinensis, P. campinensis, P. campinensis, P. campinensis, P. campinensis, P. campinensis, P. campinensis, P. campinensis, P. campinensis, P. campinensis, P. campinensis, P. campinensis, P. campinensis, P. campinensis, P. campinensis, P. campinensis, P. campinensis, P. campinensis, P. campinensis, P. campinensis, P. campinensis, P. campinensis, P. campinensis, P. campinensis, P. campinensis, P. campinensis, P. campinensis, P. campinensis, P. campinensis, P. campinensis, P. campinensis, P. campinensis, P. campinensis, P. campinensis, P. campinensis, P. campinensis, P. campinensis, P. campinensis, P. campinensis, P. campinensis, P. campinensis, P. campinensis, P. campinensis, P. campinensis, P. campinensis, P. campinensis, P. campinensis, P. campinensis, P. campinensis, P. campinensis, P. campinensis, P. campinensis, P. campinensis, P. campinensis, P. campinensis, P. campinensis, P. campinensis, P. campinensis, P. campinensis, P. campinensis, P. campinensis, P. campinensis, P. campinensis, P. campinensis, P. campinensis, P. campinensis, P. campinensis, P. campinensis, P. campinensis, P. campinensis, P. campinensis, P. campinensis, P. campinensis, P. campinensis, P. campcampinasensis), P. castaneae, P. catalpae, P. cathormii, P. cavernae, P. cellulosilyticus, P. cellulositrophicus, P. chartarius, P. chibensis, P. chinensis, P. chinjuensis, P. chitinolyticus, P. chondroitinus, P. chungangensis, P. cineris, P. cisolokensis, P. contaminans, P. cookii, P. crassostreae, P. cucumis, P. curdlanolyticus, P. daejeonensis, P. dakarensis, P. darangshiensis, P. darwinianus, P. dauci, P. dendritiformis, P. dodoensis, P. donghaensis, P. doosanensis, P. durus, P. edaphicus, P. ehimensis, P. elgii, P. elymi, P. endophyticus, P. enshidis, P. esterisolvens, P. etheri, P. eucommiae, P. faecis, P. favisporus, P. ferrarius, P. filicis, P. flagellatus, P. flavidus, P. floccincommunis, P. flocculus, P. fluorescens, P. fonticola, P. formosus, P. fortuitus, P. frigidicampi, P. funeosolvens, P. gaucho, P. gelidus, P. ginsengihumi, P. ginsengisoli, P. ginsengisoli, P. ginsengisoli, P. ginsengisoli, P. ginsengisoli, P. ginsengisoli, P. ginsengisoli, P. ginsengisoli, P. ginsengisoli, P. ginsengisoli, P. ginsengisoli, P. ginsengisoli, P. ginsengisoli, P. ginsengisoli, P. ginsengisoli, P. ginsengisoli, P. ginsengisoli, P. ginsengisoli, P. ginsengisoli, P. ginsengisoli, P. ginsengisoli, P. ginsengisoli, P. ginsengisoli, P. ginsengisoli, P. ginsengisoli, P. ginsengisoli, P. ginsengisoli, P. ginsengisoli, P. ginsengisoli, P. ginsengisoli, P. ginsengisoli, P. ginsengisoli, P. ginsengisoli, P. ginsengisoli, P. ginsengisoli, P. ginsengisoli, P. ginsengisoli, P. ginsengisoli, P. ginsengisoli, P. ginsengisoli, P. ginsengisoli, P. ginsengisoli, P. ginsengisoli, P. ginsengisoli, P. ginsengisoli, P. ginsengisoli, P. ginsengisoli, P. ginsengisoli, P. ginsengisoli, P. ginsengisoli, P. ginsengisoli, P. ginsengisoli, P. ginsengisoli, P. ginsengisoli, P. ginsengisoli, P. ginsengisoli, P. ginsengisoli, P. ginsengisoli, P. ginsengisoli, P. ginsengisoli, P. ginsengisoli, P. ginsengisoli, P. ginsengisoli, P. ginsengisoli, P. ginsengisoli, P. ginsengisoli, P. ginsengisoli, P. ginsengisoli, P. ginsengisoli, P. ginsengisoli, P. ginsengisoli, P. ginsengisoli, P. ginsengisoli, P. ginsengisoli, P. ginsengisoli, P. ginsengisoli, P. ginsengisoli, P. ginsengisoli, P. ginsengisoli, P. ginsengisoli, P. ginsengisoli, P. ginsengisoli, P. ginsengisoli, P. ginsengisoli, P. ginsengisoli, P. ginsengisoli, P. ginsengisoli, P. ginsengisoli, P. ginsengisoli, P. ginsengisoli, P. ginsengisoli, P. ginsengisoli, P. ginsengisoli, P. ginsengisoli, P. ginsengisoli, P. ginsengisoli, P. ginsengisoli, P. ginsengisoli, P. ginsengisoli, P. ginsengisoli, P. ginsengisoli, P. ginsengisoli, P. ginsengisoli, P. ginsengisoli, P. ginsengisoli, P. ginsengisoli, P. ginsengisoli, P. ginsengisoli, P. ginsengisoli, P. ginsengisoli, P. ginsengisoli, P. ginsengisoli, P. ginsengisoli, P. ginsengisoli, P. ginsengisoli, P. ginsengisoli, P. ginsengisoli, P. ginsengisoli, P. ginsengisoli, P. ginsengisoli, P. ginsengisoli, P. ginsengisoli, P. ginsengisoli, P. ginsengisoli, P. ginsengisoli, P. ginsengisoli, P. ginsengisoli, P. ginsengisoli, P. ginsengisoli, P. ginsengisoli, P. ginsengisoli, P. ginsengisoli, P. ginsengisoli, P. ginsengisoli, P. ginsengisoli, P. ginsengisoli, P. ginsengisoli, P. ginsengisoli, P. ginsengisoli, P. ginsengisoli, P. ginsengisoli, P. ginsengisoli, P. ginsengisoli, P. ginsengisoli, P. ginsengisoli, P. ginsengisoli, P. ginsengisoli, P. ginsengisoli, P. ginsengisoli, P. ginsengisoli, P. ginsengisoli, P. ginsengisoli, P. ginsengisoli, P. ginsengisoli, P. ginsengisoli, P. ginsengisoli, P. ginsengisoli, P. ginsengisoli, P. ginsengisoli, P. ginsengisoli, P. ginsengisoli, P. ginsengisoli, P. ginsengisoli, P. ginsengisoli, P. ginsengisoli, P. ginsengisoli, P. ginsengisoli, P. ginsengisoli, P. ginsengisoli, P. ginsengisoli, P. ginsengisoli, P. ginsengisoli, P. ginsengisoli, P. ginsengisoli, P. ginsengisoli, P. ginsengisoli, P. ginsengisoli, P. ginsengisoli, P. ginsengisoli, P. ginsengisoli, P. ginsengisoli, P. ginsengisoli, P. ginsengisoli, P. ginsengisoli, P. ginsengisoli, P. ginsengisoli, P. ginsengisoli, P. ginsengisoli, P. ginsengisoli, P. ginsengisoli, P. ginsengisoli, P. ginsengisoli, P. ginsengisoli, P. ginsengisoli, P. ginsengisoli, P. ginsengisoli, P. ginsengisoli, P. ginsengisoli, P. ginsengisoli, P. ginsengisoli, P. ginsengisoli, P. ginsengisoli, P. ginsengisoli, P. ginsengisoli, P. ginsengisoli, P. ginsengisoli, P. ginsengisoli, P. ginsengisoli, P. ginsengisoli, P. ginsengisoli, P. ginsengisoli,*P. flagellatus*, *P. fonticola*, *P. forsythiae*, *P. frigoriresistens*, *P. fujiensis*, *P. fukuinensis*, *P. gansuensis*, *P. gelatinilyticus*, *P. ginsengagri*, *P. ginsengarvi*, *P. ginsengihumi*, *P. ginsengiterrae*, *P. glacialis*, *P. glebae*, *P. glucanolyticus*, *P. glycanilyticus*, *P. gorillailyticus*. *P. gorillae*, *P. graminis*, *P. granivoraans*, *P. guangzhouensis*, *P. harenae*, *P. helianthi*, *P. hemerocallicola*, *P. herberti*, *P. hispanicus*, *P. hodogayensis*, *P. hordei*, *P. horti*, *P. humicus*, *P. hunanensis*, *P. ihbetae*, *P. ihuae*, *P. ihumii*, *P. Illinois* *P. insulae*, *P. intestini*, *P. jamilae*, *P. jilunlii*, *P. kobensis*, *P. koleovorans*, *P. konkukensis*, *P. konsidensis*, *P. koreensis*, *P. kribbensis*, *P. kyunghee*.kyungheensis), P. lactis, P. lacus, P. larvae, P. lautus, P. lemnae, P. lentimorbus, P. lentus, P. liaoningensis, P. limicola, P. lupini, P. luteus, P. lutimineralis, P. macerans, P. macquariensis, P. marchantiophytorum, P. marinisediminis, P. marinum, P. massiliensis, P. maysiensis, P. medicaginis, P. mendelii, P. mesophilus, P. methanolicus, P. mobilis, P. montanisoli, P. montaniterrae, P. motobuensis, P. mucilaginosus, P. nanensis, P. naphthalenovorans, P. nasutitermitis, P. nebraskensis, P. nematophilus, P. nicotianae, P. nuruki, P. oceanisediminis, P. odorifer, P. oenotherae, P. oralis, P. oryzae, P. oryzisoli, P. oryzae, P. oryzisoli, P. oryzae, P. oryzisoli, P. oryzae, P. oryzisoli, P. oryzae, P. oryzisoli, P. oryzae, P. oryzisoli, P. oryzae, P. oryzisoli, P. oryzae, P. oryzisoli, P. oryzae, P. oryzisoli, P. oryzae, P. oryzisoli, P. oryzae, P. oryzisoli, P. oryzae, P. oryzisoli, P. oryzae, P. oryzisoli, P. oryzae, P. oryzisoli, P. oryzae, P. oryzisoli, P. oryzae, P. oryzisoli, P. oryzae, P. oryzisoli, P. oryzae, P. oryzisoli, P. oryzae, P. oryzisoli, P. oryzae, P. oryzisoli, P. oryzae, P. oryzisoli, P. oryzae, P. oryzisoli, P. oryzae, P. oryzisoli, P. oryzae, P. oryzisoli, P. oryzae, P. oryzisoli, P. oryzae, P. oryzisoli, P. oryzae, P. oryzisoli, P. oryzae, P. oryzisoli, P. oryzae, P. oryzisoli, P. oryzae, P. oryzisoli, P. oryzae, P. oryzisoli, P. oryzae, P. oryzisoli, P. oryzae, P. oryzisoli, P. oryzae, P. oryzisoli, P. oryzae, P. oryzisoli, P. oryzae, P. oryzisoli, P. oryzae, P. oryzisoli, P. oryzae, P. oryzisoli, P. oryzae, P. oryzisoli, P. oryzae, P. oryzisoli, P. oryzae, P. oryzisoli, P. oryzae, P. oryzisoli, P. oryzae, P. oryzisoli, P. oryzae, P. oryzisoli, P. oryzae, P. oryzisoli, P. oryzae, P. oryzisoli, P. oryzae, P. oryzisoli, P. oryzae, P. oryzisoli, P. oryzae, P. oryzisoli, P. oryzae, P. oryzisoli, P. oryzae, P. oryzisoli, P. oryzae, P. oryzisoli, P. oryzae, P. oryzisoli, P. oryzae, P. oryzisoli, P. oryzae, P. oryzisoli, P. oryzae, P. oryzisoli, P. oryzae, P. oryzisoli, P. oryzae, P. oryzisoli, P. oryzae, P. oryzisoli, P. oryzae, P. oryzisoli, P. oryzae, P. oryzisoli, P. oryzae, P. oryzisoli, P. oryzae, P. oryzisoli, P. oryzae, P. oryzisoli, P. oryzae, P. oryzisoli, P. oryzae, P. oryzisoli, P. oryzae, P. oryzisoli, P. oryzae, P. oryzisoli, P. oryzae, P. oryzisoli, P. oryzae, P. oryzisoli, P. oryzae, P. oryzisoli, P. oryzae, P. oryzisoli, P. oryzae, P. oryzisoli, P. oryzae, P. oryzisoli, P. oryzae, P. oryzisoli, P. oryzae, P. oryzisoli, P. oryzae, P. oryzisoli, P. oryzae, P. oryzisoli, P. oryzae, P. oryzisoli, P. oryzae, P. oryzisoli, P. oryzae, P. oryzisoli, P. oryzae, P. oryzisoli, P. oryzae, P. oryzisoli, P. oryzae, P. oryzisoli, P. oryzae, P. oryzisoli, P. oryzae, P. oryzisoli, P. oryzae, P. oryzisoli, P. oryzae, P. oryzisoli, P. oryzae, P. oryzisoli, P. oryzae, P. oryzisoli, P. oryzae, P. oryzisoli, P. oryzae, P. oryzisoli, P. oryzae, P. oryzisoli, P. oryzae, P. oryzisoli, P. oryzae, P. oryzisoli, P. oryzae, P. oryzisoli, P. oryzae, P. oryzisoli, P. oryzae, P. oryzisoli, P. oryzae, P. oryzisoli, P. oryzae, P. oryzisoli, P. oryzae, P. oryzisoli, P. oryzae, P. oryzisoli, P. oryzae, P. oryzisoli, P. oryzae, P. oryzisoli, P. oryzae, P. oryzisoli, P. oryzae, P. oryzisoli, P. oryzae, P. oryzisoli, P. oryzae, P. oryzisoli, P. oryzae, P. oryzisoli, P. oryzae, P. oryzisoli, P. oryzae, P. oryzisoli, P. oryzae, P. oryzisoli, P. oryzae, P. oryzisoli, P. oryzae, P. oryzisoli, P. oryzae, P. oryzisoli, P. oryzae, P. oryzisoli, P. oryzae, P. oryzisoli, P. oryzae, P. oryzisoli, P. oryzae, P. oryzisoli, P. oryzae, P. oryzisoli, P. oryzae, P. oryzisoli, P. oryzae, P. oryzisoli, P. oryzae, P. oryzisoli, P. oryzae, P. oryzisoli, P. oryzae, P. oryzisoli, P. oryzae, P. oryzisoli, P. oryzae, P. oryzisoli, P. oryzae, P. oryzisoli, P. oryzaeP. ottowii, P. ourofinensis, P. pabuli, P. paeoniae, P. panacihumi, P. panacisoli, P. panaciterrae, P. paridis, P. pasadenensis, P. pectinilyticus, P. peoriae, P. periandrae, P. phocaensis, P. phoenicis, P. phyllosphaerae, P. physcomitrellae, P. pini, P. pinihumi, P. pinisoli, P. pinistramenti, P. pocheonensis, P. polyp myxa, P. polysaccharolyticus, P. popilliae, P. populi, P. profundus, P. prosopidis, P. protaetiae, P. provencensis, P. psychroresistens, P. pueri, P. puernese, P. puldeungensis, P. purispatii, P. qingshengii, P. qinlingensis, P. quercus, P. radicis, P. relictisesami, P. residui, P. rhizoplanae, P. rhizoryzae, P. rhizosphaerae, P. rhamnii, P. rhodinolfii, P. rhodocrous, P. rhodopurpureus, P. rhodovorus, P. rishiriensis, P. roseus, P. roseus, P. roseus, P. roseus, P. roseus, P. roseus, P. roseus, P. roseus, P. roseus, P. roseus, P. roseus, P. roseus, P. roseus, P. roseus, P. roseus, P. roseus, P. roseus, P. roseus, P. roseus, P. roseus, P. roseus, P. roseus, P. roseus, P. roseus, P. roseus, P. roseus, P. roseus, P. roseus, P. roseus, P. roseus, P. roseus, P. roseus, P. roseus, P. roseus, P. roseus, P. roseus, P. roseus, P. roseus, P. roseus, P. roseus, P. roseus, P. roseus, P. roseus, P. roseus, P. roseus, P. roseus, P. roseus, P. roseus, P. roseus, P. roseus, P. roseus, P. roseus, P. roseus, P. roseus, P. roseus, P. roseus, P. roseus, P. roseus, P. roseus, P. roseus, P. roseus, P. roseus, P. roseus, P. roseus, P. roseus, P. roseus, P. roseus, P. roseus, P. roseus, P. roseus, P. roseus, P. roseus, P. roseus, P. roseus, P. roseus, P. roseus, P. roseus, P. roseus, P. roseus, P. roseus, P. roseus, P. roseus, P. roseus, P. roseus, P. roseus, P. roseus, P. roseus, P. roseus, P. roseus, P. roseus, P. roseus, P. roseus, P. roseus, P. roseus, P. roseus, P. roseus, P. roseus, P. roseus, P. roseus, P. roseus, P. roseus, P. roseus, P. roseus, P. roseus, P. roseus, P. roseus, P. roseus, P. roseus, P. roseus, P. roseus, P. roseus, P. roseus, P. roseus, P. roseus, P. roseus, P. roseus, P. roseus, P. roseus, P. roseus, P. roseus, P. roseus, P. roseus, P. roseus, P. roseus, P. roseus, P. roseus, P. roseus, P. roseus, P. roseus, P. roseus, P. roseus, P. roseus, P. roseus, P. roseus, P. roseus, P. roseus, P. roseus, P. roseus, P. roseus, P. roseus, P. roseus, P. roseus, P. roseus, P. roseus, P. roseus, P. roseus, P. roseus, P. roseus, P. roseus, P. roseus, P. roseus, P. roseus, P. roseus, P. roseus, P. roseus, P. roseus, P. roseus, P. roseus, P. roseus, P. roseus, P. roseus, P. roseus, P. roseus, P. roseus, P. roseus, P. roseus, P. roseus, P. roseus, P. roseus, P. roseus, P. roseus, P. roseus, P. roseus, P. roseus, P. roseus, P. roseus, P. roseus, P. roseus, P. roseus, P. roseus, P. roseus, P. roseus, P. roseus, P. roseus, P. roseus, P. roseus, P. roseus, P. roseus, P. roseus, P. roseus, P. roseus, P. roseus, P. roseus, P. roseus, P. roseus, P. roseus, P. roseus, P. roseus, P. roseus, P. roseus, P. roseus, P. roseus, P. roseus, P. roseus, P. roseus, P. roseus, P. roseus, P. roseus, P. roseus, P. roseus, P. roseus, P. roseus, P. roseus, P. roseus, P. roseus, P. roseus, P. roseus, P. roseus, P. roseus, P. roseus, P. roseus, P. roseus, P. roseus, P. roseus, P. roseus, P. roseus, P. roseus, P. roseus, P. roseus, P. roseus, P. roseus, P. roseus, P. roseus, P. roseus, P. roseus, P. roseus, P. roseus, P. roseus, P. roseus, P. roseus, P. roseus, P. roseus, P. roseus, P. roseus, P. roseus, P. roseus, P. roseus, P. roseus, P. roseus, P. roseus, P. roseus, P. roseus, P. roseus, P. roseus, P. roseus, P. roseus, P. roseus, P. roseus, P. roseus, P. roseus, P. roseus, P. roseus, P. roseus, P. roseus, P. roseus, P. roseus, P. roseus, P. roseus, P. roseus, P. roseus, P. roseus, P. roseus, P. roseus, P. roseus, P. roseus, P. roseus, P. roseus, P. roseus, P. roseus, P. roseus, P. roseus, P. roseus, P. roseus, P. roseus, P. roseus, P. roseus, P. roseus, P. roseus, P. roseus, P. roseus, P. roseus, P. roseus, P. roseus, P. roseus, P. roseus, P. roseus, P. roseus, P. roseus, P. roseus, P. roseus, P. roseus, P. roseus, P. roseus, P. roseus, P. roseus, P. roseus, P. roseus, P. roseus, P. roseus, P. roseus, P. roseus, P. roseus, P. roseus, P. roseus, P. roseus, P. roseus, P. roseus, P. roseus, P. roseus, P. roseus, P. roseus, P. roseus, P. roseus, P. roseus, P. roseus, P. roseus, P. roseus, P. roseus, P. roseus, P. roseus, P. roseus, P. roseus, P. roseus, P. roseus, P. roseus, P. roseus, P. roseus, P. roseus, P. roseus, P. roseus, P. roseus, P. roseus, P. roseus*P. rigui*, *P. ripae*, *P. rubinfantis*, *P. ruminocola*, *P. sabinae*, *P. sacheonensis*, *P. salinicaeni*, *P. sanguinis*, *P. sediminis*, *P. segetis*, *P. selenii*, *P. selenitireducens*, *P. senegalensis*, *P. senegalimassiliensis*, *P. seodonensis*, *P. septentrionalis*, *P. cemetery*. *Pseudomonas sepulcri*, *P. shenyangensis*, *P. shirakamiensis*, *P. shunpengii*, *P. siamensis*, *P. silagei*, *P. silvae*, *P. sinopodophylli*, *P. solanacearum*, *P. solani*, *P. soli*, *P. sonchi* group, *P. sophorae*, *P. spiritus*, *P. sputi*, *P. stellifer*, *P. susongensis*, *P. sendum*. *Pseudomonas swuensis*, *Ps. taichungensis*, *Ps. taihuensis*, *Ps. taiwanensis*, *Ps. taohuashanense*, *Ps. tarimensis*, *Ps. telluris*, *Ps. tepidiphilus*, *Ps. terrae*, *Ps. terreus*, and *P. terreus* are all species of *Bacillus*.terregens), Paenibacillus tisoviensis, P. thailandensis, P. thermoaerophilus, P. thermophilus, P. thiaminolyticus, P. tianmuensis, P. tibetensis, P. timonensis, P. translucens, P. tritici, P. triticisoli, P. tuaregi, P. tumbae, P. tundrae, P. turicensis, P. tylopili, P. typhae, P. tyrfis, P. uliginis, P. urinalis, P. validus, P. velaei, P. vini, P. vortex, P. vorticalis, P. vulneris, P. wenxiniae, P. whitsoniae, P. wooponensis, P. xianmiancola, P. wulumuqiensis, P. wynnii, P. xanthanilyticus, P. xanthinilyticus, P. xerothermodurans, P. xinjiangensis, P. xylanexedens, P. xylaniclasticus, P. xylanilyticus, P. xylanisolvens, P. yanchengensis, P. yonginensis, P. yunnanensis, P. zanthoxyli, P. zanthoxyli, P.zeae).

[0054] Preferred species include Bacillus subtilis, Bacillus alginate-producing bacteria, Bacillus alkaline-earth-producing bacteria, Bacillus vesicularis, Bacillus amyloliquefaciens, Bacillus anaerobic bacteria, Bacillus antarcticus, Bacillus assamum, Bacillus azotobacter, Bacillus barbarus, Bacillus cerevisiae, Bacillus brasiliensis, Bacillus spp., Bacillus campinatus, and Bacillus jinnensis. Bacillus subtilis, Bacillus chitinosa, Bacillus chondroitinus, Bacillus volcanic ash, Bacillus cloacae, Bacillus oota, Bacillus dendriticus, Bacillus ehime, Bacillus egeiformis, Bacillus honeycomb, Bacillus dextran, Bacillus polysaccharide, Bacillus cerealis, Bacillus granulosus, Bacillus porphyria, Bacillus juvenileus, Bacillus spp., Bacillus juvenileus, Bacillus japonic ... Kobe Bacillus, Coleoptera Bacillus, Korean Bacillus, Krebsori Bacillus, Lactobacillus, Larval Bacillus, Brilliant Bacillus, Slow-acting Bacillus, Ephedra Bacillus, Macori Bacillus, Marseille Bacillus, Mendelian Bacillus, Motobu Bacillus, Naphthylophagocytic Bacillus, Nematophyte Bacillus, Odor Bacillus, Feed Bacillus, Pierreo Bacillus, Phenyle Bacillus Bacillus subtilis, Bacillus phylum, Bacillus polymyxa, Bacillus spp., Bacillus rhizosphere, Bacillus hemoglobinus, Bacillus stellaria, Bacillus taichungensis, Bacillus terreus, Bacillus thiamine, Bacillus timonis, Bacillus pilosa, Bacillus zurichensis, Bacillus robustus, Bacillus vortex, Bacillus wounds, Bacillus wiltii, Bacillus xylolyticus

[0055] Particularly preferred are Paenibacillus peoriae, Paenibacillus jamilae, Paenibacillus brasiliensis, Paenibacillus kribbensis, Paenibacillus koreensis, Paenibacillus rhizosphaerae, Paenibacillus polymyxa, Paenibacillus amylolyticus, Paenibacillus terrae, Paenibacillus polymyxa polymyxa, Paenibacillus polymyxa plantarum, Paenibacillus nov. spec. epiphyticus, Paenibacillus terrae, Paenibacillus macerans, Paenibacillus alvei,

[0056] More preferred are Paenibacillus polymyxa, Paenibacillus polymyxa polymyxa, Paenibacillus polymyxa plantarum, Paenibacillus nov. spec. epiphyticus, Paenibacillus terrae, Paenibacillus macerans, Paenibacillus alvei,

[0057] Even more preferred are Paenibacillus polymyxa, Paenibacillus polymyxa polymyxa, Paenibacillus polymyxa plantarum and Paenibacillus terrae.

[0058] Some suitable Paenibacillus strains are described and the deposit information is given in the following international patent applications; spores of such microorganisms or any pesticidal ly active variant thereof can be incorporated as spores of the composition according to the present application: WO 2019155253: DSM32460 (Paenibacillus polymyxa strain VMC10 / 96), NCBI Accession Number NR_117743 (Paenibacillus peoriae DSM320), NCBI Accession Number HG324076 (Paenibacillus polymyxa DSM 36, Clone 13), NCBI Accession Number AJ271157 (Paenibacillus jamilae CECT 5266), NCBI Accession Number NR_025169 (Paenibacillus cribrillicus AM49), NCBI Accession Number NR_025106 (Paenibacillus brasiliensis PB172), NCBI Accession Number AF391124 (Paenibacillus terrae AM141); WO 2020181053: Paenibacillus sp. NRRL B-67721, Paenibacillus sp. NRRL B-67723, Paenibacillus sp. NRRL B-67724, Paenibacillus sp. NRRL B-50374.

[0059] Most preferably, the microorganism is a microorganism taxonomically classified as Paenibacillus and is selected from any one of the following species: Paenibacillus polymyxa, Paenibacillus polymyxa subsp. polynyxa, Paenibacillus polymyxa subsp. plantarum and Paenibacillus terrae. According to the present application, the following type strains are preferred:

[0060]

[0061]

[0062]

[0063]

[0064]

[0065]

[0066] As mentioned above, the prevailing belief in the art, supported by evidence in several species of the whole phylum Firmicutes, is that high inoculum titers are beneficial for industrial fermentations, in particular to reduce the duration of the lag phase and to ensure a fast germination of the spores. Contrary to this belief, the inventors have found that low inoculum titers are beneficial for industrial fermentations. At the same time, the inventors found that very low inoculum titers do not have a beneficial effect in industrial fermentations of Paenibacillaceae, as shown in e.g. WO 2006096164 and WO 2017068012. While useful inoculum titers will be described herein, in particular in the example section, it is understood that the optimal inoculum titer is also influenced to some extent by the medium composition. The present application accordingly provides a method of screening the inoculum amount of a microorganism of the family Paenibacillaceae, more preferably of the genus Paenibacillus.

[0067] According to the present application, the terms "spore" and "endospore" are used interchangeably.

[0068] The present application provides a microbial culture, wherein the microorganism is of the family Paenibacillaceae, preferably of the genus Paenibacillus. The term "culture" according to the present application means a collection or aggregate of microorganisms, wherein at least 50% of all living cells and spores are of the family Paenibacillaceae, preferably of the genus Paenibacillus. More preferably, the culture is a pure culture of microorganisms of the family Paenibacillaceae, preferably of the genus Paenibacillus. Furthermore, the "culture" according to the present application can either be a liquid culture, wherein the microorganisms are suspended in a liquid culture medium suitable for their growth, or a microbial preparation in a form suitable for inoculation of a liquid culture, preferably in the form of a cryopreserved cell bank vial, preferably containing a cryoprotectant and / or a stabilizer, such as glycerol and / or a sugar. Culture media and methods for generating and maintaining cell bank vials are known to the skilled person.

[0069] The cells in the culture are metabolically coordinated and / or in an activated metabolic state. A particular advantage of the present invention is that for the first time such a culture is provided in an industrially relevant amount, i.e. in a liquid culture volume of more than 10 I, more preferably 10-1000 I, even more preferably 20-1000 I, even more preferably 50-1000 I, even more preferably 100-1000 I. However, a particular advantage of the method is that the culture volume needed to carry out the method can be small, preferably 10-500 ml, more preferably 15-250 ml, even more preferably 20-100 ml. As described herein, the present invention is advantageously applicable to industrial fermentation. Thus, the fermenter vessel preferably has a culture volume of more than 200 I, preferably 300 I, more preferably 400-1000 I, more preferably 500 I, more preferably 1000-150000 I, more preferably 2000-150000 I, more preferably 50000-150000 I. A particular advantage is that the fermenter can have a culture volume of 50-300 m 3 . Such fermenter volumes are often encountered in existing industrial fermentation facilities. Thus, the present invention advantageously avoids the need to re-equip, in particular to scale up or down, existing fermentation hardware.

[0070] The culture of metabolically coordinated and / or in an activated metabolic state microbial cells has many advantages in a fermentation process, as described in more detail below. In particular, such a culture enables a reduction of the fermentation time needed to produce a desired amount of a target substance, and / or a reduction of the viscosity during fermentation, and / or an increase of the fermentation reproducibility. It is particularly surprising that such advantages can be obtained by maintaining appropriate culture conditions for Paenibacillaceae, in particular for species belonging to the genus Paenibacillus. Such microorganisms have been used in the past for a variety of applications. However, so far it has been believed that genetic modification of such microorganisms is the only feasible way to significantly reduce the fermentation time needed to produce a desired amount of a target substance, and / or to reduce the viscosity during fermentation, and / or to increase the fermentation reproducibility relative to wild type cells. A further advantage of the present invention is that the desired properties of the microbial culture for inoculation of an industrial fermentation can easily be observed using standard laboratory practice and equipment. Furthermore, the conditions for producing the microbial culture according to the present invention are robust, such that variations in the operating aspects typical in a laboratory or production environment can be tolerated. In particular, the time between inoculation of the growth medium and harvesting of the culture can vary by several hours. Thus, the identification of the properties of the microbial culture is easily determined in practice and can be reliably obtained using standard culture media and equipment.

[0071] In the culture of the present invention, the microorganism of the Paenibacillaceae family, preferably of the Paenibacillus genus, is preferably characterized by any of the following parameters:

[0072] a) the median cell corpuscle length is 0.8-2.9 pm, more preferably 1-2.5 pm, even more preferably 1.3-2 pm, even more preferably 1.3-1.8 pm, and / or

[0073] b) the 75th percentile of the cell corpuscle length is 1.3-3 pm, more preferably 1.3-2.9 pm, more preferably 1.5-2.5 pm, and / or

[0074] c) the 25th percentile of the cell corpuscle length is 0.8-2.5 pm, more preferably 0.8-1.9 pm, more preferably 0.8-1.5 pm.

[0075] Thus, 75% of the cell corpuscles preferably have a length of at most 3 pm, more preferably of up to 2.9 pm, more preferably of at most 2.5 pm, and / or 25% of the cell corpuscles have a length of at most 2.5 pm, more preferably of 1.9 pm, even more preferably of 0.8-1.5 pm.

[0076] The microorganism of the Paenibacillaceae family forms rod-shaped corpuscles in liquid culture. According to the present invention, the "cell corpuscle length" is the extension of such a corpuscle along the longest axis. The drawings and examples depict such corpuscles in more detail and describe methods for determining the cell corpuscle length. A particular advantage of the present invention is that the microorganism of the Paenibacillaceae family produces two distinctively different growth types in liquid culture. In the common type observed in current technical fermentations, the cells are rather large with a median cell corpuscle length of 3.4 pm. However, the inventors have found that there is a second growth type that provides the advantages of the present invention. Cells growing in this growth type are referred to herein as "metabolically synergistic" or "in an activated metabolic state" because their fermentation properties in liquid fermentation are significantly and consistently different from the properties of microorganisms growing in the first growth type as described herein. A particular advantage is that the microorganism of the second growth type ("metabolically synergistic" microorganism or "in an activated metabolic state" microorganism) can be distinguished from the microorganism of the first growth type by simple microscopic examination. For a trained user, the transition from the first growth type to the second growth type in liquid culture can be easily observed due to the significant change in the median cell corpuscle length.

[0077] The Paenibacillaceae microorganism culture of the present invention is preferably obtainable or obtained by inoculating a culture medium with a concentration of microorganism that is sufficient to produce said culture, wherein the concentration is preferably determined by:

[0078] i) cultivating a series of cultures inoculated with different inoculation concentrations for a sufficient time to distinguish the cultures by a parameter indicative of metabolic activity, wherein the parameter is preferably selected from any one of the following: growth rate, cell size, viscosity, production of an indicator substance, optical density, cell count, pH process, gas formation, oxygen uptake rate, CO2 transfer rate, and variance of any of the preceding parameters; and

[0079] ii) determining the one or more parameters; and

[0080] iii) selecting an inoculation concentration based on the determined one or more parameters.

[0081] A particular advantage of the present invention is that the transition from the first growth type to the second metabolically synergistic or activated growth type can be induced by selecting an inoculation concentration that is sufficiently low. Inoculation can be performed by inoculating fresh growth medium with microbial cells and / or spores. By varying the inoculation concentration, an optimal concentration can be found that effects the transition from the first growth type to the second growth type within the desired cultivation time.

[0082] A further advantage of the present invention is that other parameters than cell pellet length can also be observed to indicate the transition of a culture to metabolically synergistic and / or to a microorganism in an activated metabolic state. As described in more detail below, the microbial culture of the present invention advantageously provides increased metabolic activity compared to wild type cells in the first growth type. Preferably, such increased metabolic activity is determined from any one of the following: growth rate, cell size, viscosity, or production of an indicator substance, optical density, cell count, pH process, gas formation, oxygen uptake rate, and CO2 transfer rate. It is also a particular advantage that the microbial culture of the present invention comprises metabolically synergistic cells and thus enables to achieve highly consistent results in repeated fermentations. Therefore, in addition to or instead of measuring any of the above parameters per se, the variance of any of the above parameters can also be determined. A low variance indicates a successful transition to the second growth type.

[0083] The production of an indicator substance is preferably determined, for example, by the concentration at a predetermined time or the time to reach a predetermined concentration. The indicator substance can be chosen at will. For example, the indicator substance can be a colored substance, a metabolic by-product (e.g. acetoin, 2,3 BDO, glutamate), or a target substance, preferably fusaricidin, paeniserine, paeniprolixine, or polymyxin. Thus, batches are preferably compared by any one of the following criteria:

[0084] - the time needed to achieve a predetermined yield of the target substance (preferably fusaricidin, pantocin, pantocinoplosin or polymyxin),

[0085] - the yield of the target substance (preferably fusaricidin, pantocin, pantocinoplosin or polymyxin) at a predetermined time after the start of step ii), preferably at 8-48 h, more preferably at 12-24 h.

[0086] In view of the above advantages, the present application also provides a fermentation process, which fermentation process comprises

[0087] i) providing a fermentation broth comprising a culture of the microorganism of the present application in a fermentation medium; and

[0088] ii) cultivating the fermentation broth under conditions suitable for the production of the target substance.

[0089] The fermentation process of the present application provides unexpected advantageous effects compared to fermentation processes not using a microorganism of the family Paenibacteriaceae in an activated and / or syntrophic metabolic state: One advantage is an increased fermentation speed. In particular, the fermentation process enables a significantly shortened lag phase after transferring the inoculum into the fermentation medium. Furthermore, the fermentation process enables the microorganism to require less time to reach the half-maximal cell and / or optical density. In addition, the fermentation process enables an increased productivity, i.e. the yield per unit time and the total yield of the target substance. For the Paenibacillus microorganism, this enables an advantageously increased yield of fusaricidin, pantocin, pantocinoplosin and / or polymyxin. In addition, the development of high viscosity can advantageously be delayed or controlled. This enables to avoid high energy input for stirring the culture medium and facilitates the purification of the produced target substance.

[0090] It is particularly advantageous and unexpected that the fermentation is performed with high reproducibility. This is preferably determined by the very low variability of the optical density at any given time of a repeated fermentation. Thus, the fermentation process according to the present application is particularly suitable for automation, as once the fermentation protocol is established, the need for user interventions to counteract unforeseen different fermentation developments can be reduced or completely avoided. Accordingly, the fermentation process of the present application enables to produce a target substance of reliable quality in several batches.

[0091] Another effect of the significantly increased reproducibility of the fermentation is that variations in the growth and metabolic behavior of the microorganism caused by small changes in the properties of the cell bank sample during the fermentation are avoided. Instead, by starting the industrial fermentation with a microorganism culture in a defined state, i.e. the microorganism culture of the present application, the fermentation is protected from such random disturbances.

[0092] In step i) the fermentation broth is prepared, preferably by inoculating a cell-free fermentation medium with the microbial culture of the present application. Thus, the fermentation medium is inoculated with a Paenibacillaceae microorganism, preferably with a pure culture of a Paenibacillus microorganism which is already in a metabolically cooperative and / or in an activated metabolic state. By using the microbial culture of the present application only a short adaptation time is required before the microorganism produces the target substance efficiently. Preferably, when the microbial culture of the present application is produced by the above-mentioned production method, the microbial culture has already grown in the fermentation medium of step i) of the fermentation method. This is further suitable to reduce the adaptation time and to achieve a particularly high productivity, i.e. the amount of recoverable target substance produced in step ii) is particularly high per given time and fermentation volume. The fermentation broth can also be prepared by inoculating the fermentation medium with purified cells of the microbial culture of the present application.

[0093] Preferably, the fermentation medium is inoculated in step i) with a sufficient amount of microorganism to maintain the metabolically cooperative and / or activated state in step ii). A particular advantage of the present application is that the optimization of the inoculation amount can be done by observing any of the above-mentioned parameters growth, cell size, viscosity and production of indicator substance and / or variance thereof. Typically, in industrial fermentations a Paenibacillus microorganism in a metabolically cooperative or activated state will be added to reach at least 2E4 cfu / ml in the fermentation medium. Preferably, the fermentation medium or medium is inoculated with 2E05 - 2E06 cfu / ml (200000 - 2000000 cfu / ml).

[0094] A particular advantage of the present application is that the microorganism maintains its metabolically cooperative and / or activated state over several passages. For example, the microbial culture of the present application can be poured as such into a new fermentation medium for inoculation or the cells of the microbial culture can be purified, preferably by centrifugation, and introduced into a new fermentation medium. In either way, the cells will continue to grow according to the second growth type described herein. Thus, for the inoculation of large industrial fermentation volumes, the present application avoids the necessity to prepare the population of microorganisms required in step i) in a single step which would otherwise likely require a large stock of inoculation material to be stored in a cell bank. Instead, the microorganisms of the cell bank vials can first be grown in small scale shake flask liquid cultures of about 50 ml - 2500 ml until they have turned into the second growth type and thus have reached the metabolically cooperative and / or activated metabolic state. Then, the cells are transferred (in the fermentation broth or after purification and preferably resuspended in a new fermentation medium) to successively larger fermentation volumes.

[0095] In step ii) the fermentation broth is fermented under conditions suitable for the production of the target substance. It is a particular advantage that the fermentation using the microbial culture of the present application is not limited to specific conditions. Instead, the skilled person can select suitable conditions in particular in view of the oxygen and nutrient requirements, the development of viscosity and / or the production of the target substance. Furthermore, additional components can be added to the fermentation medium or additional components can be extracted from the fermentation medium. Preferably, the pH of the fermentation broth is adjusted by adding an appropriate amount of an acid or a base. Preferably, the oxygen concentration is adjusted by increasing or decreasing the aeration (e.g. bubbling oxygen into the fermentation broth and / or adjusting the speed of the mixer). Preferably, the temperature is adjusted by heating or cooling the jacket of the fermenter vessel and / or by increasing or decreasing the concentration and / or type of available nutrients. Furthermore, preferably, the medium comprises or is supplied with an antifoam agent in an amount sufficient to prevent or reduce the formation of foam during the fermentation step ii).

[0096] Preferably, the fermentation process of the present application further comprises

[0097] iii) recovering the target substance from the cultivated fermentation broth of step ii).

[0098] The target substance according to the present application is preferably selected from

[0099] - microorganisms, preferably live cells and / or spores, and / or

[0100] - one or more metabolites, preferably at least one fusaricidin, pantocin, panipatolosin or polymyxin, preferably at least two or more fusaricidins, pantocins, panipatolosins or polymyxins, more preferably 3 to 120 fusaricidins, wherein the one or more fusaricidins comprise any of the following: fusaricidin A, B or D, and / or surfactin and / or iturin.

[0101] In particular, the target substance is preferably any of the following:

[0102] - a composition comprising spores and / or cells of the microorganism, preferably a composition comprising or consisting of live cells and spores in a ratio of at most 4 : 1, more preferably 3 : 1 to 0.2 : 1, even more preferably less than 0.2 : 1, even more preferably a composition containing only spores and no live cells,

[0103] - a composition comprising or consisting of at least one fusaricidin, panidarcin, panipatulin or polymyxin, preferably at least two or more fusaricidins, panidarcins, panipatulins or polymyxins, more preferably 3-120 fusaricidins, wherein the one or more fusaricidins comprise any of the following: fusaricidin A, B or D, and / or a surfactant peptide and / or iturin, and / or

[0104] - a composition comprising or consisting of at least one viscosity increasing or viscosifying substance, preferably selected from the group consisting of exopolysaccharides, proteoglycans and poly-gamma-glutamic acid.

[0105] A particular advantage of the present application is that the target substance is not limited to a particular type of substance. As shown in the examples, the use of the fermentation process of the present application enables a more reproducible higher productivity and / or a higher yield of various target substances compared to fermentation processes in which the fermentation medium is not inoculated with a population of metabolically cooperative and / or activated microorganisms.

[0106] The target material can be cells and / or spores of microorganisms. In agriculture, bacterial spores are used in plant pest control compositions to reduce or prevent plant pathogenic fungal or bacterial diseases. Spore bioformulations are also used to increase plant resistance to biotic and abiotic stresses, thereby accelerating plant growth, and increasing yield during plant, fruit, or legume harvest. Spore products are applied to leaves, shoots, fruits, roots, or plant propagation material, as well as substrates on which plants grow (Toyota K. Bacillus-related Spore Formers: Attractive Agents for Plant Growth Promotion. Microbes Environ. 2015; 30(3):205-207. doi:10.1264 / jsme2.me3003rh).Bochow, H. et al. "Use of Bacillus Subtilis as Biocontrol Agent. IV. Salt-Stress Tolerance Induction by Bacillus Subtilis FZB24 Seed Treatment in Tropical Vegetable Field Crops, and Its Mode of Action / Die Verwendung von Bacillus Subtilis zur biologischen Bekampfung. IV. Induktion einer Salzstress-Toleranz durch Applikation von Bacillus subtilis FZB24 bei tropischem Feldgemuse und sein Wirkungsmechanismus." Zeitschrift fur Pflanzenkrankheiten und Pflanzenschutz / Journal of Plant Diseases and Protection, Vol. 108, No. 1, 2001, pp. 21-30. JSTOR, www.jstor.org / stable / 43215378. Accessed December 14, 2020. (Hashem, Abeer & Tabassum, B. & Abd Allah, Elsayed. (2019). Bacillus subtilis: A plant-growth promoting rhizobacterium that also impacts biotic stress. Saudi Journal of Biological Sciences. 10.1016 / j.sjbs.2019.05.004.).

[0107] Furthermore, bacterial spores are also applied in the field of nanobiotechnology and building chemistry, such as for self-healing concrete (crack repair), mortar stability and reduction of water permeability [J. Y. Wang, H. Soens, W. Verstraete, N. De Belie, Self-healing concrete by use of microencapsulated bacterial spores, Cement and Concrete Research, Volume 56, 2014, Pages 139-152, ISSN 0008-8846, https: / / doi.org / 10.1016 / j.cemconres.2013.11.009] [Ricca E, Cutting SM. Emerging Applications of Bacterial Spores in Nanobiotechnology. J Nanobiotechnology. Published online 2003 Dec 15. doi: 10.1186 / 1477-3155-1-6].

[0108] Furthermore, bacterial spores are also applied in the field of cleaning products, such as for laundry cleaning, hard surface cleaning, hygiene and odor control in clinical and domestic environments (Caselli E. Hygiene: microbial strategies to reduce pathogens and drug resistance in clinical settings. Microb Biotechnol. 2017 Sep; 10(5): 1079-1083. doi: 10.1111 / 1751-7915.12755. Epub 2017 Jul 5). For example, spores are used in cosmetic compositions, such as skin cleansing products (US 20070048244), in dishwashing detergents (WO 2014 / 107111), in pipe de-oiling agents (DE 19850012), in laundry malodor control (WO 2017 / 157778 and EP 3430113) or in allergen removal (US 20020182184). Spores can also be embedded in non-biological matrices to catalyze their subsequent decomposition.

[0109] Further, bacterial spores are also applied in the field of human and animal nutrition and health. For example, different bacterial strains are applied in broiler chickens as part of an antibiotic replacement strategy (Neveling, D.P., Dicks, L.M. Probiotics: an Antibiotic Replacement Strategy for Healthy Broilers and Productive Rearing. Probiotics & Antimicro. Prot. 13, 1-11 (2021). https: / / doi.org / 10.1007 / s12602-020-09640-z). Other examples include aquaculture, pig farming, etc. (Nayak, S.K. (2021), Multifaceted applications of probiotic Bacillus species in aquaculture with special reference to Bacillus subtilis. Rev. Aquacult. 13: 862-906. https: / / doi.org / 10.1111 / raq.12503). Applications of bacterial spores for human health are also abundantly described (e.g. US20180289752; Lee, NK., Kim, WS. & Paik, HD. Bacillus strains as human probiotics: characterization, safety, microbiome, and probiotic carrier. Food Sci Biotechnol 28, 1297-1305 (2019). https: / / doi.org / 10.1007 / s10068-019-00691-9).

[0110] The target substance can also be a fusaricidin, a panilserine, a paniplosporin or a polymyxin. In each case, it is particularly preferred that one or more of the fusaricidins comprises any of fusaricidin A, B or D. As described below, fusaricidins, panilserines, paniplosporins or polymyxins have many applications, in particular for promoting plant health and / or plant pest control.

[0111] The target substance can also be a viscosity increasing substance or a viscosifying substance, such as Paenan and / or Levan. Microbial exopolysaccharides have many applications, such as in oil drilling, thickening agents in cosmetics, for paints, in food, as rheology modifiers, for pharmaceuticals and home care. Several applications are listed in, for example, Liyaskina EV, Rakova NA, Kitykina AA, Rusyaeva VV, Toukach PV, Fomenkov A, Vainauskas S, Roberts RJ, Revin VV. Production and characterization of the exopolysaccharide from strain Paenibacillus polymyxa 2020. PLoS One. 2021 Jul 6; 16(7): e0253482. doi: 10.1371 / journal.pone.0253482. PMID: 34228741; PMCID: PMC8259973.

[0112] Further, the microorganism or spores thereof can be the target substance. As described, for example, in Grady EN, MacDonald J, Liu L, Richman A, Yuan ZC. Current knowledge and perspectives of Paenibacillus: a review. Microb Cell Fact. 2016 Dec 1 ; 15(1):203. doi: 10.1186 / s12934-016-0603-7. PMID: 27905924; PMCID: PMC5134293, many Paenibacillus species can directly promote crop growth through biological nitrogen fixation, phosphate solubilization, production of the plant hormone indole-3-acetic acid (IAA), and release of siderophores that enable iron acquisition. They can also provide protection against insect herbivores and plant pathogens, including bacteria, fungi, nematodes, and viruses.

[0113] The microbial culture in step i) of the fermentation process of the present invention is preferably obtainable by or obtained by:

[0114] i.i) inoculating a pre-culture fermentation medium with an amount of microorganisms; and

[0115] i.ii) cultivating the culture of step i.i) to provide a culture having:

[0116] a) a median cell particulate length of 0.8-2.9 pm, more preferably of 1-2.5 pm, even more preferably of 1.3-2 pm, even more preferably of 1.3-1.8 pm, and / or

[0117] b) a 75th percentile of cell particulate length of 1.3-3 pm, more preferably of 1.3-2.9 pm, more preferably of 1.5-2.5 pm, and / or

[0118] c) a 25th percentile of cell particulate length of 0.8-2.5 pm, more preferably of 0.8-1.9 pm, more preferably of 0.8-1.5 pm.

[0119] As described above, cell particles having such a cell particle length are those cell particles belonging to the microbial culture of the present application, wherein the Paenibacillaceae, preferably the Paenibacillus genus, metabolize synergistically and / or are in an activated metabolic state. Further, as described above, such microorganisms can also be described by further parameters such as, for example, growth rate, cell size, viscosity, production of indicator substances, optical density, cell count, pH process, gas formation, oxygen uptake rate, CO2 transfer rate, and variance in any of these parameters.

[0120] The present application also provides a method for reducing the fermentation time required to produce a desired amount of a target substance, and / or for reducing the viscosity during fermentation, and / or for increasing the fermentation repeatability accuracy, the method comprising the steps of:

[0121] i) providing a culture according to the present application in a fermentation medium; and

[0122] ii) cultivating the culture in the fermentation medium.

[0123] As described herein, in particular in the figures and examples, an advantage of the microbial culture of the present application is that the microorganisms are in a metabolic state which enables the production of a target substance much faster than those microorganisms grown according to the first growth type. Thus, when used in a fermentation process, the culture of the present application enables the production of a given amount of a target substance faster, or enables the production of more target substance in a given time. A particular advantage is that such a faster fermentation can be achieved compared to a microbial culture grown according to the first growth type, while also achieving a reduction in the viscosity of the fermentation medium.

[0124] Correspondingly, the microbial culture of the present application is preferably used for reducing the fermentation time required to produce a desired amount of a target substance, and / or for reducing the viscosity during fermentation, and / or for increasing the fermentation repeatability accuracy.

[0125] As described herein, the present application particularly improves the reproducibility of fermentations using Paenibacillaceae microorganisms, preferably of the Paenibacillus genus, and thus facilitates automated fermentation. Correspondingly, the present application provides a cultivation parameter data set for controlling a fermentation, the cultivation parameter data set comprising

[0126] - the name of a set of applicable fermentation media,

[0127] - the name of a set of applicable microbial samples,

[0128] - the name of fermentation conditions which facilitate obtaining a culture according to the present application by cultivating an applicable sample in an applicable fermentation medium,

[0129] wherein the fermentation conditions preferably comprise at least one of the following: type of culture medium, amount of culture medium, required culture temperature range, required culture aeration parameter range, required amount of produced cells, required culture duration, required pH during cultivation, required yield or amount of target substance.

[0130] The cultivation parameter dataset can advantageously be used to manipulate a fermentation of Paenibacillaceae microorganisms, preferably of the genus Paenibacillus. Due to the reproducibility obtainable with the microbial culture of the present application, the cultivation parameter dataset is sufficient to manipulate such a fermentation to produce a target substance.

[0131] The present application accordingly also provides a computer-implemented fermentation method, the fermentation method comprising

[0132] - receiving, by a computing device, a cultivation parameter dataset according to the present application,

[0133] - receiving a fermentation medium in accordance with the cultivation parameter dataset,

[0134] - receiving a sample of a volume of microorganisms in accordance with the cultivation parameter dataset, and

[0135] - controlling, by the computing device, a fermenter to cultivate said microorganisms and / or spores thereof in the fermentation medium under the fermentation conditions of the cultivation parameter dataset.

[0136] As described herein, the fermentation according to the cultivation parameter dataset results in producing the microbial culture of the present application and / or maintaining such Paenibacillaceae microorganisms, preferably of the genus Paenibacillus, in a metabolically cooperative and / or activated state, thereby ensuring the advantages of the present application. In particular, the computer-implemented fermentation method enables producing a target substance reliably, faster and / or with a higher yield compared to microorganisms grown according to a first growth type.

[0137] The controlling preferably comprises

[0138] - determining one or more parameters indicative of metabolic activity, wherein the parameters are preferably selected from any of the following: growth rate, cell size, viscosity, production of an indicator substance, optical density, cell count, pH process, gas formation, oxygen uptake rate, CO2 transfer rate, and variance of any of the preceding parameters;

[0139] - comparing the determined one or more parameters to one or more parameters of a target fermentation condition, preferably the cultivation parameter dataset; and

[0140] - adjusting the fermentation conditions to promote consistency of the determined one or more parameters with the target fermentation condition.

[0141] In this regard, the present application also provides a method for producing one or more viscosity increasing substances. The method comprises:

[0142] i) inoculating a fermentation medium with a culture of microorganisms according to the present application at a concentration that is at a predetermined concentration sufficient to obtain the desired viscosity during the cultivation; and

[0143] ii) cultivating the culture obtained in step i); and

[0144] iii) preferably recovering the one or more viscosity increasing substances or viscosifying substances.

[0145] Thus, the method starts with a culture of microorganisms of the present application. However, maintaining the cells in a second growth type allows for a viscosity decrease during the fermentation. Thus, the method generally involves cultivating a culture at a higher cell density in step ii). A particular advantage of the present application is that even if the culture of microorganisms of the present application is only used to inoculate an industrial fermentation without maintaining conditions for growth according to a second growth type, the culture of microorganisms of the present application allows for an improved reproducibility and yield of the target substances, including viscosity increasing substances.

[0146] The present application is further described below by way of figures and examples, none of which are intended to limit the scope of the present application. Example

[0147] Example 1 : Effect of inoculum volume on growth of Paenibacillus polymyxa LU21132 in shake flask Figure 1 < / s

[0148] The effect of initial preculture inoculum volume on growth of Paenibacillus polymyxa was investigated in shake flask cultures using strain LU21132 of the genus Paenibacillus.

[0149] Strain LU21132 is an apramycin mutant of the wild-type isolate Paenibacillus polymyxa LU17007 and was derived from a random mutagenesis process. This strain was exemplarily chosen to demonstrate the effect of preculture inoculum volume in single-stage and two-stage cultures, but the same phenomenon was also demonstrated in the wild-type strain Paenibacillus polymyxa LU17007 and further mutants thereof (e.g. LU20754 and LU21552) as well as in public Paenibacillus strains (e.g. Paenibacillus polymyxa DM365 or Paenibacillus terrae DSM15891).

[0150] Different ratios (% v / v) (0.1% - 4.0% v / v) of the working cell bank from strain LU21132 were used for inoculation of preculture flasks to analyze their effect on biomass formation of LU21132 within 24 h of growth in PX-125 medium. The inoculum volumes tested are listed in Table 1.

[0151] Table 1 : Shake flask inoculum volume used to test the effect of inoculum density on growth of P. polymyxa in 250 ml shake flasks containing 30 ml of PX-125 medium

[0152]

[0153] WCB1041 for use in this cultivation was produced after 72 h cultivation in shake flasks using a soybean-based complex medium. Prior to filling, a microscopic evaluation was performed to identify the progress of sporulation within the culture. Under the microscope, spores as well as cells containing endospores were detected. The WCB of strain P. polymyxa LU21132 was then filled in 2 ml cryovials with 25% glycerol and 5% sucrose. The preparation procedure of all WCBs used in this study is listed in Table 5.

[0154] Culture conditions

[0155] The composition of PX-125 is listed in Table 2. The components of the stock solutions were dissolved in distilled water and either sterile filtered or autoclaved at 121 °C, 1 bar overpressure for 60 min. The sterile solutions were stored at room temperature or at 4 °C. The antifoam agent was added to the main solution shortly before starting the autoclaving process. After mixing the stock solutions, the pH of the medium was set to 6.5 with a 25% (w / w) ammonia solution or a 40% (w / w) phosphoric acid.

[0156] Table 2: Composition of the complex medium PX-125, and storage (room temperature (RT) or 4 °C) and sterilization method (sterile filtration / autoclaving, s / a) of the stock solutions.

[0157]

[0158] The cultivation was performed in 250 ml baffled shake flasks containing 30 ml of medium PX-125 sealed with a gas-permeable silicon plug. The cultivation was performed at 33 °C, 150 rpm and a 25 mm shaking frequency for 24 h.

[0159] To determine the bacterial growth, an optical density (OD) measurement at 600 nm was performed using the corresponding uninoculated medium as a blank. The OD of the cultivation experiments with different inoculum volumes is shown in Table 3. Figure 1

[0160] Example 2: Growth curves in MTP scale cultivation at different inoculum volumes Figure 2

[0161] ​​Cultivation of Paenibacillus sp. LU20754 was performed in flower-shaped 48-well microtiter plates, which were sealed with sterile membranes of m2p laboratories (F-GPR48-10, gas-permeable sealing foil with evaporation-reducing layer). The parameters for cultivation were a shaking frequency of 900 rpm, a shaking diameter of 3 mm, a total well volume of 2.4 mL and a liquid volume per well of 1.2 ml, 85% humidity, 35°C. Biomass formation was assessed by performing a scatter light analysis on each well at an excitation and emission wavelength of 620 nm, respectively. Different inoculation ratios (0.6%, 1.2% and 1.8% v / v) of spore WCB (7.95E+07 spores / ml) of the strain Paenibacillus polymyxa LU20754 were used.

[0162] Example 3: Cell size and cell arrangement depending on inoculation volume Figure 3 + Figure 4 )

[0163] Paenibacillus polymyxa strain LU21552 was cultivated in 1 L unbaffled shake flasks using 100 ml of a soy-based medium. Two different inoculation volumes of 0.2% and 1% (v / v), respectively, were tested using spore WCB 1059 produced after 72 h of fermentation (see Table 5). Cultivation was performed at 33°C, 250 rpm, 5 cm throw. After 28 h of cultivation, an optical microscopy evaluation was performed at 1000x magnification.

[0164] Cellular particle length was determined by using the analyze particles function implemented in Fiji (Schindelin J, Arganda-Carreras I, Frise E, Kaynig V, Longair M, Pietzsch T, Preibisch S, Rueden C, Saalfeld S, Schmid B, Tinevez JY, White DJ, Hartenstein V, Eliceiri K, Tomancak P, Cardona A: Fiji: an open-source platform for biological-image analysis. Nat Methods. 2012, 9: 676-682. DOI: 10.1038 / nmeth.2019) and calculating the Feret diameter of each particle identified by the particle analyzer (reference to the image with the particle outline of the selected cell). First, a threshold was applied to the RGB microscope image using the default thresholding method to produce a binary image. Subsequently, the segmented image was used as input for the analyze particles function and the cellular particle length of all analyzed cells was shown in the image showing the cell outlines as well as the numbered report in the analysis. The following table shows the cellular particle length of each cellular particle identified in Figure 3 A and Figure 3 B.

[0165]

[0166] Thus, the cutoff cellular particle length in pm for the respective percentile is:

[0167]

[0168] Example 4: Influence of the shake flask inoculation volume on the production of fusaricidin by Paenibacillus polymyxa LU21132 Figure 5

[0169] After 24 h of cultivation, the production of fusaricidin was evaluated in the cultivation of Example 1. For this, 50 mI of the cultivation broth were mixed with 950 mI of acetonitrile-water (1 : 1) mixture for extraction. The samples were treated in an ultrasonic bath for 30 min at 20 °C. Then the samples were centrifuged for 5 min at 14000 rpm and the supernatant was filtered into an HPLC vial for measurement. The fusaricidin concentration was determined by HPLC-UV-VIS as listed in Table 3 and Table 4:

[0170] ​Table 3: HPLC settings for quantification of fengicin A, B and C in broth samples

[0171]

[0172] Table 4: Solvent gradient for HPLC-based quantification of fengicin A, B and C in broth samples

[0173]

[0174] In Figure 2 the production of fengicin A, B and D in the cultivation of Example 2 is shown.

[0175] Example 5: WCB inoculum density effect independent of WCB preparation procedure Figure 6 + Figure 7 )

[0176] Different working cell bank (WCB) preparation procedures were tested to identify the strain’s sensitivity to the inoculum volume in the shake flask pre-culture. The used WCB preparation procedures are listed in Table 5.

[0177] Table 5: WCB preparation protocols

[0178]

[0179] Six different WCBs from strain LU21132 (see Table 5) of 0.3% and 0.6% (v / v, see Table 1) were taken to start the shake flask cultivation. The WCB vials were heated at 60 °C for 30 min before inoculating the pre-culture medium PX-125. The cultivation was performed in 250 ml shake flasks according to the procedure of Example 1.

[0180] Bacterial growth was evaluated after 24 h of cultivation and is shown in Figure 6 Table 6. Furthermore, the production of the secondary metabolite fengicin was quantified after 24 h of growth using the method described in Example 2. The levels of fengicin as a function of the WCB inoculum volume are shown in Figure 7 Table 7.

[0181] Example 6: Pre-culture inoculum volume does affect fengicin titers in the main culture (two-stage process) Figure 8 )

[0182] After 24 h of pre-culture cultivation in Example 3, 600 µl of each method were transferred into the main culture medium PX-143 to investigate the effect of the pre-culture inoculum volume on the performance in the two-stage process.

[0183] Main culture medium: PX-143

[0184] Table 6: Composition of complex medium PX-143, and storage (room temperature (RT) or 4°C) and sterilization method (sterile filtration / high pressure sterilization, s / a) of stock solutions.

[0185]

[0186] The production of fusaricidin in the main culture was again evaluated after 48 h of cultivation using the method described in Example 2. In Figure 5 The fusaricidin levels detected in the cultivation of Example 4 are shown in Table 2. Here, the fusaricidin levels of the main cultures inoculated with pre-culture flasks having a pre-culture inoculum volume of 0.6% are shown relative to the fusaricidin levels detected in parallel main cultures from a pre-culture inoculum of 0.3%.

[0187] Example 6: Pre-culture inoculum volume does synchronize growth in main cultures of various WCBs (two-stage process) Figure 9

[0188] Biomass formation in the main cultures of Example 5 was analyzed by OD600 measurement of the final broth samples after 48 h of cultivation. Figure 6 The range of OD600 [A.U.] values of the cultivations using the six WCBs from Table 5 at 0.3% and 0.6% inoculum volume is shown. Cultivation conditions: 250 ml baffled shake flask, 30 ml medium, 150 rpm, 2.5 cm throw, 33°C, pH 6.5, 24 h pre-culture, 48 h main culture.

[0189] Example 7: Main culture inoculum volume influences broth viscosity Figure 10

[0190] The influence of inoculum volume on broth viscosity was analyzed in a soybean-based exopolysaccharide production medium (Table 7).

[0191] Table 7: Composition of exopolysaccharide production medium, and storage (room temperature (RT) or 4°C) and sterilization method (sterile filtration / high pressure sterilization, s / a) of stock solutions.

[0192]

[0193] ​​Cultivation of Bacillus polymyxa LU21132 was performed in 250 ml baffled shake flasks filled with 30 ml of medium (Table 7) at 150 rpm, 2.5 cm throw, 33 °C and pH 6.5. After 16 h of cultivation, the viscosity was assessed via rotational rheology using a cylinder geometry with an Anton Paar rheometer (model MCR 302). The measurements were performed at T = 33 °C. At the beginning, the samples were pretreated for 100 s in a pre-shear experiment with a constant shear rate of 10 s -1 -1. 10 data points were recorded (every 10 s). After the pretreatment, the viscosity as a function of the shear rate was measured. Therefore, the shear rate was varied from 1 s -1 -1 to 100 s -1 -1. 25 data points were recorded in a logarithmic way. EPS formation was partially uncoupled from growth, especially at 2%, 4% and 6% inoculation volume, as indicated by OD600.

Claims

1. A microbial culture, wherein the microorganism belongs to the family Bacillusaceae, preferably to the genus Bacillus, wherein the cells in the culture... -Metabolic synergy and / or - In an activated metabolic state.

2. The culture as described in claim 1, wherein a) The median cell particle length is 0.8–2.9 μm, more preferably 1–2.5 μm, even more preferably 1.3–2 μm, even more preferably 1.3–1.8 µm, and / or b) The 75th percentile of cell particle length is 1.3-3 μm, more preferably 1.3-2.9 μm, even more preferably 1.5-2.5 μm, and / or c) The 25th percentile of cell particle length is 0.8-2.5 μm, more preferably 0.8-1.9 μm, and even more preferably 0.8-1.5 μm.

3. The culture as described in claim 1 or 2, wherein the culture can be obtained by inoculating a culture medium with a certain concentration of microorganisms, the concentration being sufficient to produce the culture, wherein the concentration is preferably determined by: i) Incubate a series of cultures inoculated with different inoculation concentrations for a sufficient time to distinguish the cultures by parameters indicating metabolic activity, wherein the parameters are preferably selected from any of the following: growth rate, cell size, viscosity, production of indicator substances, optical density, cell count, pH process, gas formation, oxygen uptake rate, CO2 transfer rate, and variance of any of the foregoing parameters. as well as ii) Determine one or more of the parameters; as well as iii) Select the inoculation concentration based on one or more determined parameters.

4. A fermentation method, the fermentation method comprising: i) providing a fermentation broth comprising a microbial culture as described in any one of claims 1-3 in a fermentation medium; and ii) Cultivate the fermentation broth under conditions suitable for the production of the target substance; and iii) Preferably, the target material is recovered.

5. The fermentation method of claim 4, wherein the microbial culture in step i) can be obtained by means of: ii) Inoculate the pre-cultured fermentation medium with a certain amount of microorganisms; and i.ii) Cultivate the culture from step ii) such that: a) The median cell particle length is 0.8–2.9 μm, more preferably 1–2.5 μm, even more preferably 1.3–2 μm, even more preferably 1.3–1.8 µm, and / or b) The 75th percentile of cell particle length is 1.3-3 μm, more preferably 1.3-2.9 μm, even more preferably 1.5-2.5 μm, and / or c) The 25th percentile of cell particle length is 0.8-2.5 μm, more preferably 0.8-1.9 μm, and even more preferably 0.8-1.5 μm.

6. The fermentation method according to any one of claims 4-5, wherein the target substance is selected from... - Microorganisms, preferably living cells and / or spores - One or more metabolites, preferably at least one fusarin, panisyl, panisylproline or polymyxin, preferably at least two or more fusarin, panisyl, panisylproline or polymyxin, more preferably 3-120 fusarin, wherein the one or more fusarin comprises any one of the following: fusarin A, B or D, and / or surfactant peptide and / or iturin.

7. A method for reducing the fermentation time required to produce a desired amount of target material, and / or for reducing viscosity during fermentation, and / or for increasing the accuracy of fermentation repeatability, the method comprising the following steps: i) Provide the culture as described in any one of claims 1-3 in a fermentation medium; as well as ii) Cultivate the culture in the fermentation medium.

8. The use of the culture as described in any one of claims 1-3 for reducing the fermentation time required to produce the desired amount of target material, and / or for reducing viscosity during fermentation, and / or for increasing the accuracy of fermentation repeatability.

9. A culture parameter dataset for controlling fermentation, the culture parameter dataset comprising: - A set of names for suitable fermentation media, - A set of names for applicable microbial samples, - Names of fermentation conditions that facilitate obtaining a culture as described in any one of claims 1-3 by culturing a suitable sample in a suitable fermentation medium, wherein such fermentation conditions preferably include at least one of the following: type of medium, amount of medium, desired range of culture temperature, desired range of culture aeration parameters, desired amount of cells produced, desired culture duration, desired pH during culture, desired yield or amount of target substance.

10. A computer-implemented fermentation method, the fermentation method comprising: - The computing device receives the culture parameter dataset as described in claim 9. - Receive fermentation medium that matches the dataset of culture parameters. -Receive a sample of a certain volume of microorganisms that conforms to the dataset of culture parameters, and The computing device controls the fermenter to culture the microorganisms and / or their spores in the fermentation medium under the fermentation conditions of the culture parameter dataset.

11. The computer-implemented fermentation method of claim 10, wherein control includes... - Determine one or more parameters that indicate metabolic activity, wherein the parameter is preferably selected from any of the following: growth rate, cell size, viscosity, production of indicator substances, optical density, cell count, pH process, gas formation, oxygen uptake rate, CO2 transfer rate, and variance of any of the foregoing parameters; - Compare the determined parameters (one or more) with the target fermentation conditions; as well as - Adjust fermentation conditions to promote consistency between the determined parameters (one or more) and the target fermentation conditions.

12. A method for producing one or more viscosity-increasing substances, the method comprising: i) Inoculating a fermentation medium with a microbial culture as described in any one of claims 1-3 at a predetermined concentration sufficient to achieve the desired viscosity during cultivation; and ii) Cultivating the culture obtained in step i); and iii) Preferably, the one or more viscosity-increasing substances are recycled.

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