Composition of isolated microorganisms and use thereof

Abstract

In a first aspect, the present invention relates to a composition of isolated microorganisms, wherein said microorganisms are in the form of viable microorganisms of at least the following genera: Microcoleus, Nostoc, Scytonema, Klebsormidium. This means that the mixture comprises at least cyanobacteria and a microalgae, preferably as a mixture with another microalgae. In a further aspect, the invention relates to a method for improving soils, comprising applying the composition of isolated microorganisms according to the invention to the soil. In particular, the invention relates to a method for improving the soil crust. Lastly, the present invention relates to the use of the composition of isolated microorganisms according to the invention for the improvement of soils, in particular for the (re)formation and improvement of soil crusts.

Description

[0001] Composition of isolated microorganisms and their use

[0002] In a first aspect, the present invention relates to a composition of isolated microorganisms, which are present at least in the form of viable microorganisms of the following genera: Microcoleus, Nostoc, Scytonema, and Klebsormidium. This means that the composition comprises at least the aforementioned genera of cyanobacteria and microalgae, preferably as a mixture with at least one other microalgae. In a further aspect, the invention relates to a method for improving soils, comprising applying the composition of isolated microorganisms according to the invention to the soil, in particular a method for improving the soil crust. Finally, the present invention is directed to the use of the composition of isolated microorganisms according to the invention for improving soils, in particular for the formation and improvement of soil crusts.

[0003] State of the art

[0004] Biological soil crusts are a complex community of photosynthetically active microorganisms, including heterotrophic fungi, protozoa, and bacteria, as well as mosses and lichens, that form the topsoil. This community of organisms, in conjunction with soil particles and various amounts of extracellular polymers produced by these microorganisms, such as extracellularly secreted polysaccharides, forms a microecosystem whose ecological function is particularly important for pioneer colonization of non-vegetated or degraded soils. Degraded soils are understood to mean that the soil can no longer fulfill its ecosystem services to a certain extent or at all. Soil degradation refers to a deterioration of the soil's ecosystem services, up to and including their complete loss.This soil crust, which can be understood as a three-component system of microorganisms, soil particles, and the extracellular polymer matrix produced by these microorganisms, covers the soil surface as a continuous layer and is usually a few millimeters thick. Soil crusts have various important functions, such as nitrogen fixation, carbon fixation, water retention, stabilization of soil particles, and the provision of available nutrients for plants. Soil crusts are found primarily in tropical zones worldwide, and different types of soil crusts can be distinguished based on temperature, relative humidity, solar intensity, and extent of deposition. A differentiation can also be made here in the development of the soil crust and the subsequent processes. A classification is provided by Belnap et al., 2003 (Belnap, J. and O. Lange (Eds.(2003) "Biological Soil Crusts: Structure, Function and Management" Ecological Studies 150, Analysis and Synthesis, Springer-Verlag Berlin Heidelberg GmbH), into different types: i) smooth soil crusts, ii) rough soil crusts, iii) rolling and spiny soil crusts. Furthermore, a distinction is often made between algal-fungal dominated crusts; light and dark algal dominated crusts; lichen crusts, green algal-lichen and cyanobacterial lichen dominated crusts; and moss and moss-lichen crusts.

[0005] Biological soil crusts can occur on all soil types in climate zones. However, they are particularly important in areas with low biological productivity due to extreme environmental conditions, such as cold, heat, or drought. Especially in arid regions, such as deserts, biological soil crusts are of great importance for water balance, the carbon and nitrogen cycle, and as a basis for the further development and colonization of soils. By altering the soil surface on raw soils, soil crusts can accelerate and facilitate the colonization of the soil by vascular plants. The development of soil crusts occurs through various stages until they reach the soil crust with microorganisms, mosses, and / or lichens. Soil crusts are important for various aspects, as already mentioned above.In particular, they increase soil moisture to facilitate the establishment of mosses and lichens, and later, vascular plants. They reduce soil erosion and thus desertification and soil degradation. They are therefore particularly important in arid regions.

[0006] Various approaches to establishing soil crusts have been described, particularly to prevent the impending decline or degradation of the soil crust due to climate change. An overview of biological soil crusts can be found, for example, in Antoninka A. et al., 2020, Restoration Ecology, 28 (pp. 2, 3, to 8).

[0007] Generally, attempts are made to form or strengthen the soil crust by introducing either liquid or solid components into the soil. In addition, various methods for restoring biological soil crusts are described, such as liquid or solid inoculation methods or so-called mosaic methods.

[0008] Various compositions for the formation of biological soil crusts have already been described in the literature. For example, Wu et al. (2013) (Wu, Y., et. al.; (2013), Plant Soil 370:115-124) and Peng et al. (2017) (Peng, C., et al., (2017) Journal of Applied Phycology, DOI 10.1007 / s10811-017-1061-2) describe inoculation with only two species: Microcoleus vaginatus and Scytonema javanicum, without specifying the mass ratio in Wu et al. (2013). In Peng 2017, the two species were used in a ratio of 4:3, but only in Petri dishes in the laboratory, not in the field. Wang et al. (2013) report a ratio of Microcoleus vaginatus to Scytonema javanicum of 10:1; the mixture was tested in a field trial from 2003 to 2006 (Wang, W., et al. (2013) “Feasibility of cyanobacterial inoculation for biological soil crusts formation in desert area” Soil Biology & Biochemistry 41 : 926- 929.)

[0009] To date, attempts have mostly been made to form soil crusts using local isolates of mixed cultures of natural soil crusts. Lababpour (2016) summarizes this (Lababpour, 2016, Int. J. Environm. Sci. Technol. 13: 2521-2532) that mixed taxa were used for inoculation in most published studies.

[0010] Modular stabilization and nutrient emulsions for initial, biogenic crust formation are known from DE 2010 019 420 A1. US Pat. No. 6,228,136 B1 describes carrier materials for cyanobacteria and correspondingly covered carrier materials suitable for application on degraded soils.

[0011] However, there is still a need to provide suitable inoculates for the formation of biological soil crusts, allowing rapid and progressive soil crust formation with subsequent colonization by lichen and / or moss or other higher vegetation. Brief description of the present invention

[0012] In a first aspect, to solve the above-mentioned problems, a composition of isolated microorganisms is provided, which is particularly suitable for soil improvement by forming soil crusts. This composition according to the invention comprises at least viable microorganisms of the following cyanobacterial genera: Microcoleus, Nostoc, Scytonema, and the green algae genus Klebsormidium. In one embodiment, the composition of isolated microorganisms according to the invention further comprises green algae of at least the genus Scenedesmus or Chlorella.

[0013] It has been shown that the composition according to the invention, when applied to the soil, allows for the effective formation of biological soil crusts and enables widespread distribution across the soil surface. The composition according to the invention is generally suitable for many different soil types and continents.

[0014] In a further aspect of the present invention, the use of the composition of isolated microorganisms according to the invention for soil improvement, in particular for building soil crusts, is described.

[0015] Finally, methods for improving soil crusts, in particular for building up and improving the soil crust, by applying a composition of isolated microorganisms according to the invention are described.

[0016] Short descriptions of the illustrations

[0017] Figure 1a shows a gel-like polymer matrix in the reflected light microscope (magnification 4×) formed by K. flaccidum on degraded soil from Burkina Faso.

[0018] Figures 1b and 1c show reflected light micrographs of corresponding soils (magnified 4x). In Figure 1b, microorganisms of the species Microcoleus, Nostoc, Scytonema, and Scenedesmus were applied to the soil one day after the application of K. flaccidum. Figure 1c shows the control, with the aforementioned species applied without prior treatment with K. flaccidum.

[0019] Figure 2a shows the induction of the biological soil crust on soil from Burkina Faso with different degradation status by inoculating the mix shown in Table 1 without the endemic isolates and rhizobacteria. Figure 2b shows the chlorophyll A contents determined after six months of the approaches shown in Figure 2a, showing the individual test plots before and after treatment, as well as the control.

[0020] Figure 3 shows the effect of the additive calcite on the formation of the soil crust.

[0021] Figure 4a shows the test areas in the field trial 11 months after treatment, with test areas 3 to 1 shown with increasing degrees of soil degradation.

[0022] Figure 4b shows corresponding chlorophyll A determinations in the test areas before and after treatment.

[0023] Finally, Figure 4c shows the total number of phototrophic colonies in the corresponding test areas before and after treatment.

[0024] Detailed description of the present invention

[0025] In a first aspect, a composition of isolated microorganisms is provided, in particular for soil improvement after application to the soil, comprising viable microorganisms of at least the following genera: a. Microcoleus; b. Nostoc; c. Scytonema; d. Klebsormidium.

[0026] This composition, containing at least various genera of cyanobacteria and the green algae genus Klebsormidium, enables the formation of biological soil crusts in various, climatically diverse areas. Depending on the target locations, different species of the same genus can be part of the composition. In particular, it has been shown that the selected genera of microorganisms support each other, e.g., with regard to sun protection or similar.

[0027] The term "isolated" used here describes that the microorganisms do not exist as such a composition in a natural environment, but are mixed together in isolation.

[0028] The term "viable" refers to the property of the microorganisms that they are present in the composition in such a way that, after inoculation under suitable conditions, growth, in particular multiplication, of the microorganisms occurs.

[0029] The term "soil improvement" or "improvement of the soil crust" means that at least one of the following properties is improved in the introduced soil: mechanical stabilization against wind and water erosion; an increase in water retention compared to non-inoculated soils; an increase in soil moisture; an increase in soil organic carbon content; CO2 fixation; and an increase in soil nitrogen content.

[0030] Unless otherwise stated, the terms "composition" and "mixture" are used synonymously. Both imply that the microorganisms mentioned are present in varying amounts, which may vary, usually in a mixed form.

[0031] Microorganisms of the genus Klebsormidium are particularly effective at promoting the growth and formation of the biological soil crust. In particular, this genus supports widespread distribution across the soil surface through increased production of extracellular matrix to improve the binding of soil particles and other microorganisms. This positive effect was particularly evident in soil textures with smaller particle sizes and low porosity. This can increase the desired properties of water retention and mechanical stabilization against wind and water erosion, as well as the level of soil moisture.

[0032] Suitable species of Klebsormidium include: flaccidum, sp.

[0033] In one embodiment, the microorganisms of the genus Microcoleus are those of the species steenstrupii, vaginatus, chtonoplastes, sociatus, paludosus.

[0034] In one embodiment, the microorganisms of the genus Nostoc are those of the species commune, punctiforme.

[0035] In a further embodiment, the Scytonema microorganisms according to the invention are those of the species javanicum, hyalinum, myochrous.

[0036] In a further aspect, the composition according to the invention is one which further comprises viable microorganisms of at least the genus Scenedesmus or Chlorella. These genera are single-celled green microalgae that particularly promote carbon fixation and have a high specific growth rate.

[0037] Suitable species of the genus Scenedesmus include, for example, Scenedesmus vacuolatus. Suitable species of the genus Chlorella include, for example, Chlorella vulgaris or Chlorella ohadii. Furthermore, endemic isolates of viable microorganisms may be present in the composition of isolated microorganisms according to the invention. This means that these endemic isolates of viable microorganisms are isolates from the soils or soil crusts that are representative of the environmental conditions of the target area to be treated. These allow for increased biodiversity and the success of the growth of the soil crust to be formed. The endemic isolates comprise the greatest possible number of different species; these are usually endemic / local phototrophic species of microalgae and cyanobacteria, but also include isolated rhizobacteria as non-phototrophic species.It is preferred that these local species comprise at least 1% by mass of the composition. These isolated endemic microorganisms, which include phototrophic species of microalgae and cyanobacteria and non-phototrophic species of rhizobacteria, improve biodiversity and the development of the soil crust, as well as its adaptation to environmental conditions.

[0038] In one embodiment, the composition comprises at least 20 species of different isolated microorganisms.

[0039] The composition of isolated microorganisms according to the invention can be present in various forms. Suitable compositions include those that are present as dried, usually particulate mixtures. Lyophilized compositions according to the invention are possible. Furthermore, the compositions can be provided in liquid form, e.g., as a suspension of various solids. Encapsulated forms of liquid or solid compositions according to the invention are also possible. For example, a suspension of microorganisms and medium can be present in a suitable encapsulated form. The encapsulation can, for example, be a gel-like encapsulation, such as that made of alginate. The capsule materials are usually biodegradable materials.

[0040] In a further embodiment, the composition of the isolated microorganisms is one that further comprises other components, additives, in particular additives or excipients, such as biocompatible excipients. Furthermore, the composition can contain additives such as nutrients or media components as residues from the cultivation of the cyanobacteria and microalgae (especially nitrogen sources such as nitrate or ammonium from standard culture media such as BG11 according to Rippka et al. 1979, Microbiology 111 (1), pp. 1-61. DOI: 10.1099 / 00221287-111-1-1 or Boids Basal Medium, according to Starr and Zeikus, 1993) J Phycol 29 (s2), pp. 1-106. DOI: 10.1111 / j.0022-3646.1993.00001.x). To improve growth and the formation of the soil crust, growth accelerators and fillers such as clay can be used as additives. Additionally, biopolymers such as carboxymethylcellulose, lignin or hydrogels derived from them, xanthan gum, chitosan, or alginates, as well as molasses / bagasse, can be used.Psyllium husks, apple pomace or pectin, or agar agar can also be used as fillers or excipients. These increase, for example, water retention and soil moisture. Sodium alginate as an additive increases water retention and soil moisture; the alginate can be present in the mixture in particulate or powder form. Alternatively, the alginate can also be used to encapsulate the composition.

[0041] Other additives also include plant seeds from grasses or similar.

[0042] If the composition according to the invention is in encapsulated form, it can be easily stored with high storage stability over a longer period of time and can be incorporated or applied into the raw soil.

[0043] For example, if the composition is in the form of a suspension, calcite CaCO3 can be used as an excipient to increase mechanical stability.

[0044] Suitable excipients, in particular biocompatible excipients, are known to the person skilled in the art, in particular for improving the storage stability and processability of the composition of the compositions according to the invention, but also as excipients in the production of the compositions according to the invention in the desired dosage form.

[0045] In one embodiment, the composition of isolated microorganisms according to the invention comprises the endemic isolates of viable microorganisms in a mass fraction of at least 1%, such as at least 2% and a maximum of 15%, based on the wet biomass. In one embodiment, the composition of isolated microorganisms according to the invention comprises the endemic isolates of viable microorganisms in a mass fraction of 2% to 10%, such as 4% to 6%.

[0046] The genus Klebsormidium is preferably present in a range of 20% to 40%, such as 25% to 35%, e.g., 30% mass fraction (w / w, weight per weight) based on wet biomass (cell wet weight).

[0047] The genus Microcoleus, for example, is present in a range of 35% to 55% by mass based on wet biomass. The microorganisms of the genera Nostoc, Scytonema, and Scenedesmus are present in one embodiment in the range of 4% to 15%, such as 5% to 10%, e.g., 7% by mass based on wet biomass. The same applies to the genus Chlorella, which can also be present in the corresponding mass proportions of 4% to 15%, such as 5% to 10%, e.g., 7% by mass based on wet biomass in the overall composition instead of the genus Scenedesmus. The total proportions of the respective species are selected so that the total mass makes up 100% by mass of the wet biomass. In addition to this 100% total mass, additives, including biocompatible excipients, nutrients, and fillers, as well as biopolymers, may also be present.

[0048] The composition of isolated microorganisms according to the invention can be provided as a single composition. Alternatively, the composition can also be provided in several parts, which are mixed together before use of the composition according to the invention to obtain an overall mix as the composition according to the invention. If appropriate, appropriate agents for finishing the composition to be applied, for example, dispersants, thickeners for preparing the suspension to be applied, etc., can also be used.

[0049] In addition, the isolated microorganisms can be stored as individual isolated microorganisms in separate storage devices and then mixed together depending on the method of application.

[0050] The individual storage of the individual isolated microorganisms also allows individual mass fractions of the microorganisms to be used on site in the composition according to the invention and, if necessary, for example, to provide a master mix with the microorganisms of the genus Microcoleus, Nostoc, Scytonema and Klebsormidium, optionally further comprising microorganisms of the genus Scenedesmus or Chlorella, which are then mixed with the endemic isolates in the area of ​​application before application.

[0051] In one embodiment, this master mix or standard mix contains the non-endemic isolates.

[0052] The mass fractions of the respective microorganism species can be varied in the overall composition to change desired properties and functions, for example, to improve nitrogen fixation, water retention, or similar. To do this, the proportions of the microorganisms particularly suited for this purpose are increased or the mass fractions of the others are decreased.

[0053] The composition of the isolated microorganisms according to the invention can therefore be provided as a kit with each of the individual isolated microorganisms provided for producing the composition according to the invention.

[0054] It is also possible that part of the composition is in liquid form while another part is in solid form and both are mixed together before use.

[0055] In a further aspect, the present invention relates to the use of the composition of isolated microorganisms according to the invention for soil improvement. As explained and further demonstrated in the examples below, the composition of the invention allows the formation of soil crusts on raw soils and the development of soil crusts to be promoted. In particular, the use of the composition of isolated microorganisms according to the invention makes it possible to establish or improve the function and functions of biocrusts, including: mechanical stabilization against wind and water erosion; increased water retention compared to bare soil; increased soil moisture; increased organic carbon content in the soil through CCh fixation; and increased nitrogen content in the soil. In particular, the use of the composition of the invention for improving soil crusts is therefore envisaged.The term "improvement of soil crusts" implies that the soil crust is also newly formed, e.g., on raw soils, and further the development of biocrusts, whereby the use according to the invention can accelerate the formation of biocrusts.

[0056] Finally, the present invention describes a method for improving soils, in particular raw soil without biocrust. The method comprises the step of applying and / or introducing a composition of isolated microorganisms according to the invention onto and / or into the soil. The incorporation or inoculation of the composition according to the invention onto and / or into the soil can be carried out according to conventional methods. The application and / or introduction can be carried out as a liquid composition or as a solid composition or also mixed, one part liquid, one part solid. Suitable methods for applying and / or introducing onto / into the soil are known to those skilled in the art. In one embodiment, it is a method for applying the composition according to the invention. The method is in particular one for improving the soil crust or for forming and accelerating the development and accelerating the formation of soil crust.

[0057] The application and / or introduction can be in an amount of 5 to 1000 g / m 2 in an amount of 10 to 500 g / m 2 , e.g. 40 to 300 g / m 2 , especially in a range of 100 to 200 g / m 2 based on wet biomass.

[0058] Furthermore, the additives or auxiliary materials can be applied and / or incorporated accordingly. The additives can be used in quantities of 1 to 1000 g / m 2 , such as in an amount of 5 to 500 g / m 2 , e.g. 15 to 300 g / m 2 , especially in a range of 25 to 100 g / m 2 be added.

[0059] The application and / or introduction can be carried out in such a way that the composition is applied and / or introduced as a whole, or in such a way that individual components of the composition are applied and / or introduced at different times, for example. In one embodiment, for example, microorganisms of the genus Klebsomodium are applied and / or introduced, followed by the other microorganisms of the composition at a later time.

[0060] The additives or excipients can be applied and / or introduced separately. This can occur before and / or after the application and / or introduction of the composition.

[0061] The present invention will be further explained with the aid of examples, without being limited to them. Example

[0062] A composition according to the invention is shown, for example, in Table 1: The endemic isolations listed in Table 1 are shown in Table 2:

[0063]

[0064] Test series 1:

[0065] Functionality Klebsormidium flaccidum:

[0066] In a first approach, K. flaccidum was used in an amount of 60 g / m 2 applied to degraded soil from Burkina Faso and developed for one day at 30°C (incubation conditions: T = 30°C, photon flux density of 100 pE / (m 2 s) in the day-night rhythm 12h / 12h).

[0067] Figure 1a shows the formation of a gel-like polymer matrix after incubation with K. flaccidum.

[0068] In Figure 1b and c, the other four microorganisms, as listed in Table 1 except for the endemic isolates, were then applied as a mixture. The application was carried out in an amount of 200 g of wet biomass per m 2 (or 140 g / m 2for the approach with K. flaccidum added) with a mixing ratio of the microorganisms of (70:10:10:10 for Microcoleus:Nostoc:Scytonma:Scenedesmus , then the incubation was continued. Figure 1b shows the experimental approach in which K. flaccidum was applied to the soil beforehand and incubated for one day, Figure 1c shows a corresponding comparison without prior application of K. flaccidum. The improved formation of the gel-like polymer matrix is ​​clearly visible. K. flaccidum supports the growth of the microorganisms accordingly. This was also reflected in an increased content of biomass in the soil determined by chlorophyll A (not shown). The basic mixture shown in Table 1 without the endemic isolates is therefore suitable for forming the soil crusts accordingly.

[0069] Test series 2:

[0070] Induction of biological soil crust with a mixture of five species.

[0071] The microorganisms listed in Table 1, except for the endemic isolates and rhizobacteria, were applied to soils from Burkina Faso, which exhibited varying degrees of soil degradation. Test plot 1 showed the most severe degradation, while test plot 3 showed the least. The samples were incubated for six months at 30°C and a PFD of 50 pE(m 2 s), 16 / 8 day / night. 100g wet biomass / m 2 soil used.

[0072] Figure 2a shows the corresponding formation of the soil crusts. The chlorophyll A content of these samples was determined after six months of incubation, see Figure 2b. Figure 2b shows the chlorophyll A content per gram of soil in the three test plots shown, each with different degradation status, from Burkina Faso before and after the six-month treatment. For comparison, a control of a natural biocrust of the dark cyanobacteria-microalgae-dominated type from Burkina Faso is shown. The chlorophyll A content, as a measure of the soil biomass content, is significantly increased.

[0073] It was also shown that water retention in these soils is improved (not shown).

[0074] Test series 3:

[0075] The influence of the additive calcite on the formation of the soil crust was investigated by incubating an artificial sand with a mixture listed in Table 2 for four weeks at room temperature. The amount was 200 g / m 2 Burkina Faso Mix. In addition, calcite was added as an additive in the lower test series in an amount of 25 g / m 2 added. As shown, the addition of the additive resulted in a thicker soil crust after four weeks of incubation.

[0076] Test series 4:

[0077] Field trial

[0078] On soil corresponding to test plots 1 to 3 described above, the formation of biological soil crusts was investigated in a field trial within a period of 11 months after treatment. As in the above series of experiments, test plot 1 exhibited a high degree of soil degradation, test plot 2 a moderate degree of soil degradation, and test plot 3 no soil degradation. Accordingly, Figure 4a shows plots after 11 months of treatment. The treatment was carried out by applying the compositions according to Table 2, and then post-treating the plots with irrigation and nutrient additions. On the plots with high degradation, the soil crusts are more pronounced than the crusts on test plot 2; test plot 3, which showed no significant degradation, showed little or no further soil crust formation due to the presence of taller vegetation in the form of grasses and shrubs.The area with high soil degradation, on the other hand, showed the formation of the soil crust with almost complete coverage.

[0079] The formation of the soil crust is also confirmed by the determination of the chlorophyll A content before and after treatment of the test plots shown in Figure 2a. While the chlorophyll A content remained constant in test plots 2 and 3, or decreased slightly after treatment, the chlorophyll A content per gram of soil in test plot 1 increased significantly and is now comparable to the chlorophyll A content of a naturally grown biological soil crust from the test area. The low chlorophyll A content in test plots 2 and 3 can be explained by the fact that these test plots are predominantly covered with grasses and shrubs and therefore have less biological soil crust with a concentration of chlorophyll A in the uppermost soil layers.

[0080] Figure 4c shows the phototrophic colonies in the topsoil layer as an indicator of the concentration of living biomass in the photosynthetic crust component, as well as the number of different phototrophic species and thus the biodiversity. The improvements after treatment are clearly visible.

[0081] In all degraded test plots (test plot 1), the formation of biological soil crusts was observed 11 months after treatment. These biological soil crusts exhibited a comparable chlorophyll A content to their natural counterparts in the test area. Furthermore, the biodiversity of phototrophic soil organisms in the corresponding layers was increased after treatment.

[0082] The determination of the chlorophyll A content was carried out as follows:

[0083] The determination of the chlorophyll A content was carried out according to Park et al 2015 (Park, Chan-Ho; Li, Xinrong; Jia, Rong Liang; Hur, Jae-Seoun (2015): Effects of Superabsorbent Polymer on Cyanobacterial Biological Soil Crust Formation in Laboratory. In Arid Land Research and Management 29 (1), pp. 55-71. DOI: 10.1080 / 15324982.2014.928835.) At least 2 g of the soil sample (or biocrust sample) were added to 5 mL of 99.8% (v / v) ethanol in a 50 mL Falcon tube, mixed three times by inversion and then incubated in a water bath at 80°C for 5 min. After inverting three times again, the samples were cooled at room temperature for 30 min. After centrifugation for 5 minutes at 4°C and 4000 rpm, the supernatant is measured in a UV-Vis spectrophotometer against 99.8% ethanol as a blank. The absorbance is measured at a wavelength of 665 nm. The Chi a concentration per g of soil is calculated using a formula by Ritchie (2006) (Ritchie, Raymond J.(2006): Consistent sets of spectrophotometric chlorophyll equations for acetone, methanol and ethanol solvents. In Photosynthesis research 89 (1), pp. 27-41. DOI: 10.1007 / s11120-006- 906 / - i I Chl with.

[0084] Chl a: Chlorophyll A content [pg / g soil]

[0085] A665: Absorption at 665 nm [AU]

[0086] V: Volume of ethanol [mL] ms: Weight of soil sample, initial weight [g] d: Path length of cuvette [cm], 1 cm

[0087] £, 665: Specific extinction coefficient of chlorophyll A in ethanol [pg x cm / Au x mL]

[0088] The total number of phototrophic colonies was determined as follows:

[0089] 1 g of the biocrust is mixed in 10 mL of Butterfield's buffer in a suitably sized Falcon tube. The Falcon tube (or alternatively a sufficiently large Petri dish) is incubated for 7 days under a low photon flux density of 40-50 pE / (m 2s) and natural day / night rhythm at room temperature.

[0090] The suspensions of the slurried biocrust are then diluted 1 / 10 and 1 / 100 using Butterfield's buffer and spread on suitable agar plates (e.g., BG11, BG11o, and BBM medium). The spread agar plates are then incubated for at least one week under the same conditions under illumination. The grown phototrophic colonies are then counted.

[0091] Individual components in the above-mentioned mixtures according to the invention can be varied. This can have different reasons. For example, a different species can be used instead of Klebsormidium flaccidum or Klebssormidium sp. to accommodate geographical peculiarities. Microcoleus-specific species such as Microcoleus steenstruppii for higher temperatures, Microcoleus vaginatus for temperate zone temperatures, or Microcoleus chtonoplastes for saline soils, or Microcoleus sociatus for sandy soils, can be selected accordingly based on local conditions. For geographical adaptation, Scytonema javanicum or Scytonema hyalinum can also be selected. The same applies when selecting the species Scenedesmus vacuolatus or Chlorella vulgaris or Chlorella ohadii (very high temperatures and sunlight). Depending on the desired results, the above-mentioned mix can also be used in its proportions, for example, toTo influence carbon fixation, nitrogen fixation, etc. N fixation can be achieved by increasing the proportion of bacteria of the genus Nostoc in general, e.g., Nostoc commune, or of Scytonema. Water runoff can be altered by increasing the proportion of Scytonema, which influences water runoff (due to its specific morphology, which results in increased surface structure).

[0092] The compositions according to the invention may also contain further additives as mentioned in the description. It has been shown that the compositions according to the invention

[0093] Promote the formation and development of soil crusts. The compositions according to the invention are thus suitable for use in soil improvement, in particular the formation of soil crusts in raw soils, and for preventing desertification or desertification.

Claims

Patent claims 1. Composition of isolated microorganisms, in particular for Soil improvement comprising at least viable microorganisms of the following genera: a. Microcoleus, b. Nostoc, c. Scytonema and d. Klebsormidium.

2. A composition of isolated microorganisms according to claim 1, further comprising viable microorganisms of the genus Scenedesmus or Chlorella.

3. A composition of isolated microorganisms according to any one of the preceding claims, comprising endemic isolates of viable microorganisms from soils onto which the mixture of isolated microorganisms according to any one of claims 1 to 2 is introduced.

4. Composition of isolated microorganisms according to one of the preceding claims, wherein this composition is in the form of a suspension, dried particles, lyophilized composition, granules, or capsules, in particular capsules containing liquid suspension.

5. Composition of isolated microorganisms according to one of the previous Claims, wherein the ratios of the microorganisms are as follows: Microcoleus 35% - 55%; Nostoc 4%-15%; Scytonema 4% - 15%; Klebsormidium 20% - 40%; Scenedesmus or Chlorella 4% - 15%; Endemic isolations >1% mass fraction; with a total mass of 100% based on total wet biomass, optionally further containing additives.

6. Composition of isolated microorganisms according to one of the previous Claims, further comprising further, in particular biocompatible additives, in particular nutrients including sugars such as trehalose or sucrose, media components, growth accelerators, fillers such as clay, biopolymers or grass seeds.

7. A composition of isolated microorganisms according to any one of the preceding claims, wherein said mixture comprises at least twenty species of different isolated microorganisms.

8. Use of a composition of isolated microorganisms according to any one of claims 1 to 7 for soil improvement.

9. Use of the composition of isolated microorganisms according to claim 8 for improving soil crusts.

10. A method for improving a soil, comprising the step of applying to the soil a composition of isolated microorganisms according to any one of claims 1 to 8.

11. A method for improving soils according to claim 10, which is a method for improving the soil crust.

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