PL247621B1 - Composition containing siderophores produced by the Pseudomonas H12B strain, method of producing a composition containing siderophores and use of siderophores produced by the Pseudomonas H12B strain - Google Patents

Abstract

The subject of the application is a composition containing siderophores produced by the Pseudomonas H12B strain, deposited under number B/00321 in the Polish Collection of Microorganisms of the Institute of Immunology and Experimental Therapy of the Polish Academy of Sciences in Wrocław, Poland, intended for improving the microbiological quality of soil, increasing the bioavailability of micro- and macronutrients necessary for the proper growth and development of soil microorganisms and plants, increasing the mobility of heavy metals in soil, accelerating the biodegradation of petroleum compounds or promoting the germination of crop plants, a method for producing a composition containing siderophores and a substrate for producing such a composition.

Description

Description of the invention

The subject of the invention is a method for producing a metabolite composition containing siderophores, the composition and its use.

STATE OF THE ART

Iron is an essential element for the proper functioning of plants, animals, and bacteria. Thanks to its chemical properties, it participates in numerous oxidation and reduction reactions crucial for cells, such as electron transport in the respiratory chain. Iron occurs in the Earth's crust primarily in the form of minerals containing oxides (magnetite, hematite) or hydroxides (goethite). Despite its widespread occurrence in the environment, iron(III) is not bioavailable due to its low solubility. For this reason, many organisms have evolved various mechanisms to enable the uptake of this crucial element from the environment. One of the most common strategies for increasing iron bioavailability by organisms is the synthesis of siderophores—compounds that efficiently chelate this element.

Siderophores are a group of low-molecular-weight compounds characterized by a high affinity for iron (III). They are highly diverse in terms of structure, functionality, and biological functions. Siderophores are produced by bacteria, fungi, and plants under iron-deficient conditions and then secreted into the environment. In the environment, they form complexes with iron ions, and the resulting complexes are then taken back into the cell via appropriate surface receptors. The high affinity of siderophores for iron is related to the presence of negatively charged oxygen atoms. They can function as electron donors for iron (III), a strong acid according to Lewis theory, allowing for the formation of complex compounds.

Due to their significant structural and functional diversity, siderophores have particularly high application potential in various fields of biotechnology. They are the subject of numerous studies aimed at better understanding their biology and increasing the potential for their practical application. Due to their high affinity for metal ions such as iron, as well as other divalent and trivalent ions, siderophores may be useful in the bioremediation of soils contaminated with heavy metals. Thanks to their affinity for various ions, siderophores can chelate heavy metals present in soil and effectively extract them from contaminated soils. These processes are conducted ex situ, for example, in remediation piles.

By increasing the bioavailability of iron in the soil, siderophores directly contribute to improving soil microbiological quality. The increased supply of bioavailable iron in the soil increases the activity and abundance of soil microorganisms. Improving soil biological quality is influenced not only by the siderophores themselves but also by the metabolites co-produced by microorganisms during siderophore synthesis. This metabolite composition containing siderophores can be applied to the soil and further contribute to improving its microbiological quality. Among the metabolites co-produced during siderophore synthesis can be various organic acids, ligands, short-chain peptides, alcohols, and unsaturated fatty acids, which, in a synergistic relationship with siderophores, jointly contribute to increasing the abundance and activity of soil microbiota, improving soil microbiological quality. Improving the microbiological quality of soil (the number and activity of microorganisms) may also be useful in the context of increasing the efficiency of biodegradation of organic compounds in contaminated soils (in situ bioremediation).

Another potential application of siderophores may be promoting plant growth. Studies have shown that bacterial siderophores not only positively influence the bioavailability of iron for the microorganisms that produce them, but also contribute to improving the iron supply of plants. Increasing the supply of iron to plants (by increasing the bioavailability of this element) is crucial for proper chlorophyll synthesis. It has been shown, for example, that siderophores produced by bacteria of the genera Pseudomonas (pyoverdine) and Streptomyces positively influence plant biomass growth thanks to the increased iron supply. Increased iron content in plant cells also increases plant photosynthetic activity. Therefore, numerous studies are currently underway to develop biotechnological processes that will enable the use of siderophore activity as a means of supporting plant fertilization.

Various secondary metabolites produced by microorganisms during siderophore production can also positively impact plant growth. As mentioned above, these can be various organic compounds with high bioavailability for both soil microorganisms and plants. The effects of metabolites accompanying siderophore synthesis on plants can be both direct and indirect. Metabolites such as amino acids, short-chain peptides, organic acids, and vitamins can be natural plant growth stimulants, significantly contributing to increased biomass and improved overall health. Organic acids can also further enhance the availability of other nutrients, such as phosphorus. The indirect effects of metabolites accompanying siderophore production involve stimulating the growth and activity of soil microbiota, which, by entering into symbiotic interactions with plants, contribute to improved growth and/or increased resistance to phytopathogens.

Bacterial siderophores are produced in microbiological media typically containing an organic carbon source and an organic or inorganic nitrogen source. This medium must be iron-free. The best-studied and most frequently described medium in the literature is GASN, intended for siderophore production. It contains glucose and asparagine as carbon and nitrogen sources, respectively. It also contains mineral salts. This medium supports relatively high siderophore production by bacteria, but producing them on a larger (industrial) scale, as required in bioremediation or agricultural research, is economically unviable. Furthermore, metabolites, including siderophores, produced in GASN medium are typically characterized by a neutral pH, which is undesirable from the perspective of the efficiency of heavy metal extraction from contaminated soils. In the bioremediation of soils contaminated with heavy metals, the pH of the added metabolites should be acidic to increase the mobility of heavy metals in the soil.

From the perspective of promoting plant growth by supplementing soil with a mixture of bacterial metabolites containing siderophores, the optimal pH for such a bacterial culture should be acidic or alkaline, depending on the plants' nutritional and environmental preferences. The varying pH of metabolites added to the soil influences its pH, which in turn has a key impact on the bioavailability and efficiency of macro- and micronutrient uptake by microorganisms and plants. This means that acidifying or alkalizing the soil with a mixture of metabolites containing siderophores can contribute to improved plant nutrition.

The production of a metabolite composition containing acidic siderophores produced on a GSA substrate and its use in the bioremediation of soils contaminated with heavy metals were disclosed in Polish patent application No. P.440597.

The present application relates to the production of a metabolite composition containing siderophores of both acidic and alkaline nature on media other than GSA, as well as the possibility of their use in (i) bioremediation of soils contaminated with heavy metals, (ii) improvement of the microbiological quality of soil, and (iii) promotion of crop germination. The present application also covers a new use of the previously described metabolite composition produced on a GSA medium for promoting crop germination, which was not disclosed in application P.440597 (PL 440597 A1).

The present application therefore presents a new method for producing a composition of metabolites of bacterial origin containing siderophores on new microbiological media containing suitable carbon and nitrogen sources, which were not disclosed in the earlier application P.440597.

The bacterial strain used in the research presented in this disclosure is Pseudomonas ANT_H12B. This strain was the subject of patent application No. P.437824 concerning the use of ANT_H12B as a plant growth promoting agent (composition of metabolites produced on GASN medium) and patent application No. P.440597 concerning the use of this strain as a producer of siderophores used in the bioremediation of soil contaminated with heavy metals (composition of metabolites produced on GSA medium). This strain was deposited on March 30, 2021, in the Polish Collection of Microorganisms of the Institute of Immunology and Experimental Therapy of the Polish Academy of Sciences in Wrocław, Poland under number B/00321.

THE ESSENCE OF THE INVENTION

The subject of the invention is a composition containing siderophores, characterized in that the siderophores are produced by the Pseudomonas H12B strain deposited under number B/00321 in the Polish Collection of Microorganisms of the Institute of Immunology and Experimental Therapy of the Polish Academy of Sciences in Wrocław, Poland, intended for:

a) improving the microbiological quality of the soil,

b) increasing the bioavailability of micro- and macronutrients necessary for the proper growth and development of soil microorganisms and plants, such as iron and phosphorus,

c) accelerating the biodegradation of petroleum compounds, useful in the processes of cleaning contaminated soils or

d) promoting the germination of crop plants.

The composition according to the invention is preferably characterized in that it is obtained by culturing the Pseudomonas H12B strain on a substrate selected from GCl, GlSA, CSA or GlASN and is additionally intended to increase the mobility of heavy metals in the soil, useful in the processes of cleaning up contaminated land, wherein:

- GCl medium has the following composition: glucose 7 g/l, NH4Cl 1.35 g/l, Na2HPO4 0.96 g/l, KH2PO4 0.44 g/l, MgSO4 x 7H2O 0.2 g/l; pH 7.0;

- GlSA medium has the following composition: glycerol 50% 3.5 g/l, (NH4)2SO4 1 g/l, Na2HPO4 .96 g/l, KH2PO4 0.44 g/l, MgSCUX 7H2O 0.2 g/l; pH 7.0;

- CSA medium has the following composition: citric acid 4 g/l, (NH4)2SC4 1 g/l, Na2HPC4

0.96 g/l, KH2PC4 0.44 g/l, MgSc4 x 7H2C 0.2 g/l; pH 7.0;

- GlASN medium has the following composition: glycerol 50% 3.5 g/l, L-asparagine 2 g/l, Na2HPC4 0.96 g/l, KH2PC4 0.44 g/l, MgSC4X 7H2C 0.2 g/l; pH 7.0.

Preferably, the composition according to the invention is characterized in that it is obtained by culturing the Pseudomonas H12B strain on a GSA medium.

Preferably, the composition according to the invention is characterized in that the improvement of the microbiological quality of the soil includes an increase in the number of soil microorganisms - heterotrophic bacteria.

Preferably, the composition according to the invention is characterized in that the improvement of the microbiological quality of the soil comprises an increase in the activity of dehydrogenases of soil microorganisms.

Preferably, the composition according to the invention is characterized in that the crop plants are selected from beet, pea and tobacco.

Another subject of the invention is a method for preparing the above-defined composition according to the invention containing siderophores, comprising the following steps:

a) an inoculum of the Pseudomonas sp. ANT_H12B bacterial strain, deposited under number B/00321 in the Polish Collection of Microorganisms of the Institute of Immunology and Experimental Therapy of the Polish Academy of Sciences in Wrocław, Poland, is obtained on a liquid complete medium intended for the multiplication of bacterial cells, preferably LB medium, cultivating the bacteria at a temperature of 20-25°C until the logarithmic phase of growth is achieved;

b) the culture obtained in step a) is centrifuged and the cell biomass is then suspended in a physiological saline solution, preferably centrifuged twice;

c) inoculating the culture medium selected from: GCl, GlSA, CSA or GlASN with the bacterial suspension obtained in point b), preferably in an amount giving an initial optical density of the culture of 0.06, measured at a wavelength of 600 nm;

d) the bacterial culture obtained in step c) is carried out at a temperature of 10-20°C;

e) the culture broth obtained in point d) is centrifuged and the supernatant is separated, thus obtaining a composition containing siderophores;

wherein:

- as the GCl medium, the following medium is used: glucose 7 g/l, NH4Cl 1.35 g/l, Na2HPC4 0.96 g/l, KH2PC4 0.44 g/l, MgSC4 x 7H2C 0.2 g/l; pH 7.0;

- as a GlSA medium, the following medium is used: glycerol 50% 3.5 g/l, (NH4)2SC4 1 g/l,

Na2HPC4 0.96 g/l, KH2PC4 0.44 g/l, MgSC4 x 7H2C 0.2 g/l; pH 7.0;

- as a CSA substrate, a medium with the following composition is used: citric acid 4 g/l, (NH4)2SC4 1 g/l,

Na2HPC4 0.96 g/l, KH2PC4 0.44 g/l, MgSC4 x 7H2C 0.2 g/l; pH 7.0;

- as the GlASN medium, the following medium is used: glycerol 50% 3.5 g/l, L-asparagine 2 g/l, Na2HPC4 0.96 g/l, KH2PC4 0.44 g/l, MgSC4 x 7H2C 0.2 g/l; pH 7.0.

Preferably, in the method according to the invention, the bacterial culturing in step d) is carried out for 48-72 hours.

Preferably, in the method according to the invention, the bacterial culturing in step d) is carried out with shaking at 120-150 rpm.

Preferably, in the method according to the invention, in step c) a GCl medium or a GISA medium is used as a culture medium for the production of a composition containing siderophores with acidifying properties.

Preferably, in the method according to the invention, in step c) a CSA medium or a GIASN medium is used as a medium for preparing the composition containing siderophores with alkalizing properties.

Another subject of the invention is the use of siderophores produced by the Pseudomonas H12B strain deposited under number B/00321 in the Polish Collection of Microorganisms of the Institute of Immunology and Experimental Therapy of the Polish Academy of Sciences in Wrocław, Poland, to improve the microbiological quality of soil, increase the bioavailability of micro- and macronutrients necessary for the proper growth and development of soil microorganisms and plants, such as iron and phosphorus; accelerate the biodegradation of petroleum compounds, useful in the processes of cleaning contaminated land or promoting the germination of crop plants.

The substrate for preparing the composition containing siderophores is selected from the following:

a. GCl medium with the following composition: glucose 7 g/l, NIW 1.35 g/l, Na2HPO4 0.96 g/l, KH2PO4 0.44 g/l, MgSO4 χ 7H2O 0.2 g/l; pH 7.0;

b. GlSA medium with the following composition: glycerol 50% 3.5 g/l, (NH4)2SO4 1 g/l, Na2HPO4 0.96 g/l, KH2PO4 0.44 g/l, MgSO4 χ 7H2O 0.2 g/l; pH 7.0;

c. CSA medium with the following composition: citric acid 4 g/l, (NH4)2SO4 1 g/l, Na2HPO4 0.96 g/l, KH2PO4 0.44 g/l, MgSO4 χ 7H2O 0.2 g/l; pH 7.0;

d. GlASN medium with the following composition: glycerol 50% 3.5 g/l, L-asparagine 2 g/l, Na2HPO4 0.96 g/l,

KH2PO4 0.44 g/l, MgSO4 χ 7H2O 0.2 g/l; pH 7.0.

The substrate may be a GCl or GlSA substrate for the production of a composition containing siderophores with acidifying properties.

The substrate may be a CSA or GlASN substrate for the preparation of a composition containing siderophores with alkalizing properties.

DETAILED DESCRIPTION OF THE INVENTION

The present inventors have developed a new method for producing metabolite compositions containing siderophores, characterized by varying pH depending on the nutrients provided in the microbiological medium. The new method for producing metabolite compositions containing siderophores involves culturing the Pseudomonas sp. ANT_H12B strain on microbiological media containing various sources and contents of carbon and nitrogen, which have not been previously reported. By culturing the microorganisms on media containing various nutrient substrates, the bacteria produce significant levels of siderophores and other co-metabolites, which contribute to the acidification or alkalization of the microbiological medium. The production of bacterial metabolite compositions containing siderophores involves the following steps:

a) preparation of an inoculum of the Pseudomonas sp. ANT_H12B bacterium deposited as B/00321 on a liquid medium intended for the multiplication of bacterial cells and culturing the bacteria at a temperature of 20-25°C until the logarithmic growth phase is achieved.

b) Preparation of the bacterial inoculum by centrifuging the bacterial culture obtained in a medium intended for bacterial cell multiplication, and then suspending the cell biomass in physiological saline solution. This procedure should be performed twice.

c) inoculation of one of the developed liquid media with the obtained and prepared inoculum - liquid media allowing the production of metabolites with acidifying properties - GCl medium and GlSA medium or CSA or GlASN media allowing the production of metabolites with alkalizing properties.

d) the inoculation of one of the developed media with the obtained and prepared bacterial inoculum should be performed in such a way that the initial optical density of the culture is 0.06 at a wavelength of 600 nm.

e) the inoculated medium for the production of metabolites, including siderophores with acidifying or alkalizing properties, is a bacterial culture which, in order to obtain the appropriate concentration of metabolites, including siderophores, should be maintained for 48-72 hours at a temperature of 10-20°C, with shaking at 120-150 rpm.

The subject of the application also includes new methods of using the produced compositions of metabolites, including siderophores, on the developed microbiological media: GCl, GlSA, CSA and GlASN.

New ways of using the produced metabolite compositions, including siderophores, include their positive effects in the following aspects:

a) improving the microbiological quality of the soil

b) increasing the bioavailability of micro- and macronutrients necessary for the proper growth and development of soil microorganisms and plants, such as iron and phosphorus

c) increasing the mobility of heavy metals in the soil, useful in the processes of cleaning up contaminated land

d) acceleration of biodegradation of petroleum compounds, useful in the processes of cleaning contaminated land

e) promoting the germination of crop plants.

The subject of the application is also a new use of the composition of bacterial metabolites, including siderophores, produced by the Pseudomonas sp. ANT_H12B strain on a GSA medium, described in the earlier application P.440597. The new use includes:

a) improving the microbiological quality of the soil

b) increasing the bioavailability of micro- and macronutrients necessary for the proper growth and development of soil microorganisms and plants, such as iron and phosphorus,

c) acceleration of biodegradation of petroleum compounds, useful in the processes of cleaning contaminated land

d) promoting the germination of crop plants.

The obtained metabolites, including siderophores produced on GCl, GISA, CSA, GlASN or GSA substrates, can be supplemented to different types of soils and show various positive effects:

a) in the case of agricultural soil supplementation, nutrient compounds such as Fe and P are mobilized, which increases their bioavailability for microorganisms and plants,

b) in the case of agricultural soil supplementation, the increased bioavailability of Fe and P stimulates the number and activity of soil microorganisms, which may have a positive impact on the growth and development of crop plants,

c) in the case of supplementation of soils contaminated with petroleum compounds, increasing the number of soil microorganisms contributes to the increased efficiency of biodegradation of these compounds, which may also be useful in the bioremediation processes of such soils.

Additionally, metabolites, including siderophores produced on GCl, GlSA, CSA or GlASN substrates, can be supplemented to soils contaminated with heavy metals, which increases the mobility of heavy metals such as Cu, Zn and Ni, which may be useful in the bioremediation processes of such soils.

The resulting metabolite composition, including siderophores, may also be useful in promoting germination and growth of crop plants. The data obtained demonstrate that metabolites produced on GCl, GlSA, CSA, GlASN, or GSA media, including siderophores, contribute to increased germination efficiency in crop plants such as peas and beets.

Unless otherwise indicated, the following examples use standard materials and methods known to one of ordinary skill in the art or follow the manufacturers' recommendations for the materials and methods provided.

Example 1. Production of acidic and basic metabolites, including siderophores, on GCl, GlSA, GlASN and CSA media by Pseudomonas H12B strain

A. Cultivation on GASN medium

Siderophores produced by the Pseudomonas H12B strain under iron-deficient conditions can provide a friendly environment, a biological agent for soil bioremediation, and plant growth promotion. The Pseudomonas H12B strain grows on complete media such as LB (Luria Bertani), however, due to the presence of iron in their composition, siderophores are not produced. Therefore, the strain was cultured on a well-known liquid medium, GASN, intended for siderophore production (iron-deficient), with the following composition: glucose 7 g/l, asparagine 2 g/l, Na2HPO4 0.96 g/l, KH2PO4 0.44 g/l, MgSO4 χ 7H2O 0.2 g/l [Bultreys A., Gheysen I. 2000]. The standard procedure for siderophore production first included the preparation of a bacterial inoculum. For this purpose, liquid LB medium (50 ml) was inoculated with a colony of Pseudomonas H12B strain taken from a plate containing a pure culture. Cultivation in LB was carried out for about 12-24 hours at room temperature (18-25°C) with shaking (approx.

150 rpm) until the logarithmic growth phase of the microorganisms was reached. Then, the obtained culture was centrifuged twice (8000 rpm, 5 minutes), and the resulting pellet, after decanting the supernatant, was suspended in physiological saline (0.85% NaCl) to remove residual iron from the complete LB medium. The actual siderophore-producing culture was prepared by inoculating 200 ml of appropriate media (iron-free), e.g., GASN, with the inoculum prepared according to the described procedure. The amount of inoculum was selected to obtain an initial optical density of the culture OD600nm = 0.06, which corresponds to approximately ¹⁰⁶ CFU/ml. The cultures were grown for 5 days in glass flasks, with oxygenation by shaking (150 rpm) at 10°C. Each day of cultivation, a sample was collected and the pH, microbial count (CFU/ml), and siderophore concentration were determined using a spectrophotometric method with a CAS reagent ([2] Arora, NK, & Verma, 2017). Sterile media for siderophore production were used as chemical controls. After cultivation, the resulting culture broth was centrifuged, and the final product was the supernatant separated from the biomass, containing bacterial metabolites, including siderophores. The use of GASN medium allowed for high siderophore production (concentration ~450 μM), however, the pH of the obtained metabolite solution was close to neutral (~7.3). To increase the effectiveness of siderophore use in both bioremediation and agriculture, it would be beneficial to be able to obtain a solution with a wider pH range, both acidic and alkaline.

B. Development of GCl, GlSA, CSA and GlASN culture media and a culture method for the production of metabolites containing acidic and alkaline siderophores

To obtain metabolite compositions, including siderophores, at varying pH levels, studies were conducted to develop media compositions for their production. Various carbon and nitrogen sources were tested, with the goal of achieving high siderophore production efficiency by the Pseudomonas H12B strain (at least similar to GASN) at varying final pH values of the culture broth. All studies were conducted according to the procedure described in section A, varying the media used. As a result of the studies, four media compositions were identified, divided into two groups:

(i) substrates with acidifying properties:

GCl: glucose 7 g/l (as C source), NH4Cl 1.35 g/l (as N source), Na2HPO4 0.96 g/l, KH2PO4 0.44 g/l, MgSO4 χ 7H2O 0.2 g/l. The pH of the medium after preparation was 7.0, and was adjusted with 0.1 M NaOH and 0.1 M HCl.

GlSA: glycerol 50% 3.5 g/l (as C source), (NH4)2SO4 1 g/l (as N source), Na2HPO4 0.96 g/l, KH2PO4 0.44 g/l, MgSO4 χ 7H2O 0.2 g/l. The pH of the medium after preparation was 7.0, and was adjusted with 0.1 M NaOH and 0.1 M HCl.

(ii) substrates with alkalizing properties:

CSA: citric acid 4 g/l (as C source), (NH4)2SO4 1 g/l (as N source), Na2HPO4 0.96 g/l, KH2PO4 0.44 g/l, MgSO4 χ 7H2O 0.2 g/l. The pH of the medium after preparation was 7.0, and was adjusted with 0.1 M NaOH and 0.1 M HCl.

GlASN: glycerol 50% 3.5 g/l (as C source), L-asparagine 2 g/l (as N source), Na2HPO4 0.96 g/l, KH2PO4 0.44 g/l, MgSO4 χ 7H2O 0.2 g/l. The pH of the medium after preparation was 7.0, and it was adjusted with 0.1 M NaOH and 0.1 M HCl.

The examples also used a GSA substrate, the composition of which was disclosed in application P.440597.

GSA: glucose 7 g/l (as C source), (NH4)2SO4 1 g/l (as N source), Na2HPO4 0.96 g/l, KH2PO4 0.44 g/l, MgSO4 χ 7H2O 0.2 g/l. The pH of the medium after preparation was 7.0, and it was adjusted with 0.1 M NaOH and 0.1 M HCl.

C. Comparison of metabolite production on different media, siderophore production and pH of the obtained solution

In the presented example, the production of siderophores and the pH of the obtained solution of bacterial metabolites produced by the Pseudomonas H12B strain on GASN medium, GSA with the composition of glucose 7 g/l, (NH4)2SO4 1 g/l, Na2HPO4 0.96 g/l, KH2PO4 0.44 g/l, MgSO4 χ 7H2O 0.2 g/l (patent application P.440597) and the newly developed GCl, GlSA, CSA and GlASN media were compared. The experiments were carried out according to the procedure described in point A. The obtained results are presented in Table 1.

PL 247621 Β1

Table 1

Change in pH of bacterial culture and change in siderophore concentration [μM] during cultivation of Pseudomonas H12B strain and in metabolites finally obtained after centrifugation on GASN (M-GASN), GSA ("M-GSA"), GCI (M-GCI), GISA (M-GISA), CSA (M-CSA) and GIASN (M-GIASN) media as well as on GASN (K-GASN), GSA ("K-GSA"), GCI (K-GCI), GISA (K-GISA), CSA (K-CSA) and GIASN (K-GIASN) media alone

Changes in pH during cultivation Variant Day 0 Day 1 Day 2 Day 3 Day 4 Obtained metabolites M-GASN 6.96 6.55 6.26 7.15 7.24 7.28 K-GASN 7.00 6.99 6.99 7.00 7.00 7.02 M-GCI 7.13 6.78 6.40 5.79 4.87 4.66 K-GCI 7.09 7.19 7.14 7.18 7.17 7.08 M-GISA 7.31 6.52 5.92 5.60 4.95 4.88 K-GISA 7.34 7.33 7.34 7.32 7.31 7.29 M-CSA 7.34 7.46 7.84 8.18 8.64 8.72 K-CSA 7.34 7.29 7.27 7.26 7.26 7.15 M-GIASN 7.06 7.74 8.39 8.37 8.81 8.94 K-GIASN 7.08 7.06 7.07 7.14 7.32 7.44 M-GSA 7,H 6.74 6.34 5.79 4.92 4.53 K-GSA 7.09 7.10 7.07 7.11 7.10 7.08 Changes in siderophore values during cultivation |pM| Variant Day 0 Day 1 Day 2 Day 3 Day 4 Obtained metabolites M-GASN 0 90.25 322.89 327.41 430.85 463.82 K-GASN 0 0 0 0 0 0 M-GCI 0 91.95 361.29 546.91 571.11 573.37 K-GCI 0 0 0 0 0 0 M-GISA 0 59.43 110.76 215.09 287.84 386.14 K-GISA 0 0 0 0 0 0 M-CSA 0 76.72 135.13 257.99 311.89 376.95 K-CSA 0 0 0 0 0 0 M-GIASN 0 77.66 165.22 326.56 390.95 389.11 K-GIASN 0 0 0 0 0 0 M-GSA 0 100.23 329.35 496.13 512.22 533.23 K-GSA 0 0 0 0 0 0

The obtained results showed that all newly developed media intended for siderophore production allowed for efficient production of these metabolites by the Pseudomonas H12B strain at similar levels, ranging from 300 to 600 μM. GCl medium produced siderophores over 20% higher than GASN medium, while the remaining media produced approximately 20% lower production. However, these differences are not significant from the perspective of potential applications. It was also demonstrated that achieving high siderophore concentrations (above 100 μM) requires at least a two-day culture, preferably 3-4 days. Further extension of the culture time did not increase metabolite production. Differences in the pH of the culture and the final metabolite solution were observed between the media tested. Cultivation of Pseudomonas H12B strain on GASN medium resulted in the production of metabolite solutions with a neutral pH (~7.3). GCl, GSA, and GlSA media allowed for the production of siderophores in acidic solutions (pH below 5), while CSA and GlASN media allowed for the production of alkaline siderophore solutions (pH above 8). In many applications of metabolites, both in agriculture and bioremediation, the pH of the solution used has a significant impact on the effectiveness of the method. Therefore, the ability to obtain siderophores in solutions with varying pH is a significant advantage of the proposed solution.

Example 2. Increasing the bioavailability of iron in soil using metabolites produced by culturing the Pseudomonas H12B strain on GCl, GlSA and CSA, GlASN and GSA media

The presented example concerns the effect of metabolites produced by the Pseudomonas H12B strain on GCl, GlSA, GlASN, CSA, and GSA culture media on increasing iron bioavailability in soil. Cultivation of Pseudomonas sp. H12B on these media and preparation of metabolites were performed as in Example 1. The effect on iron bioavailability was examined using:

(i) bacterial metabolites produced on GCl ("M-GCL"), GlSA ("M-GlSA"), CSA ("M-CSA"), GlASN ("M-GlASN") and GSA ("M-GSA") media (ii) sterile GCl ("K-GCl"), GlSA ("K-GlSA"), CSA ("K-CSA"), GlASN ("K-GlASN") and GSA ("K-GSA") media as chemical controls (iii) distilled water as an additional control ("K-W")

The effect of metabolites was tested in environmental soil of agricultural origin. 200 g of air-dried soil was sieved through a 2 mm mesh and placed in a PVC container, followed by the addition of 20 ml of the appropriate metabolites/control (10:1 (w/v)). Three replicates were prepared for each treatment. The mixture was supplemented with distilled water to obtain soil moisture of 40% of maximum water holding capacity and mixed by hand. The containers were placed in a thermal cabinet set at 25°C. The experiment was conducted for 14 days, with constant soil moisture maintained using distilled water. Soil samples were collected after the experiment was initiated, on day 7 and day 14. To determine the bioavailability of iron in soil, 5 g of dried soil sample was extracted in 10 ml of extraction solution (0.005 M DTPA, 0.01 M calcium chloride, 0.1 M triethylamine, pH 7.3) for 2 hours and then filtered through filter paper. Iron concentration in the resulting post-extraction fluids was determined by flame atomic absorption spectrometry (FA-AAS) using a Thermo Fisher ICE 3300 spectrometer. Based on these data, the amount of bioavailable iron in the sample (mg/kg soil) was calculated. For each variant, the percentage change in iron bioavailability was calculated on days 7 and 14 of the experiment (the difference in the amount of bioavailable iron (mg/kg) between day 7 or 14 and the beginning of the experiment was calculated, and then the percentage of the initial amount of bioavailable iron was calculated). The results are presented in Table 2.

PL 247621 Β1

Table 2

Change in iron bioavailability in the tested soil as a result of adding a solution of metabolites produced on GCI (‘,M-GCI’), GISA (‘M-GISA’), CSA (‘M-CSA’), GIASN (‘M-GIASN’) and GSA (‘M-GSA’) media and on GCI (‘K-GCI’), GISA (‘K-GISA’), CSA (‘K-CSA’), GIASN (‘K-GIASN’) and GSA (‘K-GSA’) media alone, as well as on water alone (‘K-W’). The “TO [mg/kg]” and T14 [mg/kg]” columns present the amount of bioavailable iron in the soil at the beginning (TO) and at the end of the experiment (T14), and the “T14 [%]” column presents the percentage change in iron bioavailability during the experiment.

Changes in iron bioavailability in soil Variant TO [mg/kg] T14[mg/kg] Tl 4 [%] M-GCI 50.62 71.79 41.82 K-GCI 47.58 59.86 25.80 M-GISA 53.15 72.84 37.04 K-GISA 48.60 60.32 24.13 M-CSA 52.14 69.51 33,32 K-CSA 52.14 60.43 15.90 M-GIASN 52.05 72.24 38.80 K-GIASN 47.96 59.00 23.03 M-GSA 50.41 69.52 37.92 K-GSA 47.96 59.00 23.03 K-W 46.63 49.22 5.55

The use of a metabolite solution containing siderophores had a positive effect on increasing iron bioavailability in the tested agricultural soil, which increased by over 30% compared to the initial value. This increase was significantly greater than when using the sterile medium control and water alone. Iron bioavailability was increased using both acidic (M-GCI, M-GSA, and M-GISA) and alkaline (M-CSA, M-GIASN) preparations, demonstrating that all newly developed media, i.e., GCI, GISA, GIASN, and CSA, regardless of their chemical nature, as well as the acidic GSA medium, enable the production of a metabolite solution that influences iron mobilization and increases iron bioavailability.

Example 3. Increasing the bioavailability of phosphorus in soil using metabolites produced by culturing the Pseudomonas H12B strain on GCI, GISA, CSA, GIASN and GSA media

This example examines the effect of metabolites produced by the Pseudomonas H12B strain on GCI, GISA, GIASN, CSA, and GSA culture media on increasing phosphorus bioavailability in soil. Cultivation of Pseudomonas sp. H12B on these media and preparation of metabolites were performed as in Example 1. The effect on phosphorus bioavailability was examined using:

(i) bacterial metabolites produced on GCI ("M-GCL"), GISA ("M-GISA"), CSA ("M-CSA"), GIASN ("M-GIASN") and GSA ("M-GSA") media (ii) sterile GCI ("K-GCI"), GISA ("K-GISA"), CSA ("K-CSA"), GIASN ("K-GIASN") and GSA ("K-GSA") media as chemical controls (iii) distilled water as an additional control ("K-W").

PL 247621 Β1

Metabolite activity was tested in environmental soil of agricultural origin similarly to Example 2, with the difference being the method of determining phosphorus content in the sample, which was performed photometrically. NANOCOLOR tube kits (Macherey-Nagel) were used for this purpose, and measurements were taken with a Pf-12 Plus photometer (Macherey-Nagel). The results are presented in Table 3.

Table 3

Change in phosphorus bioavailability in the tested soil as a result of adding a solution of metabolites produced on GCI ("M-GCI"), GISA ("M-GISA"), and CSA ("M-CSA"), GIASN ("M-GIASN") and GSA ("M-GSA") media, and on GCI ("K-GCI"), GISA ("K-GISA"), CSA ("K-CSA"), GIASN ("K-GIASN") and GSA ("K-GSA") media alone, as well as water alone ("K-W"). The columns "TO [mg/kg]" and "T14 [mg/kg]" present the amount of bioavailable phosphorus in the soil at the beginning (TO) and at the end of the experiment (T14), and the column "T14 [%]" presents the percentage change in phosphorus bioavailability during the experiment.

Changes in phosphorus bioavailability in soil Variant TO [mg/kg] T14 [mg/kg] T14 [%] M-GCI 14.95 18.28 22.31 K-GCI 13.82 15.56 12.61 M-GISA 14.10 18,20 29.08 K-GISA 13.82 15.04 8.84 M-CSA 13.54 16.92 25.00 K-CSA 13.96 14.60 4.59 M-GIASN 14.91 18.38 23.28 K-GIASN 14.01 15.56 11.13 M-GSA 15.32 18.54 21.03 K-GSA 13.17 14.62 10.95 K-W 15.54 15.76 1.44

As with iron bioavailability, the use of a metabolite solution containing siderophores had a positive effect on increasing phosphorus bioavailability in the tested agricultural soil. This increase was approximately 20-30% and was significantly greater than the control of sterile medium and water alone. Phosphorus bioavailability was also increased with both acidic (M-GCI, M-GSA, and M-GISA) and alkaline (M-CSA and M-GIASN) formulations. The results obtained in Examples 2 and 3 demonstrate that all newly developed substrates, i.e., GCI, GISA, GIASN, and CSA, regardless of their chemical nature, as well as the acidic GSA medium, enable the production of a metabolite solution that influences iron and phosphorus mobilization, thus improving soil chemical quality.

Example 4. Increasing the mobility of heavy metals in contaminated industrial soils using metabolites produced by culturing the Pseudomonas H12B strain on GCI, GISA, GIASN and CSA media

The presented example demonstrates the effect of metabolites produced by the Pseudomonas H12B strain on GCI, GISA, GIASN, and CSA culture media on increasing the mobility of heavy metal contaminants (zinc, copper, and nickel) in soil. This example intentionally omitted the GSA medium, whose production and use in heavy metal bioremediation

PL 247621 Β1 is the subject of patent application P.440597. The cultivation of Pseudomonas sp. H12B on the above-mentioned media and the preparation of metabolites were carried out as in Example 1. The effect on the mobility of heavy metals was examined using:

(i) bacterial metabolites produced on GCI ("M-GCI"), GISA ("M-GISA"), GIASN ("M-GIASN") and CSA ("M-CSA") media (ii) sterile GCI ("K-GCI"), GISA ("K-GISA"), GIASN ("K-GIASN") and CSA ("K-CSA") media as chemical controls (iii) distilled water as an additional control ("K-W").

The activity of metabolites and the determination of the mobility of elements were tested on industrial soil contaminated with heavy metals in the same way as in Example 2. The obtained results are presented in Table 4.

Table 4

Change in the mobility of zinc, copper, and nickel in the tested soil as a result of adding a solution of metabolites produced on GCI ("M-GCL"), GISA ("M-GISA"), GIASN ("M-GIASN"), and CSA ("M-CSA") substrates, as well as on GCI ("K-GCI"), GISA ("K-GISA"), GIASN ("K-GIASN"), and CSA ("K-CSA") substrates alone, and also on water alone ("K-W"). The columns "TO [mg/kg]" and "T14 [mg/kg]" present the amount of bioavailable element in the soil at the beginning (TO) and at the end of the experiment (T14), while the columns "T14 [%]" present the percentage change in the bioavailability of a given element during the experiment.

Variant Changes in zinc mobility Changes in copper mobility Change in nickel mobility TO [mg/kg] T14 [mg/kg] T14 [%] TO [mg/kg] T14 [mg/kg] T14 [%] TO [mg/kg] T14 [mg/kg] T14 [%] M-GCI 36.67 49.87 36.00 24.65 32.79 33.02 34.57 46.67 35.00 K-GCI 35.94 38.45 6.98 23.67 25.56 7.98 33.18 35.51 7.02 M-CSA 34.11 41.95 22.98 24.16 30.2 25.00 36.3 45.37 24.99 K-CSA 38.14 41.57 8.99 23.92 25.35 5.98 35.6 38.1 7.02 M-GISA 35.04 47.29 34.97 24.41 32.87 34.66 32.60 43.02 31.96 K-GISA 34.82 40.01 14.91 22.13 23.91 8.04 33.36 35.23 5.61 M-GIASN 32.45 40.11 23.61 24.65 30.09 22.07 33.80 41.64 23.20 K-GLASN 34.48 38.01 10.22 21.15 22.36 5.73 30.98 33.17 7.06 K-W 37.04 37.04 0 23.67 23.2 -1.99 35.26 34.55 -2.01

The mobility of heavy metals in soil was increased by the application of metabolite solutions containing siderophores. The increase in mobility was significantly greater for all preparations than for all control variants. The acidic solution (M-GCI and M-GISA) showed the greatest increase in elemental mobility, exceeding 30% for all metals tested. Alkaline metabolite solutions (M-CSA and M-GIASN) increased metal mobility to a slightly lesser extent (22-25%). This difference may be due to the acidic pH of the M-GCI and M-GISA preparations, which further increases the solubility of metals such as zinc, copper, and nickel. These results demonstrate the potential of using both alkaline and acidic metabolite solutions produced on newly developed substrates for the remediation of contaminated soils.

Example 5. Improving the biological quality of soil by increasing the number and activity of microorganisms using metabolites obtained by culturing the Pseudomonas H12B strain on GCl, GlSA and CSA, GlASN and GSA media

The presented example concerns the effect of metabolites produced by the Pseudomonas H12B strain on GCl, GISA, GlASN, CSA, and GSA culture media on increasing (a) the abundance and (b) the activity of soil microorganisms – dehydrogenase activity. Cultivation of Pseudomonas sp. H12B on these media and preparation of metabolites were performed as in Example 1.

The experiment examined:

(a) influence on the number of soil microorganisms (heterotrophs) by means of:

(i) bacterial metabolites produced on GCl (‘M-GCL’), GlSA (‘M-GlSA’), GlASN (‘M-GlASN’), GSA (‘M-GSA’) and CSA (‘M-CSA’) media (ii) sterile GCl (‘K-GCl’), GlSA (‘K-GlSA’), GlASN (‘K-GlASN’), GSA (‘K-GSA’) and CSA (‘K-CSA’) media as chemical controls (iii) distilled water as an additional control (‘W’).

The activity of metabolites was tested in environmental agricultural soil and industrial soil contaminated with heavy metals and organic compounds. Experimental conditions were analogous to those in Example 2. The number of microorganisms in all variants was determined in samples collected at the beginning and then on days 3, 5, 7, 10, 12, and 14 of the experiment. The method used was to plate dilutions of the soil extract onto plates with solid LB medium. 1 g of the collected soil sample was placed in a glass flask, and 10 ml of physiological saline (0.85% NaCl) was added and shaken (150 rpm) for 24 hours. The next day, dilutions were prepared from the obtained soil extract and plated onto plates with solid LB medium. After 2 days of incubation at room temperature, the grown colonies were counted and the number of heterotrophic microorganisms was calculated, expressed as CFU/ml. The results are presented in Table 5.

(b) influence on the activity of soil microorganisms (dehydrogenase activity) by means of:

(i) bacterial metabolites produced on GCl (‘M-GCL’), GlSA (‘M-GlSA’), GlASN (‘M-GlASN’), GSA (‘M-GSA’) and CSA (‘M-CSA’) media (ii) sterile GCl (‘K-GCl’), GlSA (‘K-GlSA’), GlASN (‘K-GlASN’), GSA (‘K-GSA’) and CSA (‘K-CSA’) media as chemical controls (iii) distilled water as an additional control (‘W’).

The activity of metabolites was tested on environmental agricultural soil and industrial soil contaminated with heavy metals and organic compounds. Experimental conditions were analogous to those in Example 2. Dehydrogenase activity was determined using the 2-(4-iodophenyl)-3-(4-nitrophenyl)-5-phenyltetrazolium chloride (INT) method. A soil sample (0.5 g) was placed in a test tube, and 2 ml of 0.2% INT solution was added. The sample was incubated in the dark with shaking for 2 hours. After this time, 4 ml of extraction solution (DMF : ethanol 1 : 1 (v/v)) was added, and the samples were incubated for another hour in the dark with shaking. After incubation, the samples were centrifuged (2 minutes/8000 rpm), and the absorbance of the supernatant was measured in a spectrophotometer at 464 nm. Measurements were taken at the beginning and end of the experiment (after 14 days). The results are presented in Table 5.

PL 247621 Β1

Table 5

Change in the number of heterotrophic microorganisms in the tested agricultural soil and industrial soil as a result of adding a solution of metabolites produced on GCI ("M-GCI"), GISA ("M-GISA"), GIASN ("M-GIASN"), GSA ("M-GSA") and CSA ("M-CSA") media, as well as on GCI ("K-GCI"), GISA ("M-GISA"), GIASN ("K-GIASN"), GSA ("K-GSA") and CSA ("K-CSA") media alone, and also on water alone ("K-W"). The following columns present the number at the beginning of the experiment and after 3, 5, 7, 10, 12 and 14 days

Number of heterotrophic microorganisms in agricultural soil Variant THIS T3 T5 T7 T10 T12 T14 M-GCI 3.45E+05 7.84E+05 l,60E+06 4.42E+06 8.71E+06 K43E+07 2.86E+07 K-GCI 3.52E+05 5.07E+05 l,06E+06 2.75E+06 5.13E+06 8.79E+06 l.85E+07 M-GISA 4.54E+05 7.76E+05 E55E+06 4.33E+06 8.71E+06 L50E+07 2.95E+07 K-G1SA 4.40EJ 05 5.12E 105 9.13E+05 2.63E and 06 5.64E and 06 9.42Et06 l,87Ei07 M-CSA 4.21E+05 7.76E+05 l,57E+06 4.46E+06 9.15E+06 l,52E+07 2.98E+07 K-CSA 3.96E+05 5.02E+05 9.32E+05 2.89E+06 5.62E+06 l,02E+07 l.96E+07 M-GLASN 4.27E+05 7.94E+05 l.45E+06 4.58E+06 9.74E+06 l,51E+07 3.02E+07 K-GLASN 3.92E+05 5.02E+05 9.68E+05 2.70E+06 5.08E+06 8.81E+06 l,86E+07 M-GSA 3.82E+05 7.48E+05 l,71E+06 4.80E+06 9.48E+06 l,39E+07 3.16E+07 K-GSA 3.96E+05 4.92E+05 9J0E+05 2J6E+06 5J4E+06 9J9E+06 C87E+07 K-W 3.10E+05 3.31E+O5 3.38E+05 3.24E+05 3 _; 55E+05 3.20E+05 3 _? 58E+05 Number of heterotrophic microorganisms in industrial soil Variant THIS T3 T5 T7 T10 T12 T14 M-GCI 6.15E+04 7.02E+04 l,02E+05 l,40E+05 4.10E+05 8.01E+05 8.55E+05 K-GCI 5.48E+04 7.02E+04 l,01E+05 l,32E+05 3.69E+05 7.53E+05 6.09E+05 M-CSA 6.03E+04 8.70E+04 l,30E+05 l,82E+05 5.31E+05 l,12E+06 l,05E+06 K-CSA 5.65E+04 7.08E+04 9.67E+04 l,28E+05 4.06E+05 8.14E+05 7.50E+05 M-GISA 6.39E+04 7.15E+04 l,24E+05 l,67E+05 4.42E+05 9.61E+05 l,20E+06 K-G1SA 5.18E+04 6.42E+04 9.49E+04 l.25E+05 3.76E+05 7.68E+05 6.25E+05 M-GLASN 5.72E+04 8.49E+04 l,16E+05 l.45E+05 4.89E+05 9.99E+05 l,05E+06 K-GLASN 5.62E+04 6.63E+04 9.79E+04 K22E+05 3.60E+05 7.84E+05 6.66E+05 M-GSA 5.54E+04 8.41E+04 l,19E+05 l.50E+05 5.18E+05 l ₅ 06E+06 9.91E+06 K-GSA 5.18E+04 6.35E+04 9.79E+04 1J7E+05 3.57E+05 7.60E+05 5.46E+05 K-W 5.15E+04 5.75E+04 6.49E+04 7.21E+04 6.59E+04 5.78E+04 5.97E+04

PL 247621 Β1

Table 6

Change in the activity of dehydrogenases in the tested agricultural soil and industrial soil as a result of adding a solution of metabolites produced on the GCI ("M-GCI"), GISA ("M-GISA"), GIASN ("M-GIASN"), GSA ("M-GSA") and CSA ("M-CSA") media and on the GCI ("K-GCI"), GISA ("M-GISA"), GIASN ("K-GIASN"), GSA ("K-GSA") and CSA ("K-CSA") media alone, as well as water alone ("K-W").

The following columns show the dehydrogenase activity at the beginning of the experiment, after 14 days and the percentage change in dehydrogenase activity.

Changes in dehydrogenase activity in agricultural soil Variant TO [nmol INTF/ml] Tl 4 [nmol INTF/ml] T14 |%] M-GCI 2,491 3,791 34.30 K-GCI 2,441 3,295 25.92 M-GISA 2,491 3,402 26.79 K-G1SA 2,441 2,957 17.45 M-CSA 2,466 3,611 31.72 K-CSA 2,466 3,206 23.10 M-GIASN 2,458 3,281 33.48 K-GIASN 2,449 3,027 23.57 M-GSA 2,334 2,833 21.38 K-GSA 2,400 2,684 11.83 K-W 2,491 3,791 34.30 Changes in dehydrogenase activity in contaminated soil Variant TO [nmol INTF/ml] T14 [nmol INTF/ml] T14 l%] M-GCI 5,126 7,882 53.76 K-GCI 5,280 7,015 32.85 M-CSA 5,376 7,419 38 K-CSA 5,178 6,708 29.56 M-GISA 5,409 7,804 44.28 K-G1SA 4,968 6,062 22.02 M-GIASN 5,834 8,656 48.37 K-GIASN 4,731 5,944 25.65 M-GSA 5,262 7,885 49.83 K-GSA 4,711 5,944 26.18 K-W 5,118 4,576 -10

Both the metabolite and sterile media application resulted in an increase in the number of microorganisms compared to the water control. This is due to the provision of nutrients that can be used by microorganisms for growth. However, the metabolite application resulted in a higher bacterial population growth rate in each treatment, and the determined number of microorganisms was 40-50% higher compared to the sterile media in all treatments and soils/grounds tested.

Dehydrogenase activity was increased by supplementing the soil with both sterile media and bacterial metabolites. However, the use of both acidic (M-GCl, M-GlSA, and M-GSA) and alkaline (M-CSA, M-GlASN) metabolites significantly increased dehydrogenase activity compared to the corresponding control variants. In agricultural soil, the use of all metabolites increased dehydrogenase activity by 8-10% compared to the control variant. For industrial soil, most substrates showed an increase in dehydrogenase activity of over 20% compared to the control variant, while in the case of the M-CSA substrate, the difference compared to the control variant was 8.5%, favoring the use of metabolites.

The results indicate that the use of newly developed substrates (both acidic and alkaline) and acidic GSA substrate is a significant factor stimulating the autochthonous soil microbiota in the context of agricultural soils and contaminated industrial lands, increasing both its abundance and activity.

Example 6. Increasing the germination efficiency of beet, pea and tobacco seeds using metabolites obtained by culturing the Pseudomonas H12B strain on GCl, GlSA, GlASN, CSA and GSA media

In the presented example, the effect of metabolite solutions produced on GCl, GlSA, GlASN, CSA, and GSA media on the germination of beet, pea, and tobacco seeds was examined. Pseudomonas sp. H12B was cultured on GCl, GlSA, GlASN, and CSA media and metabolite preparation was performed as in Example 1. Pseudomonas sp. H12B was cultured on GSA media and metabolite preparation was performed as described in patent application no. P.440597. The effect on the number of soil microorganisms was examined using:

(i) bacterial metabolites produced on GCl ("M-GCL"), GlSA ("M-GlSA"), GlASN ("M-GlASN"), GSA ("M-GSA") and CSA ("M-CSA") substrates. Metabolites for germination tests were diluted tenfold and then combined in a 1 : 1 (v/v) ratio with plant medium (KNOPP - 3 mM Ca(NO3)2, 1.5 mM KNO3, 1.2 mM MgSO4, 1.1 mM KH2PO4, 0.1 mM C10H12N2O8FeNa, 5 μM CuSO4, 2 μM MnSO4 χ 5 H2O, 2 μM ZnSO4 χ 7 H2O, 15 nM (NH4)6Mo7O2 χ 4 H2O) (ii) plant medium - KNOPP mixed with water in a 1 : 1 (v/v) ratio ("Control")

A 2-cm-high layer of lignin was placed in glass dishes (200 mm diameter). It was soaked with 125 ml of the metabolite/control solution appropriate for the given variant. An 18-cm-diameter filter paper disc was then placed on the lignin layer. Seeds were conditioned by soaking them for 30 minutes in 100 ml of a tenfold diluted solution of the appropriate metabolites. After this time, the excess liquid was poured off, and 100 seeds of a given species were placed on the filter paper placed in the dish. The seeds were incubated in the dark for 5 days at 20°C. The number of germinating seeds in each variant was counted on each day of the experiment. The results are presented in Table 7.

PL 247621 Β1

Table 7

Number of germinating beet, pea, and tobacco seeds after priming seeds with metabolite solutions produced on GCI ("M-GCI"), GISA ("M-GISA"), CSA ("M-CSA"), GSA ("M-GSA"), and GIASN ("M-GIASN") medium, as well as priming in Knopp medium alone ("Control"). The following columns present the number of germinating seeds at the beginning of the experiment and after 1, 2, 3, 4, 5, and 6 days of the experiment.

Number of seeds germinated Beetroot THIS Tl T2 T3 T4 T5 T6 M-GCI 0 5 44 68 79 79 80 M-GISA 0 5 46 60 68 75 78 M-CSA 0 5 51 68 77 84 84 M-GIASN 0 9 55 71 82 85 86 M-GSA 0 10 58 76 81 83 84 Control 0 5 44 64 70 72 75 Pea THIS Tl T2 T3 T4 T5 T6 M-GCI 2 6 50 98 98 98 98 M-GISA 0 2 31 86 100 100 100 M-CSA 0 0 39 97 100 100 100 M-GIASN 0 3 45 95 99 100 100 M-GSA 0 14 98 100 100 100 100 Control 0 0 12 62 95 99 100 Tobacco THIS Tl T2 T3 T4 T5 T6 M-GCI 0 0 0 70 95 96 96 M-GISA 0 0 2 86 95 95 95 M-CSA 0 0 0 63 70 83 83 M-GIASN 0 0 2 88 95 95 97 M-GSA 0 0 0 89 98 98 98 Control 0 0 2 70 94 94 94

The use of metabolites produced in the newly developed GCl, GISA, CSA, and GIASN media, as well as in GSA medium, accelerated the germination rate of all tested seeds compared to the Knopp control. In the case of pea, the first seeds began germinating one day earlier than in the control. In the case of beet seeds, a higher number of germinating seeds was observed in the experiment than in the control. In the case of tobacco seeds, a higher seed germination rate was observed in the experiment than in the control. The results indicate a positive effect of the tested metabolites on the rate and efficiency of germination, regardless of the initial pH of the metabolites produced. The studies also indicate a lack of toxic effects of the metabolites on plants.

Example 7. Increasing the efficiency of remediation of soils contaminated with organic compounds using metabolites produced by culturing the Pseudomonas H12B strain on GCl, GISA, GlASN, CSA and GSA media

The presented example uses the results obtained in Example 5.

The effectiveness of microbial bioremediation of soils contaminated with organic compounds is directly and positively related to the abundance and activity of soil microbiota. Appropriate stimulation of microorganisms, for example, through soil supplementation, increases the abundance of indigenous bacteria, which results in an increased rate of biodegradation of organic compounds, which are sources of important bioelements, such as carbon. This increased biodegradation rate is reflected in the activity of dehydrogenases, as these enzymes are essential for the decomposition of organic compounds. As shown in Example 5, supplementation of contaminated industrial soils with metabolites produced on GCl, GlSA, GlASN, CSA, and GSA substrates both increases the abundance of microorganisms in the soil and stimulates their activity. Therefore, metabolites can be used in various bioremediation treatments to support the effective biodegradation of organic pollutants in soil.

Claims (12)

1. A composition containing siderophores, characterized in that the siderophores are produced by the Pseudomonas H12B strain deposited under number B/00321 in the Polish Collection of Microorganisms of the Institute of Immunology and Experimental Therapy of the Polish Academy of Sciences in Wrocław, Poland, intended for: a) improving the microbiological quality of the soil, b) increasing the bioavailability of micro- and macronutrients necessary for the proper growth and development of soil microorganisms and plants, such as iron and phosphorus, c) accelerating the biodegradation of petroleum compounds, useful in the processes of cleaning up contaminated land or d) promoting the germination of crop plants. 2. The composition according to claim 1, characterized in that it is obtained by culturing the Pseudomonas H12B strain on a substrate selected from GCl, GlSA, CSA or GlASN and is additionally intended to increase the mobility of heavy metals in soil, useful in the processes of cleaning up contaminated land, wherein: - GCl medium has the following composition: glucose 7 g/l, NH4Cl 1.35 g/l, Na2HPO4 0.96 g/l, KH2PO4 0.44 g/l, MgSO4 X 7H2C 0.2 g/l; pH 7.0; - GlSA medium has the following composition: glycerol 50% 3.5 g/l, (NH4)2SO4 1 g/l, Na2HPO4 0.96 g/l, KH2PO4 0.44 g/l, MgSc4 χ 7H2O 0.2 g/l; pH 7.0; - CSA medium has the following composition: citric acid 4 g/l, (NH4)2SC4 1 g/l, Na2HPC4 0.96 g/l, KH2PC4 0.44 g/l, MgSC4 χ 7H2C 0.2 g/l; pH 7.0; - GlASN medium has the following composition: glycerol 50% 3.5 g/l, L-asparagine 2 g/l, Na2HPC4 0.96 g/l, KH2PC4 0.44 g/l, MgSC4 χ 7H2C 0.2 g/l; pH 7.0. 3. The composition according to claim 1, characterized in that it is obtained by culturing the Pseudomonas H12B strain on a GSA medium. 4. The composition according to claim 1, characterized in that the improvement of the microbiological quality of the soil comprises an increase in the number of soil microorganisms - heterotrophic bacteria. 5. The composition of claim 1, wherein the improvement in the microbiological quality of the soil comprises increasing the activity of dehydrogenases of soil microorganisms. 6. The composition according to claim 1, characterized in that the crop plants are selected from beet, pea and tobacco. 7. A method for producing a composition containing siderophores as defined in claim 1, characterized in that it comprises the following steps: a) an inoculum of the Pseudomonas sp. ANT_H12B bacterial strain, deposited under number B/00321 in the Polish Collection of Microorganisms of the Institute of Immunology and Experimental Therapy of the Polish Academy of Sciences in Wrocław, Poland, is obtained on a liquid complete medium intended for the multiplication of bacterial cells, preferably LB medium, cultivating the bacteria at a temperature of 20-25°C until the logarithmic phase of growth is achieved; b) the culture obtained in step a) is centrifuged and the cell biomass is then suspended in a physiological saline solution, preferably centrifuged twice; c) inoculating the culture medium selected from: GCl, GlSA, CSA or GlASN with the bacterial suspension obtained in point b), preferably in an amount giving an initial optical density of the culture of 0.06, measured at a wavelength of 600 nm; d) the bacterial culture obtained in step c) is carried out at a temperature of 10-20°C; e) the culture broth obtained in point d) is centrifuged and the supernatant is separated, thus obtaining a composition containing siderophores; wherein: - as the GCl medium, the following medium is used: glucose 7 g/l, NH4Cl 1.35 g/l, Na2HPO4 0.96 g/l, KH2PO4 0.44 g/l, MgSO4 x 7H2O 0.2 g/l; pH 7.0; - as the GlSA medium, the following medium is used: glycerol 50% 3.5 g/l, (NH4)2SO4 1 g/l, Na2HPO4 0.96 g/l, KH2PO4 0.44 g/l, MgSO4 x 7H2O 0.2 g/l; pH 7.0; - as a CSA medium, the following medium is used: citric acid 4 g/l, (NH4)2SO4 1 g/l, Na2HPO4 0.96 g/l, KH2PO4 0.44 g/l, MgSO4 x 7H2O 0.2 g/l; pH 7.0; - as the GlASN medium, the following medium is used: glycerol 50% 3.5 g/l, L-asparagine 2 g/l, Na2HPO4 0.96 g/l, KH2PO4 0.44 g/l, MgSO4 x 7H2O 0.2 g/l; pH 7.0. 8. The method according to claim 8, characterized in that the bacterial culturing in step d) is carried out for 48-72 hours. 9. The method according to claim 8, characterized in that the bacterial culturing in step d) is carried out with shaking at 120-150 rpm. 10. The method according to claim 8, characterized in that in step c) a GCl medium or a GlSA medium is used as a culture medium for the production of a composition containing siderophores with acidifying properties. 11. The method according to claim 8, characterized in that in step c) a CSA substrate or a GlASN substrate is used as a substrate for preparing the composition containing siderophores with alkalizing properties. 12. Use of siderophores produced by the Pseudomonas H12B strain deposited under number B/00321 in the Polish Collection of Microorganisms of the Institute of Immunology and Experimental Therapy of the Polish Academy of Sciences in Wrocław, Poland, to improve the microbiological quality of soil, increase the bioavailability of micro- and macronutrients necessary for the proper growth and development of soil microorganisms and plants, such as iron and phosphorus; accelerate the biodegradation of petroleum compounds, useful in the processes of cleaning up contaminated land or promoting the germination of crop plants.