Method and use for increasing the production yield of a plant being cultivated in a hydroponic system

Abstract

The present invention relates to a method for increasing the production yield of a plant being cultivated in a hydroponic system, comprising (a) adding biochar into the hydroponic system, wherein the biochar is coated with at least one microorganism that along with the biochar promotes the growth of the plant, and (b) culturing the plant in the hydroponic system (a) to obtain products from the plant as well as the use of biochar being coated with at least one microorganism that along with the biochar promotes the growth of a plant for increasing the production yield of the plant when grown in a hydroponic system.

Description

[0001] Method and use for increasing the production yield of a plant being cultivated in a hydroponic system

[0002] The present invention relates to a method for increasing the production yield of a plant being cultivated in a hydroponic system, comprising (a) adding biochar into the hydroponic system, wherein the biochar is coated with at least one microorganism that along with the biochar promotes the growth of the plant, and (b) culturing the plant in the hydroponic system (a) to obtain products from the plant as well as the use of biochar being coated with at least one microorganism that along with the biochar promotes the growth of a plant for increasing the production yield of the plant when grown in a hydroponic system.

[0003] In this specification, a number of documents including patent applications and manufacturer's manuals are cited. The disclosure of these documents, while not considered relevant for the patentability of this invention, is herewith incorporated by reference in its entirety. More specifically, all referenced documents are incorporated by reference to the same extent as if each individual document was specifically and individually indicated to be incorporated by reference.

[0004] Hydroponic cultivation, an innovative method of growing plants without soil, leverages nutrient-rich water solutions to deliver macro- und micronutrients directly to plant roots. By eliminating soil, hydroponics offer precise control over growing conditions, leading to faster growth rates and higher yields (Fussy & Papenbrock 2021, Plants 11(9), 1153. DOI: 10.3390 / plantsll091153).

[0005] A hydroponic system is a system of growing plants using mineral solution, in water, without soil. This can be extremely helpful to countries that have poor land, which is not able to sustain agriculture. Due to rapid urbanization and industrialization as well as rising sea levels, for example by melting of icebergs (as an obvious impact of global warming), arable land under cultivation is further going to decrease. Soil fertility status may have attained a saturation level, and productivity may not increase further with an increased level of fertilizer application. Besides, poor soil fertility in some of the cultivable areas, less chance of natural soil fertility buildup by microbes due to continuous cultivation, frequent drought conditions and unpredictability of climate and weather patterns, rise in temperature, river pollution, poor water management and wastage of huge amount of water, decline in ground water level, etc. are threatening food production under conventional soil-based agriculture. Hence, in near future it might become challenging to feed the entire population of the world based on soil-based agriculture only (see Jain et al. (2019), book: Recent Advances in Chemical Sciences & Biotechnology; Publisher: New Delhi Publication, Chapter 11: A review on hydroponic system: hope and hype).

[0006] For the above reasons there is an urgent need for a method and use for the efficient production of plants being cultivated in a hydroponic system. This need is addressed by the present invention.

[0007] Accordingly, the present invention relates in first aspect to a method for increasing the production yield of a plant being cultivated in a hydroponic system, comprising (a) adding biochar into the hydroponic system, wherein the biochar is coated with at least one microorganism that along with the biochar promotes the growth of the plant, and (b) culturing the plant in the hydroponic system (a) to obtain products from the plant.

[0008] The present invention relates in a second aspect the use of biochar being coated with at least one microorganism that along with the biochar promotes the growth of a plant for increasing the production yield of the plant when grown in a hydroponic system.

[0009] It is to be understood that depending on the nature of plant that is to be used in connection with the first and second aspect of the nature of the desired production yield and products from the plant may be different. For instance, in the case crop plants either seedlings may be produced in order to sustain and / or further expand the overall production, or mature plants from which grain, corn, another food or metabolites can be harvested. Hence, the increase in production yield can be, for example, an increase of plant biomass, of fruits and / or seeds, or of plant metabolites.

[0010] The nature of the plant to be employed is not particularly limited. Preferred but non-limiting examples of plants will be described herein below.

[0011] As mentioned, as used herein a hydroponic system is a system of growing plants using mineral solution, in water, without soil. Preferred but non-limiting examples of hydroponic systems will be provided herein below. Hydroponic nutrient solutions are designed to provide macronutrients, including nitrogen, phosphorus, sulfur, potassium, calcium, and magnesium as well as micronutrients, like boron, zinc, manganese, iron among others. By delivering these nutrients directly to the roots of the plants, hydroponic systems allow for more efficient absorption and faster growth than on soil. Nitrogen is responsible for promoting healthy leaf growth, as well as aiding in the production of proteins and enzymes. Phosphorus plays a crucial role in root development and flower formation. Sulfur helps with protein synthesis and enzyme activity. Potassium is needed for the overall plant health and disease resistance. Calcium is essential for cell wall structure and stability. Magnesium is involved in chlorophyll production.

[0012] Micronutrients that are preferably additionally used in hydroponic systems include iron, manganese, zinc, copper, boron, molybdenum, and chlorine. Each of these elements plays a vital role in the plant's metabolic processes. Iron (Fe) is essential for chlorophyll production and energy transfer within cells, Manganese (Mn) is involved in enzyme activation and helps plants withstand stress conditions. Zinc (Zn) is needed for the synthesis of growth hormones and promotes root development. Copper (Cu) aids in the formation of lignin, which strengthens cell walls. Boron (B) influences cell division and carbohydrate metabolism. Molybdenum (Mo) is necessary for nitrogen fixation and helps convert nitrate into ammonia. Chlorine (Cl) is involved in photosynthesis and osmotic regulation.

[0013] Hence, a hydroponic system as used herein preferably employs a nutrient solution that comprises solved N, P, K, Ca, Mg and S and preferably further one or more of solved Fe, Mn, Zn, Co, B, Mo and Cl. The one or more of solved Fe, Mn, Zn, Co, B, Mo and Cl are with increasing preference two or more, three or more, four or more, five or more, six or more or all seven of Fe, Mn, Zn, Co, B, Mo and Cl.

[0014] Biochar is the lightweight black residue, consisting of carbon and ashes, remaining after the pyrolysis of biomass. Preferred sources of the biomass will be provided herein below. Biochar-based fertilizers are used in the art for plant growth promotion and protection (see for review: Agarwal et al. (2022), Biotech, 12:136 and Martinez-Gomez et al. (2022), Front. Plant Sci., Vol 13). Also the possibility to coat biochar with at least one microorganism for conventional soil-based agriculture is known from the prior art (DE102022206064).

[0015] The at least one microorganism coating of the biochar is not particularly limited as long as it helps the biochar to promote the growth of the plants.

[0016] The surface area of biochar is porous, and the microorganism can be placed into these pores, thereby coating the biochar. This can be achieved, for example, by incubating the microorganism with the biochar in a suspension. While the beneficial effects of biochar on conventional soil-based agriculture are well-established, the present invention specifically uses biochar in hydroponic systems that do not use soil. In addition, the biochar for hydroponic systems is coated with at least one microorganism which further ensures that the plants grow well in the hydroponic system, especially in early phases of seedling adaptation to the hydroponic environment. Biochar, a carbon-rich material produced from organic matter through pyrolysis, can be used as an additive to hydroponic substrates (Hansen et al. (2023), J Environ Manage 348, 119271. https: / / doi.Org / 10.1016 / j.jenvman.2023.119271). Biochar has stable electrical conductivity (EC) properties, which help to maintain ionic balance in hydroponic systems and facilitate efficient nutrient uptake by plants (Awad et al. (2017), J Clean Prod, 156, 581-588). Its porous structure improves water retention, relevant in substrate-based cultivation systems, and provides a habitat for beneficial microorganisms (Downie et al. (2012), Physical properties of biochar. In Biochar for environmental management (pp. 45-64). Routledge). These microorganisms can enhance nutrient availability, suppress pathogens, and promote plant health, for example by releasing hormones, organic acids and other metabolites. It is demonstrated in the appended examples that combining hydroponics with biochar and microorganisms creates a synergistic environment where plants thrive. The biochar improves and stabilizes the chemical properties of the growing medium, while microorganisms contribute to nutrient availability and cycling, and disease prevention. This integrated approach not only boosts plant growth and productivity but also enhances the sustainability and resilience of hydroponic systems. In the appended examples several plant species were cultivated in different hydroponic systems to test the influence of biochar and microorganisms, different bacteria and fungi, on plant growth. Different experiments were conducted exemplary with several promising plant species: the glycophytes water cress (Nasturtium officinale), mangold (Beta vulgaris var. Lucullus), strawberry (Fragaria x ananassa 'Mara Des Bois') and white cabbage (Brassica oleracea var. Capitata) were grown in nutrient film technique (NFT) system. The halophyte Salicornia europaea cultivated in a nutrient solution using a deep water culture hydroponics system, and for comparison in substrate, with and without biochar. The presence of biochar coated with at least one microorganism species in the nutrient solution improves plant growth drastically, especially during establishment of the culture in the adaptation phase. As can be seen from the composition of the analyzed plant material biochar coated with at least one microorganism species induces a better availability and therefore uptake of macro- and micronutrients. The increase in biomass by the addition of coated biochar was found to be statistically significant for all tested plant species, with p-values indicating high levels of significance (p < 0.05 to p < 0.0001). Mangold exhibited the greatest percentage increase in biomass (1076%), followed by white cabbage (707%), and strawberry (310%) (Figure 3). Next to the promotion of the growth of a plant for increasing the production yield of the plant, it was found that when grown in a hydroponic system in accordance with the method of the invention also the contents of specialized molecules, such as phenolic compounds, gluosinolates and other metabolites increase in the plants. Specialised molecules (also called specialised metabolites or secondary metabolites) are organic compounds produced by lifeforms, e.g. bacteria, archaea, fungi, animals, or plants, which are not directly involved in the normal growth, development, or reproduction of the organism. Instead, they play crucial ecological roles, such as defense against herbivores or pathogens, or in mediating interactions with the environment.

[0017] Based on the results obtained with different types of soil and plant growth substrates it was observed that highly powdered versions of biochar support growth of plants cultivated in hydroponics systems without blocking pumps and tubes.

[0018] In accordance with a preferred embodiment of the first and second aspect of the invention the plant is a vegetable plant, a horticultural plant, a medicinal and spice plant, a fruit plant or a crop plant.

[0019] In accordance with a more preferred embodiment of the first and second aspect of the invention the vegetable plant species is / are carrot, broccoli, spinach, tomato, bell pepper, cucumber, cauliflower, zucchini, onion, potato, green bean, pea, eggplant, sweet potato, cabbage, radish, beet, asparagus, artichoke, lettuce, water cress or celery or a combination thereof; the horticultural plants species is / are begonias, orchids, iris, roses or a combination thereof; the medicinal and spice plant species is / are basil, cannabis, nasturtium, sage, mints, parsley, thyme, rosemary or a combination thereof; the fruit plant species is / are apple, banana, orange, lemon, lime, melon, watermelon, grape, grapefruit, plum, mango, pineapple, kiwi, peach, avocado, blueberry, blackberry, strawberry, raspberry, mandarin, durian, passion fruit, persimmon, dragon fruit, papaya, pomegranate or a combination thereof; and / or the crop plant species is / are quinoa, amaranth, sugarcane, maize, rice, hop, oat, sorghum, millet, barely, rye, canary grass, wheat or a combination thereof.

[0020] The above plants are preferred because they are of commercial interest. Vegetable plants, medicinal and spice plants, fruit plant and crop plants are food plants and / or plants of medical interest. In particular for these plants high production yields are needed.

[0021] In accordance with a preferred embodiment the biochar has been obtained from sewage sludge, wood chips, and other sources by pyrolysis, preferably at 700°C to 850°C for lh to 2h without air and optionally by crushing the biochar into particles.

[0022] Examples of other sources are shells of plant seeds and kernels, preferably nut shells, switchgrass, miscanthus, corn, end-of-life biomass (ELB) such as animal manure, garden waste, forestry and agricultural residues.

[0023] Sewage sludge is preferred as source of biomass for biochar but other sources are also suitable. Sewage sludge can be produced in the treatment of household or municipal waste, and can be industrial sewage.

[0024] Pyrolysis is the thermal decomposition of materials at elevated temperatures, often in an inert atmosphere. Pyrolysis differs from other processes like combustion and hydrolysis in that it usually does not involve the addition of other reagents such as oxygen (O2, in combustion) or water (in hydrolysis).

[0025] The biochar is preferably crushed into particles by milling the biochar. The particle size can be adjusted as needed, for example, by milling and / or sieving the biochar (e.g. on a 0.315 mm mesh).

[0026] In accordance with a further preferred embodiment the biochar is made of particles having a diameter of 1 mm or less, preferably 0.8 mm, more preferably less or 0.5 mm and most preferably between 0.2 mm and 0.4 mm.

[0027] The particle size of biochar is to expose the inner pores and their functional groups to enhance lead adsorption The above small particle sizes are particular suitable for the water-based hydroponic system while.

[0028] In accordance with another preferred embodiment the weight ratio of coated biochar and solution in the hydroponic system is between 0.5 / 100 to 5 / 100 by weight, preferably between 1 / 100 and 3 / 100 by weight and most preferably about 2 / 100.

[0029] The term "about" as used herein means with increasing preference ±20%, ±10% and ±50%. The weight ratio of coated biochar and solution in the hydroponic system ensures that enough biochar is present for increasing the production yield of a plant being cultivated in a hydroponic system. In accordance with a preferred embodiment the at least one microorganism is or comprises a bacterium and / or a fungus, preferably a bacterium and a fungus.

[0030] In accordance with a more preferred embodiment the bacterium is selected from Azotobacter chroococcum, Azospirillum brasilense, Bacillus velezensis, Bacillus megaterium, Bacillus subtilis, Paenibacillus spp., Pseudomonas spp., Serratia spp., Sinorhizobium meliloti, Gluconacetobacter diazotrophicus, and Pseudomonas fluorescens; and / or the fungus is selected from Serendipita indica, Talaromyces allahabadensis and Trichoderma simmonsii, and other Trichoderma species.

[0031] The bacterium is most preferably Pseudomonas fluorescens as used in the appended examples.

[0032] The combination of particular microorganisms with crop plants were shown to be particularly suitable: Paenibacillus spp. / maize, Talaromyces allahabadensis / maize and barley, Pseudomonas fluorescens for all vegetable and horticultural plant species. Hence, for maize Paenibacillus spp. and Talaromyces allahabadensis are most preferred.

[0033] In accordance with a preferred embodiment the hydroponic system is a static solution culture, a continuous-flow solution culture, nutrient film technique culture (NFT), deep flow technique (DFT) culture, aeroponics culture, fogponics culture, passive sub-irrigation culture, ebb and flow subirrigation culture, run-to-waste culture, deep water culture, or top-fed deep-water culture.

[0034] While hydroponic systems can be set up in different ways it is believed that the biochar according to the invention can be used in all formats of hydroponic systems for increasing the production yield of a plant being cultivated in the respective hydroponic system. The above hydroponic systems are nonlimiting but preferred examples.

[0035] In accordance with a preferred embodiment the hydroponic system does not comprise any other growing support materials than the biochar. However, future results could also involve additional inert material.

[0036] As discussed above, the biochar alone is sufficient for increasing the production yield of a plant being cultivated in a hydroponic system.

[0037] In accordance with a preferred embodiment the plant is water cress, mangold, strawberry or white cabbage and the at least one microorganism is Pseudomonas fluorescens.

[0038] For water cress, mangold, strawberry and white cabbage Pseudomonas fluorescens proved to be particularly suitable; see appended examples.

[0039] In accordance with a preferred embodiment the at least one microorganism along with the biochar promotes the growth of the plant by increasing the availability of nutrients, in particular phosphate for the plant in the hydroponic system, stabilizes the pH and the electrical conductivity.

[0040] Phosphorus is one of the most frequently used fertilizers worldwide; German agriculture alone uses 115,000 tons annually (BMLEH, recent publications, https: / / www.bmel.de / EN / topics / farming / plant- production / fertilisation.html). While natural phosphorus reserves are shrinking biochar can be used as source of nutrients, in particular phosphate. The at least one microorganism helps to release the phosphate load from the biochar, so that it becomes accessible to the plants. Further technical advantages of the at least one microorganism along with the biochar are pH stabilization and electrical conductivity stabilization.

[0041] In accordance with a related preferred embodiment the at least one microorganism along with the biochar reduces the amount of mineral fertilizer applied in the hydroponic system.

[0042] Since the biochar is available as source of nutrients, in particular phosphate, the amount of mineral fertilizer applied in the hydroponic system; i.e. generally as solved mineral fertilizer in the solution of the hydroponic system can be reduced.

[0043] In accordance with a preferred embodiment comprises prior to step (a)

[0044] (a') coating biochar with at least one microorganism that along with the biochar promotes the growth of the plant by mixing a liquid medium with the at least one microorganism with the biochar and drying the biochar, preferably at about 18°C to about 22°C and / or to a final moisture of 0.4% to 1.5% water and minimum of 106colony forming units (CFU) for bacteria and spore-forming fungi or respective amount of fungal mycelium per kg of biochar, and

[0045] (a") optionally prior to (a') producing biochar by pyrolyzing sewage sludge, wood ships or other sources preferably at 700°C to 850°C for 1 and 2h without air and optionally crushing the biochar into particles.

[0046] Herein above the characteristics of the biochar according to the invention have been described. The above preferred embodiment comprises the preparation of this biochar as active steps of the claimed methods. Hence, the preferred characteristics of the biochar such a particle sizes or the nature of the at least one microorganism also apply to this preferred embodiment.

[0047] In accordance with a preferred embodiment, the biochar being coated with at least one microorganism that along with the biochar promotes the growth of the plant is biochar (preferably having a particle size of about 0.3 mm) being coated with Pseudomonas fluorescens (preferably at least at a CFU (colony forming units) of 5 x 108per g).

[0048] The above preferred embodiment and the below more preferred embodiments are particularly suitable for increasing the production yield of water cress, mangold, strawberry and white cabbage; see appended examples. Mangold and white cabbage are more preferred and mangold is most preferred.

[0049] The term "about" as used herein is with increasing preference ±20%, ±10% and ±5%.

[0050] In microbiology, a colony-forming unit (CFU, cfu or Cfu) is a unit which estimates the number of microbial cells (bacteria, fungi etc.) in a sample that are viable, and hence able to multiply. Counting with colony-forming units requires culturing the microbes and counts only viable cells, in contrast with microscopic examination which counts all cells, living or dead.

[0051] In accordance with a more preferred embodiment, the method comprises prior to step (a) keeping the biochar under an aluminum sheet and heat sterilization of the biochar at 1809C for 90 min, and cooling down the biochar before taking of the aluminum sheet and coating the biochar with with Pseudomonas fluorescens.

[0052] Covering the tray with aluminum foil is advantageous as it limits the availability of air / oxygen.

[0053] In a further preferred embodiment, the biochar is (homogeneously) coated with Pseudomonas fluorescens by homogeneously mixing the biochar with the Pseudomonas fluorescens, and evenly spreading the biochar in trays, and allowing the biochar to dry, preferably for 48 h under a laminar flow hood, preferably having an airflow ranging from about 0.3 m / s to about 0.5 m / s.

[0054] Drying is preferably stopped when the moisture content of the biochar is about 10%.

[0055] In accordance with a yet further more preferred embodiment the biochar is added into the hydroponic system by mixing the biochar into the nutrient solution of the hydroponic system.

[0056] The nutrient solution is preferably set to and maintained with an EC value of 1.6 mS cm1. Necessary fertilization is preferably provided on bi-weekly basis (until the harvest of the plant).

[0057] The average nutrient solution flow of the hydroponic system is preferably about 7.67 ml / s (volume flow) or 0.61 cm / s (velocity). An average pH of about 7.5 is preferably maintained in the hydroponic system throughout the method.

[0058] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skills in the art to which this invention belongs. In case of conflict, the patent specification including definitions, will prevail.

[0059] Regarding the embodiments characterized in this specification, in particular in the claims, it is intended that each embodiment mentioned in a dependent claim is combined with each embodiment of each claim (independent or dependent) said dependent claim depends from. For example, in case of an independent claim 1 reciting 3 alternatives A, B and C, a dependent claim 2 reciting 3 alternatives D, E and F and a claim 3 depending from claims 1 and 2 and reciting 3 alternatives G, H and I, it is to be understood that the specification unambiguously discloses embodiments corresponding to combinations A, D, G; A, D, H; A, D, I; A, E, G; A, E, H; A, E, I; A, F, G; A, F, H; A, F, I; B, D, G; B, D, H; B, D, I; B, E, G; B, E, H; B, E, I; B, F, G; B, F, H; B, F, I; C, D, G; C, D, H; C, D, I; C, E, G; C, E, H; C, E, I; C, F, G; C, F, H; C, F, I, unless specifically mentioned otherwise.

[0060] Similarly, and also in those cases where independent and / or dependent claims do not recite alternatives, it is understood that if dependent claims refer back to a plurality of preceding claims, any combination of subject-matter covered thereby is considered to be explicitly disclosed. For example, in case of an independent claim 1, a dependent claim 2 referring back to claim 1, and a dependent claim 3 referring back to both claims 2 and 1, it follows that the combination of the subject-matter of claims 3 and 1 is clearly and unambiguously disclosed as is the combination of the subject-matter of claims 3, 2 and 1. In case a further dependent claim 4 is present which refers to any one of claims 1 to

[0061] 3, it follows that the combination of the subject-matter of claims 4 and 1, of claims 4, 2 and 1, of claims

[0062] 4, 3 and 1, as well as of claims 4, 3, 2 and 1 is clearly and unambiguously disclosed.

[0063] This also holds true for alternatives in different claims that depend from each other. Thus, if claim 1 recites three alternatives of the same category and claim 2 recites three alternatives of a different category as recited in claim 1, and refers back to claim 1, all combinations of the alternatives as recited in claims 1 and 2 are explicitly disclosed herein.

[0064] The above considerations apply mutatis mutandis to all appended claims.

[0065] The figures show.

[0066] Figure 1: Pictures of strawberry in the hydroponic system after 4 and 9 weeks.

[0067] Figure 2: Fresh weight and dry weight of water cress with different amounts of biochar (BC) and microorganism (MO).

[0068] Figure 3: Effect of P3 treatment on the dry weight biomass of (A) Mangold (n=12), (B) white Cabbage (n=4), and (C) Strawberry (n=5). Plants were cultivated in a nutrient film technique (NFT) hydroponics system in greenhouses. The system consisted of a nutrient solution container (60 L) with an EC value of 1.6 mS cm-1using Ferty Mega 2. Control groups received 100% nutrition, while P3 treatment groups received an additional 3% (v / v) biochar inoculated.

[0069] The examples illustrate the invention.

[0070] Example 1 - Material and Methods

[0071] Plant material

[0072] For the experiments plant species from different sources were used: Strawberry (Fragaria x ananassa 'Mara Des Bais') plants were obtained from Hornbach, Germany, water cress (Nasturtium officinale) was originally obtained from Steffen Gbckemeyer, Poggenhagen, and further propagated in the greenhouse, seeds of mangold (Beta vulgaris var. Lucullus) and white cabbage (Brassica oleracea var. capitataf alba), seed type Langendjiker Dauer, were bought from Weigelt Samen (product no. 3780, Grolsheim, Germany). Seeds of Salicornia europaea L. var. aprica were obtained from Serra Maris bvba, Belgium. The seeds were sown and stored at 4°C for two days to boost germination.

[0073] Cultivation of microorganisms

[0074] For cultivation of P. fluorescens DSM 23471 inoculant, 1.8 L of CASO broth (Carl Roth, Germany, Art. No. X938.1) was autoclaved in 3 L conical flasks. The autoclaved media was inoculated at OD600 0.1 using a primary culture of P. fluorescens. The inoculant was further incubated at 30gC on a shaker (KS 4000 i control, I KA, Freiburg, Germany) at 180 rpm for 18 h. After 18 h, colony forming units (CFU) of the inoculant were quantified

[0075] Production of biochar

[0076] The foundational material for the biochar is dry sewage sludge. The sewage sludge was collected from the Danish wastewater treatment plant Farevejle. The sewage sludge was dried at 120 °C and then pyrolyzed at controlled temperature of 665°C in an in-house slow pyrolysis continuous screw reactor at Aquagreen, Denmark (https: / / aquagreen.dk / ) to ensure uniform heating and treatment. This high- temperature treatment ensures the complete elimination of contaminants and microorganisms, resulting in a sanitized and stable biochar. The samples came from the same batch to secure consistency in its property. The process also balances the pH of the resulting biochar. Before the biochar was used for the pot experiment, the biochar was milled on a cross-beater mill (Retsch SKI, Haan, Germany), followed by sieving on a 0.315 mm mesh. This fine particulate nature allows for a larger surface area, facilitating effective microbial inoculation. Specific microorganisms are introduced onto the surface of the biochar, with the selection of MO tailored to suit different crops and environmental conditions, such as marginal soils and regions with high salt or sand concentrations. The inoculated biochar is then subjected to a controlled drying process under sterile conditions to ensure the preservation of microbial viability and product integrity. Hansen et al. (2023), J Environ Manage 348, 119271. https: / / doi.Org / 10.1016 / j.jenvman.2023.119271 have reported the physicochemical characteristics of the biochar used. pH measurement of biochar

[0077] The pH of biochar samples was measured by suspending 5.0 g (ground to < 2 mm) biochar in deionised water in a 1:10 ratio (Singh et al. (2017), Biochar pH, electrical conductivity and liming potential. Biochar: A guide to analytical methods, 23.). After 1 hour of shaking, suspension was allowed to stand for 30 minutes before pH measurements were taken using pH meter (Dostmann pH 50, Rodgau, Germany) calibrated using pH 7 and pH 10 buffers.

[0078] Heat sterilization and inoculation of biochar

[0079] A total of 1.8 kg of finely sieved biochar (0.3 mm particle size) was distributed in two metallic trays (24 cm diameter) each consisting of 900 g. Trays were covered with aluminum foil and later heat sterilized at 180gC for 90 min. After 90 min, biochar was cooled down at room temperature while keeping the aluminum sheet covered on the trays. Covering the tray with aluminum foil is crucial as it limits the availability of air / oxygen. After cooling down freshly prepared P. fluorescens culture was poured in 1:1 ratio (v / w) in each tray. Biochar was homogeneously mixed with the MOs and evenly spread in the trays and allowed to dry for 48 h under the laminar flow hood with airflow ranging from 0.3 m / s to 0.5 m / s (Thermo Scientific Safe S2020 1.8 Safety Cabinet, Langenselbold, Germany). When moisture content of biochar was below 10%, CFU of P. fluorescens was quantified on the surface of biochar. This mixture is called Plantilizer prototype (P3) (with at least a CFU of 5 x 108per g).

[0080] Germination of seeds / propagation of cuttings

[0081] Seeds of Strawberry were germinated in soil (Einheitserde TOY 800 Art. No. 101025) for 4 weeks and then transferred to the hydroponic culture. Seeds of mangold were germinated in soil (Einheitserde TOY 800 Art. No. 101025) and transferred in hydroponics system at the age of 2 weeks. Seeds of white cabbage were germinated for 2 weeks in soil (Einheitserde TOY 800 Art. No. 101025) and then transferred to the hydroponic culture. Cuttings from water cress (Nasturtium officinale) were propagated in the greenhouse. Propagated cuttings were transferred into the hydroponics systems once size of 10 to 15 cm was reached. Cuttings of Cannabis sativa mother plants were propagated in a climatic chamber and transferred into hydroponics system once a size of 8 to 10 cm was reached.

[0082] Seeds of S. europaea L. var. aprica were sown and stored at 4°C for two days to boost germination. Then the plants were transferred to the greenhouse and after one month they were transplanted to individual pots.

[0083] Transfer of the plants

[0084] Seedlings of white cabbage, mangold, strawberry and cuttings of watercress were transferred into mesh pots (height of 5 cm and top and bottom diameter of 5 cm and 3.5 cm, respectively) consisting of granulates (a hydroponic substrate of size 0.5 to 1 cm in diameter). Salicornia plants were cultivated in hydroponics as described (Hulkko et al. (2022), Sci Rep 12, 20507. https: / / doi.org / 10.1038 / s41598- 022-24865-4; Turcios et al. (2023), Plants 12, 2472. DOI: 10.3390 / plantsl2132472).

[0085] Cultivation of plants

[0086] The plant experiment was conducted in greenhouses at Leibniz University Hannover, Germany (52°23'42" N; 9°42'13" E). The glycophytic plants were cultivated in an NFT system. The hydroponics system, with an area of 300 cm x 5.5 cm x 8.5 cm (LxHxW) was arranged vertically in four layers, consisted nutrient solution container (60 L) for growing 12 plants of mangold and five plants of strawberry, 24 cuttings of watercress and four plants of white cabbage in each horizontal line. Control treatment received 100% nutrition of EC value 1.6 mS cm1using Ferty Mega 2 (Hauert MANNA Dungerwerke GmbH, Nurnberg, Germany). To check the effect of the combination of BC and MO, an additional 3% (v / v) concentration of the P3 as described above was mixed into the nutrient solution (60 L) with EC value of 1.6 (mS cm1) of the system for even distribution. The EC value of 1.6 mS cm'1was maintained and necessary fertilization was provided on bi-weekly basis until the harvest. The average nutrient solution flow was 7.67 ml / s (volume flow) or 0.61 cm / s (velocity). Average pH of 7.5 was maintained throughout the experiment in both treatments.

[0087] For cultivation of mangold, strawberry and white cabbage in the NFT system, the temperature in the greenhouse was between 22 to 23gC during the day (10 h) and 17 to 18gC during the night (14 h). The temperature in the solution was maintained between 18 to 20gC. The average light intensity was 255.9 pmol / m2 / s ± 6.77 pmol / m2 / s.

[0088] The cultivation of cannabis in a deep water culture system was performed in climatic chambers. The temperature was between 25gC during the day (18 h) and 22gC during the night (6 h). The average light intensity was 350 pmol / m2 / s.

[0089] In the S. europaea experiment, the treatments were two different salt concentrations (7.5 g L1and 15 g L1NaCI). This salt range was selected because this is where the Salicornia grows best. Light was for 12 h of artificial light (sodium vapor lamps, SON-T Agro 400, Philips, Amsterdam, Netherlands). Light intensity ranged from 114 pmol m’2s’1- 640 pmol m’2s’1depending on the time of the day and the weather conditions. Plastic boxes containing 2 L of Hoagland solution were used, the boxes had a plastic lid with three holes, and each box contained three plants as described (Turcios et al. (2023), Plants 12, 2472. DOI: 10.3390 / plantsl2132472). The two salinities were reached at two different time points; first, 7.5 g L’1NaCI were added to the nutrient solution of all the experimental units, reaching the first salt concentration evaluated (128.34 mM NaCI); after 2 days of adaptation, the second group was added with another 7.5 g L’1NaCI, reaching the second salt concentration (256.6 mM). The water was aerated constantly by small compressors and one air stone in the middle of each tank (Eheim, Deizisau, Germany). The water level was adjusted constantly in each tank with tap water to compensate the evapotranspiration and therefore the salinity was kept constant.

[0090] Harvest of the plants and biomass determination

[0091] Plants in the NFT system were harvested after 9 weeks of growth. The shoot of the strawberry, mangold and white cabbage was harvested, and fresh weight was recorded. Samples were numbered, placed in aluminum trays and later dried at 70gC in a drying chamber (Memmert, Modell 600) until consistent weight was reached. The Salicornia plants were harvested after 25 days in hydroponics. For this, plants were cut at the base of the above-ground part, separating the roots from the shoots. The fresh weights of the above-ground biomass and roots were determined per experimental unit. After harvesting, a representative sample of shoots and roots was taken from each experimental unit, frozen immediately in liquid nitrogen and stored at -80°C before analysis. The left-over plant material was dried in a drying chamber (Memmert, Modell 600) at 70 °C until a constant weight is reached and used for the elemental analysis through ICP-OES. The water content in the plant material was determined using both values, dry weight and fresh weight of each sample.

[0092] Water sample collection

[0093] Water samples were collected at different levels. Nutrients and BC were added to the hydroponic system and first water sampling was done before the transfer of the plants into the system. Water samples were taken from the exit point of each horizontal as well as the first entry point of the nutrient solution. Second sampling of water was done after one week of transfer. Third and fourth water sampling was done at the time of harvests (two harvesting points).

[0094] ICP-OES analysis

[0095] Elemental composition of biochar was determined as described (Hansen et al. (2023), J Environ Manage 348, 119271. https: / / doi.Org / 10.1016 / j.jenvman.2023.119271).

[0096] Statistical analysis

[0097] The statistical analysis was performed in GraphPad Prism 10.2.1 software using one-way analysis of variance (ANOVA) followed by Tu key's t-test to compare the dry weight biomass of the control and P3 treatment groups. The differences in biomass were found to be statistically significant for all plant species, with p-values indicating high levels of significance (p < 0.05 to p < 0.0001).

[0098] Example 2 - Results

[0099] CPU count of Pseudomonas fluorescens on Biochar

[0100] The successful inoculation of biochar with P. fluorescens resulted in a high CFU count of 5.2 x 108per g (Table 1), demonstrating effective colonization and survival of the microorganisms on the biochar surface.

[0101] Table 1. CFU of Pseudomonas fluorescens

[0102] Effect of BC and MO on plant biomass

[0103] Combination of BC and MO had a significant effect on the growth of the plants consequently resulting in higher biomass in water cress, mangold, strawberry and white cabbage when compared to controls without BC and MO. As an example, the gain in biomass of strawberry is shown without and with P3 (Figure 1).

[0104] In Figure 2 the gain in biomass of water cress is shown. Unfortunately, the light conditions were not suitable for the growth of water cress and they only grew well till the first harvest.

[0105] The dry weight biomass of mangold significantly increased from 1.03 g in the control group to 12.11 g in the P3 treatment group, corresponding to an increase of approximately 1076%. This substantial enhancement in biomass is statistically significant (p < 0.0001), as indicated by the four asterisks in Figure 1. Similar results were observed in white cabbage and strawberry as well. Among the three plant species, mangold exhibited the greatest percentage increase in biomass (1076%), followed by white cabbage (707%), and strawberry (310%) (Figure 3).

[0106] The cannabis plants cultivated in climatic chambers without about with biochar / MO differ in their morphology, especially in the root morphology. In general, the plants treated with biochar / MO looked healthier.

[0107] In the hydroponic system where the halophyte plant species was cultivated in the presence of salt and biochar it was demonstrated that salt has an impact on the uptake of other elements such as Ca, K, and Mg, where increasing the concentration of NaCI in the different culture media decreased the uptake of those elements in the plant tissues. This could be an antagonist effect of salt on other ions, as the plants uptake more Na+excluding other cations. Biochar doesn't show an effect on Ca, K and Mg, however, it increased the uptake of S and microelements such as Fe and Zn (data not shown). Example 3 - Discussion

[0108] The high survival rate of Pseudomonas fluorescence indicates the potential for biochar to serve as a robust carrier for beneficial microorganisms in hydroponic systems, contributing to the enhanced plant growth observed in the treated groups. The increase in biomass of all plant species cultivated in the hydroponic systems suggests that the application of BC inoculated with P. fluorescens significantly enhances plant biomass, with varying degrees of effectiveness across different plant species. Especially, in the adaptation phase after transfer from soil or substrate to the hydroponic system the presence of BC and MO were very beneficial, showing a high potential for mitigating the stress effects in various systems. With respect to cannabis at the time of submission final biomass and, more important, the influence on flowering and on the content of cannabinoids could not yet be determined.

[0109] When the Salicornia plants were cultivated at different salinities in the presence of BC the access to important nutrients was improved. Further additions of MO might contribute to an increased availability of all necessary nutrients. Experiments are in progress.

Claims

CLAIMS1. Method for increasing the production yield of a plant being cultivated in a hydroponic system, comprising(a) adding biochar into the hydroponic system, wherein the biochar is coated with at least one microorganism that along with the biochar promotes the growth of the plant, and(b) culturing the plant in the hydroponic system of (a) to obtain products from the plant.

2. Use of biochar being coated with at least one microorganism that along with the biochar promotes the growth of a plant for increasing the production yield of the plant when grown in a hydroponic system.

3. The method of claim 1 or the use of claim 2, wherein the plant is a vegetable plant, a horticultural plant, a medicinal and spice plant, a fruit plant or a crop plant.

4. The method or the use of claim 3, wherein the vegetable plant species is / are carrot, broccoli, spinach, tomato, bell pepper, cucumber, cauliflower, zucchini, onion, potato, green bean, pea, eggplant, sweet potato, cabbage, radish, beet, asparagus, artichoke, lettuce, water cress or celery or a combination thereof; the horticultural plants species is / are begonias, orchids, iris, roses or a combination thereof; the medicinal and spice plant species is / are cannabis, basil, nasturtium, sage, mints, parsley, thyme, rosemary or a combination thereof; the fruit plant species is / are apple, banana, orange, lemon, lime, melon, watermelon, grape, grapefruit, plum, mango, pineapple, kiwi, peach, avocado, blueberry, blackberry, strawberry, raspberry, mandarin, durian, passion fruit, persimmon, dragon fruit, papaya, pomegranate or a combination thereof; and / or the crop plant species is / are quinoa, amaranth, sugarcane, maize, rice, hop, oat, sorghum, millet, barely, rye, canary grass, wheat or a combination thereof.

5. The method or the use of any preceding claim, wherein the biochar has been obtained from sewage sludge, wood chips, and other sources by pyrolysis, preferably at 700°C to 850°C for lh to 2h without air and optionally by crushing the biochar into particles.

6. The method or the use of any preceding claim, wherein the biochar is made of particles having a diameter of 1 mm or less, preferably 0.8 mm, more preferably less or 0.5 mm and most preferably between 0.2 mm and 0.4 mm.

7. The method or the use of any preceding claim, wherein the weight ratio of coated biochar and solution in the hydroponic system is between 0.5 / 100 to 5 / 100 by weight, preferably between 1 / 100 and 3 / 100 by weight and most preferably about 2 / 100.

8. The method or the use of any preceding claim, wherein at least one microorganism is or comprises a bacterium and / or a fungus, preferably a bacterium and a fungus.

9. The method or the use of any preceding claim, wherein the bacterium is selected from Azotobacter chroococcum, Azospirillum brasilense, Bacillus velezensis, Bacillus megaterium, Bacillus subtilis, Paenibacillus spp., Pseudomonas spp. Serratia spp., Sinorhizobium meliloti, Gluconacetobacter diazotrophicus, and Pseudomonas fluorescens; and / or the fungus is selected from Serendipita indica, Talaromyces allahabadensis and Trichoderma simmonsii, and other Trichoderma species.

10. The method or the use of any preceding claim, wherein the hydroponic system is a static solution culture, a continuous-flow solution culture, nutrient film technique culture (NFT), deep flow technique (DFT) culture, aeroponics culture, fogponics culture, passive sub-irrigation culture, ebb and flow sub-irrigation culture, run-to-waste culture, deep water culture, or top-fed deepwater culture.

11. The method or the use of any preceding claim, wherein the hydroponic system does not comprise any other growing support materials than the biochar.

12. The method or the use of any preceding claim, wherein the plant is water cress, mangold, strawberry or white cabbage and the at least one microorganism is Pseudomonas fluorescens.

13. The method or the use of any preceding claim, wherein t least one microorganism along with the biochar promotes the growth of the plant by increasing the availability of nutrients, in particular phosphate for the plant in the hydroponic system, stabilizes the pH and the electrical conductivity.

14. The method or the use of any preceding claim, wherein the at least one microorganism along with the biochar reduces the amount of mineral fertilizer applied in the hydroponic system.

15. The method of any preceding claim, wherein the method comprises prior to step (a)(a') coating biochar with at least one microorganism that along with the biochar promotes the growth of the plant by mixing a liquid medium with the at least one microorganism with the biochar and drying the biochar, preferably at about 18°C to about 22°C and / or to a final moisture of 0.4% to 1.5% water and minimum of 106colony forming units (CFU) for bacteria and spore-forming fungi or respective amount of fungal mycelium per kg of biochar, and(a") optionally prior to (a') producing biochar by pyrolyzing sewage sludge, wood ships or other sources preferably at 700°C to 850°C for 1 and 2h without air and optionally crushing the biochar into particles.

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