Microorganism and nutrient delivery system

Abstract

The present invention relates to a method of producing a microbe and nutrient delivery system, the method comprising hydrothermal carbonization of suitable biomass to form a slurry comprising hydrothermal char and HTC process water, cooling the slurry, inoculating the cooled slurry with a microbial inoculant, fermenting the inoculated slurry, and drying the fermented slurry to obtain the microbe and nutrient delivery system. And obtaining or recovering the microorganism and nutrient delivery system from the fermentation product, wherein the cooling step and the inoculating step are carried out under sterile conditions. The invention also relates to microbial and nutrient delivery systems obtainable by said method and uses thereof, including agricultural and environmental applications.

Description

Technical Field

[0001] The present invention relates to the valorization of products from a hydrothermal carbonization process. Specifically, a method for producing a microorganism and nutrient delivery system is provided, which includes integrating a hydrothermal carbonization process with a fermentation process. The present invention also relates to the microorganism and nutrient delivery system and its uses, including agricultural and environmental applications. Background Art

[0002] In view of the ever-increasing world population and the increasing environmental damage caused by increasing levels of industrialization, there is a need for improved agricultural plants that meet food production needs with more environmentally sustainable inputs.

[0003] Agricultural fertilizers are widely used to promote crop growth and yield, and avoid soil impoverishment. In general, fertilizers deliver nutrients to the soil, which allows crops to grow and develop better in the soil. Various fertilizer formulations are known in the field of agricultural science. The most commonly used fertilizers are raw animal feces and mineral fertilizers (mineral fertilizers) produced by chemical processes, such as nitrogen fertilizers manufactured by the Haber-Bosch process. Both of these fertilizers are the sources of quickly available nutrients, but the shortcoming is the significant risk of nutrient loss, which not only causes economic losses, but also destroys aquatic ecosystems (water pollution, eutrophication, biodiversity loss, etc.). Another significant shortcoming of raw feces is that it may contain bacterial pathogens that can cause disease in humans and livestock.

[0004] Organic fertilizers derived from living organisms, such as compost, and biofertilizers containing beneficial microorganisms, have been proposed as more environmentally sustainable solutions, but their use is not yet widespread. The main challenges in using and developing biofertilizers are reliability, improper formulation, high contamination levels, low quality, short shelf life, and consistency of inoculants under field conditions.

[0005] Appropriate formulations can ensure microbial survival during storage and application. Formulations can be broadly categorized as either solid carriers or liquid formulations. A disadvantage of liquid formulations is that the metabolic activity of beneficial microorganisms decreases rapidly after manufacture, and they can pose a high risk of contamination. Peat is a commonly used solid carrier material for both seed coating and soil application. To prevent contamination, peat must be sterilized before use, an energy-intensive process. Furthermore, because it is a fossil resource, its use in agricultural applications is being phased out.

[0006] Hydrochar has been proposed as a promising carrier for beneficial microorganisms (Thunshirn et al., 2022, Critical Reviews in Environmental Science and Technology 52: 4147-4171). Hydrochar is a solid product of the hydrothermal carbonization (HTC) process of wet biomass, such as digestate from anaerobic digestion processes. Its porous structure and high carbon content provide a suitable microenvironment for beneficial microorganisms. However, hydrochar has been found to exhibit some phytotoxicity (Celletti et al., 2021, Journal of Environmental Management 280: 111635), which may hinder its use as a carrier for biofertilizer formulations. In addition, a large amount of process water is obtained as a by-product of the HTC process that must be regulated and handled.

[0007] There remains a need in the art for additional and / or improved biostimulants such as biofertilizers that are stable and have an improved shelf life, that are environmentally sustainable, and that are produced with minimal impact on the environment. Summary of the Invention

[0008] The present invention is based, at least in part, on the inventors' discovery that process water produced during hydrothermal carbonization (HTC) performs unexpectedly well as a growth medium for microorganisms. The hydrothermal carbon, along with (residual) process water, can support and promote the growth and expansion of microorganisms and can therefore be used as a substrate for fermentation.

[0009] Thus, one aspect of the present invention provides a method or process for producing a microorganism and nutrient delivery system, the method comprising:

[0010] -Provide biomass;

[0011] - subjecting the biomass to a hydrothermal carbonization process (HTC) to form a slurry comprising hydrothermal char and HTC process water;

[0012] - cooling the slurry to a temperature suitable for the growth of the microbial inoculant;

[0013] - inoculating the cooled slurry with the microbial inoculant;

[0014] - subjecting the inoculated slurry to a fermentation process to form a fermentation product; and

[0015] - obtaining or recovering the microorganism and nutrient delivery system from the fermentation product,

[0016] The inoculation step and the cooling step are performed under sterile conditions.

[0017] By combining the HTC process with the fermentation process, the process water of the HTC process is valued, i.e., used as a growth medium for microorganisms. In addition, the microorganism can convert water-soluble nutrients (such as biochemical oxygen demand (BOD) nutrients, nitrate / ammonium) in the process water into biomass, thereby improving nutrient recovery from the biomass substrate. The integration of the HTC process and the fermentation process is advantageous because a separate microbial cultivation step is not required, which will reduce operating costs. In addition, the HTC process that occurs under conditions suitable for killing or inactivating microorganisms eliminates the need for a sterilization step before the fermentation process. The method is also energy-efficient because at least a portion of the heat required for the HTC process can be recovered from the cooling step before the fermentation step.

[0018] Another aspect relates to a microorganism and / or nutrient delivery system that can be obtained by the method of the present invention. The microorganism and nutrient delivery system provided herein comprises:

[0019] - Fermented hydrochar; and

[0020] - Microorganisms attached to the surface or pores of the fermented hydrochar.

[0021] Due to the sterile conditions in at least the cooling and inoculation steps of the method, the microorganism and nutrient delivery system is characterized in that it is minimally contaminated with unwanted microorganisms, so that the microorganisms of the system essentially consist of the microbial species contained in the microbial inoculant (the desired microorganisms).

[0022] The fermented hydrochar provides nucleation sites for microorganisms (a carrier / vehicle function). Furthermore, the porous structure of the hydrochar protects desired or beneficial microorganisms from environmental conditions (drought, heavy rain, soil type, etc.) and / or reduces predation by, for example, nematodes and protozoa in the soil. Without wishing to be bound by any theory, due to the growth and expansion of microorganisms on the hydrochar, attachment and / or pore colonization may be improved compared to using the hydrochar as a carrier / support (where the microorganisms are dried on the surface and / or mixed with the hydrochar). Also advantageously, due to the at least partial fermentation of the hydrochar by the beneficial microorganisms, the hydrochar is more stable (less fermentable material), resulting in a product with an improved shelf life. Also advantageously, due to the at least partial fermentation of the hydrochar, the growth and phytotoxicity of unwanted and / or pathogenic microorganisms can be reduced. Furthermore, certain nutrients (e.g., phosphorus) with low bioavailability in the hydrochar may be dissolved by the beneficial microorganisms, resulting in a product with enhanced nutrient supply.

[0023] Further aspects relate to the use of the microorganisms and nutrient delivery systems of the present invention as biostimulants, in particular as biofertilizers and / or biocontrol agents, bioremediation agents, or as microbial inoculants, for example for anaerobic digesters, septic systems, water treatment systems or in methods according to the present invention.

[0024] These and other aspects and preferred embodiments of the present invention are described in the following sections and in the appended claims. The subject matter of the appended claims is hereby incorporated into this specification. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] The following description of the drawings, which illustrate certain embodiments of the invention, is merely exemplary in nature and is in no way intended to limit the present teachings, their application, or uses.

[0026] Figure 1 A schematic diagram of an embodiment of the method according to the invention is shown.

[0027] Figure 2 : Mycelial growth of Trichoderma harzianum after 7 days in fermented peat (A) or fermented hydrothermal charcoal from pig manure (fermented HC) (B).

[0028] Figure 3 : Comparison of growth of microbial species in control (non-fermented) (A, B) and fermented (C, D) hydrochars exposed to non-sterile conditions (B, D) or kept sterile (A, C) on nutrient agar (NA) plates.

[0029] Figure 4 : Comparison of Trichoderma harzianum growth in hydrothermal charcoal (HC-C) from fermentation of swine manure on yeast mannitol agar (YMA) plates, where inoculation was performed under sterile (A) or non-sterile (B) conditions. DETAILED DESCRIPTION

[0030] As used herein, references indefinitely include both the singular and the plural unless the context clearly dictates otherwise.

[0031] As used herein, the term "comprising" is synonymous with "including" or "containing" and is inclusive or open-ended and does not exclude other unrecited members, elements, or method steps. The term also encompasses "consisting of" and "composed of," as well as the term "consisting essentially of," which has its recognized meaning in patent terminology.

[0032] The recitation of numerical ranges by endpoints includes all integers and, where appropriate, fractions encompassed within the respective ranges, as well as the recited endpoints. This applies to numerical ranges regardless of whether they are introduced by the expression "from ... to ..." or the expression "between ... and ..." or another expression. Any numerical range recited herein is intended to include all subranges subsumed therein.

[0033] As used herein, the terms "about" or "approximately" when referring to a measurable value such as a parameter, amount, duration, etc., are intended to encompass variations from the specified value, for example, + / - 10% or less, preferably + / - 5% or less, more preferably + / - 1% or less, more preferably + / - 0.1% or less, as long as such variations are suitable for performing the disclosed invention. It should be understood that the value to which the modifier "about" or "approximately" refers is itself also specifically and preferably disclosed.

[0034] Furthermore, the terms first, second, third, etc. in this specification and claims are used to distinguish similar elements and are not necessarily used to describe a sequential or chronological order unless otherwise indicated. It is to be understood that the terms so used are interchangeable under appropriate circumstances, and that the embodiments of the invention described herein are capable of operation in other sequences than described or illustrated herein.

[0035] While the terms "one or more" or "at least one," e.g., one or more members or at least one member of a group of members, are themselves explicit, by way of further example, the terms specifically encompass reference to any one of the members, or to any two or more of the members, e.g., any ≥3, ≥4, ≥5, ≥6, or ≥7 of the members, etc., and up to all of the members. In another example, "one or more" or "at least one" can refer to 1, 2, 3, 4, 5, 6, 7, or more.

[0036] As used herein, the term "and / or" when used in connection with a list of two or more items means that any one of the listed items may be used alone, or any combination of two or more of the listed items may also be used. For example, if a list is described as including the group A, B, and / or C, the list may include A alone, B alone, C alone, a combination of A and B, a combination of A and C, a combination of B and C, or a combination of A, B, and C.

[0037] This discussion of the background to the invention is included to explain the context of the invention. It should not be taken as an admission that any of the material referred to was published, known or part of the common general knowledge in any country before the priority date of any claim.

[0038] Throughout this disclosure, various publications, patents, and published patent specifications are referenced by identifying references. All documents cited in this specification are incorporated herein by reference in their entirety. Specifically, the teachings or portions of such documents specifically mentioned herein are incorporated herein by reference.

[0039] Unless otherwise defined, all terms used in disclosing the present invention, including technical and scientific terms, have the meanings commonly understood by those skilled in the art to which the present invention belongs. For further guidance, term definitions are included to better understand the teachings of the present invention. When a particular term is defined in conjunction with a particular aspect of the present invention or a specific embodiment of the present invention, unless otherwise defined, such connotation or meaning is intended to apply throughout this specification, i.e., also in the context of other aspects or embodiments of the present invention.

[0040] In the following paragraphs, different aspects or embodiments of the present invention are defined in more detail. Unless explicitly stated otherwise, each aspect or embodiment so defined may be combined with any other aspect or embodiment. In particular, any feature indicated as preferred or advantageous may be combined with any other feature indicated as preferred or advantageous.

[0041] References throughout this specification to "one embodiment," "an embodiment," or "an embodiment" mean that a particular feature, structure, or characteristic described in connection with the embodiment is included in at least one embodiment of the present invention. Thus, appearances of the phrases "in one embodiment" or "in an embodiment" in various places throughout this specification are not necessarily all referring to the same embodiment, but may be different. Furthermore, the particular features, structures, or characteristics may be combined in any suitable manner, as would be apparent to one skilled in the art from one or more embodiments of the present disclosure. Furthermore, although some embodiments described herein include some features included in other embodiments but not other features, combinations of features from different embodiments are meant to be within the scope of the present invention and to form different embodiments, as would be understood by one skilled in the art. For example, in the appended claims, any of the claimed embodiments may be used in any combination.

[0042] Similarly, it should be appreciated that in the description of exemplary embodiments of the invention, various features of the invention are sometimes grouped together in a single embodiment, figure, or description thereof to simplify the disclosure and aid in the understanding of one or more different inventive aspects.

[0043] In one aspect, the present invention provides a method of producing a microorganism and a nutrient delivery system, the method comprising:

[0044] -Provide biomass;

[0045] - subjecting the biomass to a hydrothermal carbonization process to form a slurry comprising hydrothermal char and HTC process water;

[0046] - cooling the slurry to a temperature suitable for the growth of the microbial inoculant;

[0047] - inoculating the cooled slurry with the microbial inoculant;

[0048] - subjecting the inoculated slurry to a fermentation process to form a fermentation product; and

[0049] - obtaining or recovering the microorganism and nutrient delivery system from the fermentation product,

[0050] The inoculation step and the cooling step are performed under sterile conditions.

[0051] Providing biomass

[0052] As used herein, the term "biomass" refers to biodegradable organic material.

[0053] The biomass is not particularly limited and may include, but is not limited to, products, by-products, and residues (including waste streams) from agriculture, forestry, and related industries, as well as industrial waste (e.g., sewage sludge) and household waste, and mixtures thereof. Non-limiting examples of biomass suitable for use in the methods of the present invention include food scraps, agricultural residues (e.g., straw, bark, sawdust, etc.), animal by-products (e.g., feces, feathers, wool, exoskeletons, etc.), organic fractions of household waste, organic fractions of industrial waste and by-products, sewage sludge, digestate from anaerobic digestion, and the like.

[0054] In certain embodiments, the biomass is animal by-products such as manure. Advantageously, animal by-products can provide high concentrations of nutrients (nitrogen, phosphorus, potassium, micronutrients, etc.).

[0055] The method of the present invention is particularly suitable for biomass with high water content, because the HTC process uses water as reaction medium. Compared with pyrolysis, wet biomass does not need to be dried before or during the process, saving a large amount of energy. In a specific embodiment, the biomass is wet biomass, such as water content between 5 weight % and 95 weight %, preferably between 10 weight % and 95%, more preferably between 10 weight % and 90 weight %, for example, biomass between 10 weight % and 80 weight %. The non-limiting examples of suitable wet biomass include excrement and the digestate from anaerobic digestion, which inherently contains water. If the biomass is too dry (such as water content is less than 10 weight % or 5 weight %), water can be added before it is used as the substrate of the HTC process for the method of the present invention. In order to make the HTC process sustainable from the perspective of energy, the water content of the substrate is preferably less than 95 weight %, more preferably less than 90 weight %, for example, less than 80 weight %.

[0056] The biomass, also referred to herein as feed or substrate, can be a single feedstock (eg, animal manure) or a mixture of two or more feedstock types.

[0057] The biomass can be used as is in the HTC process, or can be subjected to one or more pretreatment steps before being used as a substrate in the HTC process. The purpose of such pretreatment may be to mix different feeds, liquefy the biomass, remove unwanted materials such as large items and / or inert, non-biodegradable materials (e.g., plastic, glass) (separation), reduce the particle size of the biomass (e.g., breaking up lumps, chopping, grinding, milling, etc.), add additives (e.g., acids, bases, salts), etc., to allow a more efficient HTC process and / or adjust the properties / composition of the final product. There are various pretreatment processes that can be used, and the choice depends on, among other things, the type of biomass.

[0058] hydrothermal carbonization

[0059] As used herein, "hydrothermal carbonization" or "HTC" refers to a wet thermochemical process involving the application of heat and pressure in the presence of water to convert biodegradable materials, particularly biomass as defined herein, into carbonaceous hydrothermal char and aqueous and gaseous byproducts. Without wishing to be bound by any theory, a series of hydrolysis, condensation, decarboxylation, dehydration, aromatization, and / or polymerization reactions may occur during HTC.

[0060] Reaction temperatures in the range of about 160°C to about 300°C, preferably between about 180°C and about 250°C, more preferably between about 200°C and about 220°C, and corresponding pressures to ensure that the water remains in a liquid state, such as between about 10 bar and about 88 bar, preferably between about 10 bar and 50, 40 or 30 bar, more preferably between about 15 bar and about 25 bar, can be applied. The treatment time can vary from a few minutes to a few hours or even a few days, preferably the HTC process is carried out for at least 30 minutes, such as between 30 minutes and 20, 19, 18, 17, 16, 15, 14 or 13 hours, preferably between about 30 minutes and about 12, 11, 10 or 9 hours or between about 30 minutes and about 8, 7, 6 or 5 hours, more preferably between about 30 minutes and about 4 hours, such as between 2 hours and about 3 hours. The selected temperature, pressure and time conditions may depend on the desired yield and / or properties of the biomass substrate and the hydrochar product. In certain embodiments, the HTC process is carried out at a temperature between about 180° C. and about 250° C. and a pressure between about 10 bar and 50 bar for at least 30 minutes. In certain embodiments, the HTC process is carried out at a temperature between about 200° C. and about 220° C. and a pressure between about 15 bar and about 25 bar for about 2 hours to 3 hours.

[0061] As used herein, the term "hydrothermal char" refers to the solid, carbon-rich product of the HTC process. It contains most of the organic compounds originally present in the biomass. Its composition and yield depend on the biomass substrate and the HTC process parameters.

[0062] As used herein, the term "process water" or "HTC process water" refers to the liquid or aqueous byproduct of the HTC process. Process water is rich in dissolved organic components and inorganic salts. As with hydrothermal carbon, the amount and composition of process water depends largely on the biomass being processed and the HTC process parameters.

[0063] The hydrochar forms a solid-liquid "slurry" together with the process water. Due to the heat treatment, the slurry (including the hydrochar and the process water) is biologically sterilized.

[0064] As used herein, the expression "subjecting the biomass to a hydrothermal carbonization process to form a slurry comprising hydrothermal char and process water" means that the biomass is converted into a slurry comprising hydrothermal char and process water by a HTC process.

[0065] cool down

[0066] The slurry is typically at a temperature above 160° C., for example, at a temperature between about 200° C. and about 220° C. In order to allow fermentation with the desired microbial inoculant, the slurry or wet hydrochar needs to be cooled to a temperature suitable for the growth of the microbial inoculant, for example, between about 4° C. and about 65° C. In embodiments, the slurry or wet hydrochar is cooled to a temperature between about 20° C. and about 50° C., preferably between about 20° C. and about 30° C., depending on the microbial inoculant.

[0067] The cooling step is performed under aseptic conditions. "Aseptic conditions" herein means conditions that prevent microbial contamination of the slurry.

[0068] The cooling or aseptic cooling of the slurry or wet hydrochar can be achieved using a heat exchanger.

[0069] Advantageously, the heat released during the cooling step can be used in the HTC process.

[0070] The cooling can be performed in one or more stages. For example, the slurry can be partially cooled to a temperature between about 80°C and about 120°C, and then the partially cooled slurry can be further cooled to a temperature suitable for the growth of the microbial inoculant, for example, a temperature between about 4°C and about 65°C. In embodiments including a solid-liquid separation step, the slurry is preferably first cooled before being subjected to solid-liquid separation. Alternatively, the slurry can be subjected to solid-liquid separation to form a wet hydrochar, and then the wet hydrochar can be cooled, or the slurry can be partially cooled (preferably to a temperature below 100°C) and subjected to solid-liquid separation, and then the wet hydrochar can be further cooled to a temperature suitable for the growth of the microbial inoculant.

[0071] Solid-liquid separation

[0072] In certain embodiments, it is preferred to utilize solid-liquid separation technology to reduce the process water content of the slurry or to at least partially dehydrate or dry the slurry to form a wet hydrochar. As used herein, "wet hydrochar" refers to a solid hydrochar containing some residual process water. In embodiments, the process water content of the wet hydrochar is less than 90% by weight, preferably less than 80% by weight or less than 70% by weight, and more preferably less than 60% by weight. In embodiments, the process water content of the wet hydrochar is between about 40% by weight and about 60% by weight, for example, between about 50% by weight and about 55% by weight. Solid-liquid separation techniques are known to those skilled in the art and include, but are not limited to, gravity decantation, centrifugation (various types of stacked disc, basket, decanter centrifuges, etc.) and filtration (filter press, tangential flow filtration, belt filter, screw press, etc.).

[0073] In a preferred embodiment, the step of reducing the HTC process water content of the slurry or the dewatering step is performed under aseptic conditions.

[0074] When the slurry is subjected to solid-liquid separation, the wet hydrochar can be washed with (sterile) water. Therefore, in embodiments, the method may further comprise a washing step of the wet hydrochar. This may be particularly advantageous when the process water contains water-soluble compounds that inhibit the beneficial microorganisms.

[0075] The solid-liquid separation step also allows for easy addition of nutrients (e.g., salts and / or carbon sources) and / or acids or bases (to adjust pH) to the wet hydrochar, for example to promote the growth of the beneficial microorganisms. Furthermore, it also allows for the addition of the microbial inoculant to the wet hydrochar in a convenient manner.

[0076] Inoculation with microbial inoculants

[0077] After cooling, the slurry or wet hydrothermal charcoal is inoculated with a microbial inoculant.

[0078] "Microbial inoculant" refers to a composition or concentrate of specific microorganisms. When used herein, it refers to a composition or concentrate of specific microorganisms that is added to a slurry or wet hydrochar for fermentation of the hydrochar. A microbial inoculant may contain one living microorganism or a combination of living microorganisms. A microbial inoculant may contain 10 6 to 10 12 cfu / g or cfu / ml, preferably 10 8 to 10 12 cfu / g or cfu / ml. The composition or concentrate may be in solid form, liquid form or other form (eg suspension) or a mixture thereof.

[0079] As used herein, "colony forming unit" or "CFU" refers to a measure of viable microorganisms in a sample. A CFU is a single living cell capable of forming a visible colony on a solid culture medium, each cell of which is derived by cell division from a single parent cell. Where the microorganism is suitable for administration in the form of spores, the phrases "CFU," "CFU / ml," and "CFU / g" also encompass reference to "spores," "spores / ml," or "spores / g," respectively.

[0080] The term "microorganism" as used throughout this specification refers to any strain, genus, or phylum of microorganisms, including but not limited to archaea, bacteria, microalgae, fungi (including mold and yeast species), mycoplasmas, microspores, nanobacteria, oomycetes, and protozoa. In some embodiments, the microorganism is a bacterial strain. In some embodiments, the microorganism is a fungal strain such as a yeast strain or a filamentous fungus strain. In some embodiments, the microorganism encompasses a single cell (e.g., a unicellular microorganism) or more than one cell (e.g., a multicellular microorganism).

[0081] As used herein, the term "bacteria" generally refers to any prokaryotic organism and can refer to organisms from the kingdom Eubacteria (bacteria), the kingdom Archaea (archaea), or both. In some cases, bacterial genera have been reassigned for various reasons (such as, but not limited to, the evolving field of whole genome sequencing), and it is understood that such nomenclature reassignment is within the scope of any claimed genus.

[0082] The term "fungus" refers broadly to a wide variety of nucleated (eukaryotic) spore-forming organisms that do not contain chlorophyll (i.e., fungi do not photosynthesize and are heterotrophs). These organisms are classified in the kingdom Fungi, separate from the other kingdoms Eukaryota. Examples of fungi include multicellular filamentous fungi and unicellular fungi. Examples of fungi include yeasts, molds, mildews, rusts, smuts, and mushrooms. Many fungi live freely in soil or water; others form parasitic or symbiotic relationships with plants or animals. The term "fungal cell" includes any cell of a fungal organism at any stage in the life cycle of the organism, for example, encompassing fungal cells of any ploidy, such as haploid, diploid, and polyploid fungal cells; and encompassing vegetative cells and fungal spores.

[0083] As used herein, the term "microalgae" refers to microscopic algae. "Microalgae" includes, but is not limited to, organisms within the following categories: (i) several eukaryotic phyla, including Rhodophyta (red algae), Chlorophyta (green algae), Dinophyta, and Haustophyta, (ii) several classes of the eukaryotic phyla, including, but not limited to, Bacillariophyta (diatoms), Oculophyta, Phaeophyta (brown algae), Xanthophyta (yellow-green algae), and Chrysophyta (gold algae), and (iii) the prokaryotic phylum Cyanobacteria (blue-green algae).The term "microalgae" includes, for example, genera selected from the group consisting of Achnanthes, Amphora, Anabaena, Anikstrodesmis, Arachnoidicus, Aster, Botryococcus, Chaetoceros, Chlamydomonas, Chlorella, Chlorococcum, Chorethron, Cocconeis, Coscinodicus, , Crypthecodinium, Cyclotella, Cylindrotheca, Desmodesmus, Dunaliella, Emiliana, Euglena, Fistulifera, Fragilariopsis, Gyrosigma, Hematococcus, Isochrysis, Lampriscus, Monochrysis, Monoraphidium, Nannochloris, Nannochloropsis, Navicula, Neochloris, Nephrochloris, Nephroselmis, Nitzschia, Nodularia, Nostoc, Odontella, Oochromonas, Oocystis, Oscillartoria, Pavlova, Phaeoda ctylum, Playtmonas, Pleurochrysis, Porhyra, Pseudoanabaena, Pyramimonas, Scenedesmus, Schyzochitrium, Stichococcus, Synechococcus, Synechocystis, Tetraselmis, Thalassiosira, and Trichodesmium.

[0084] The term "strain" (e.g., in the phrases "fungal strain" and "bacterial strain"), as the basic operational unit of microbial taxonomy (e.g., fungal or bacterial taxonomy), is generally used to refer to a population consisting of the descendants of a single isolate in pure culture, usually consisting of a series of cultures ultimately derived from an initial single fungal or bacterial colony. When a species contains two or more different isolates, the term "strain" may be used to refer to an isolate or a group of isolates that can be distinguished from other isolates of the same genus and species by phenotypic or genotypic characteristics, or both.

[0085] In embodiments, the microbial inoculant may comprise one or more bacterial strains, yeast strains, and / or filamentous fungi strains, preferably bacterial strains and / or filamentous fungi strains. The microorganisms may or may not be genetically modified. For example, one or more microorganisms of the genera listed below may be used in the present invention.

[0086] bacteria

[0087] Acetobacter, Achromobacter, Acinetocacter, Actinomyces, Actinoplanes, Actinomadura, Aerococcus, Aeromonas, Alcaligenes, Alcanivorax, Alloiococcus, Alteromonas, Amycolatops sis), Anabaena, Arthrobacter, Arthrospira, Atopobium, Azoarcus, Azobacter, Azorhizobium, Azospirillum, Azotobacter, Bacillus, Beijerinckia, Bifidobacterium, Bradyrhizobium, Bradyrhizobium, Brevibacterium, Brevundimonas, Carnobacterium, Catenisphaera, Cellulomonas, Chryseobacterium, Citrobacter, Clostridium, Corynebacterium, Cyanobacteria, Dermatophilus, Desulfotomaculum, Dietz Dietzia, Enterobacter, Enterococcus, Escherichia, Frankia, Flavobacterium, Geobacillus, Gluconacetobacter, Gluconobacter, Gordonia, Herbaspirillum, Humicola, Janthinobacterium,Lactobacillus, Lactococcus, Leuconostoc, Klebsiella, Marinobacter, Microbacterium, Micromonospora, Microtetraspora, Moraxella, Mycobacterium, Blastomyces ococcus), Micrococcus, Nocardia, Oenococcus, Paenibacillus, Pediococcus, Phormidium, Phyllobacterium, Propionibacterium, Pseudomonas, Raoultella, Ralstonia alstonia), Rhizobia, Rhizobium, Rhodococcus, Saccharopolyspora, Serratia, Shigella, Sinorhizobium, Sphingomonas, Staphylococcus, Streptococcus, Streptomyces reptomyces), Symbiobacterium, Synechococcus, Synechocystis, Tetragenococcus, Thermoactinomyces, Thermomonospora, Vagococcus, Vibrio, Weissella, Xanthomonas.

[0088] Preferably, the microbial inoculant comprises one or more bacterial strains belonging to a genus selected from the group consisting of Achromobacter, Aeromonas, Azospirillum, Bacillus, Bradyrhizobium, Enterobacter, Gluconacetobacter, Klebsiella, Pseudomonas, Rhizobia, Rhizobium and Streptomyces, or selected from the group consisting of Achromobacter, Aeromonas, Azospirillum, Bacillus, Bradyrhizobium, Enterobacter, Gluconacetobacter, Klebsiella, Pseudomonas, Rhizobia, Rhizobium and Streptomyces. illus), Bradyrhizobium, Enterobacter, Gluconacetobacter, Klebsiella, Pseudomonas, Rhizobia, Rhizobium and Streptomyces, more preferably a genus selected from the group consisting of Streptomyces, Bacillus, Azospirillum and Bradyrhizobium or a genus selected from the group consisting of Azotobacter, Streptomyces, Bacillus, Azospirillum and Bradyrhizobium.

[0089] yeast

[0090] Arxula, Aureobasidum, Blastobotrys, Brettanomyces (whose sexual stage is Dekkera), Candida, Citeromyces, Cryptococcus, Cystofilobasidium, Debaryomyces, Endomycopsis, Filobasidiella ), Galactomyces, Geotrichum, Glaciozyma, Guehomyces, Hansenula, Hanseniaspora (whose asexual counterpart is Kloeckera), Hyphopichia, Kluyveromyces, Kodamaea, Komagataella, Lachaise ncea), Lipomyces, Metschnikowia, Meyerozyma, Moniella, Mrakia, Ogataea, Pichia, Phaffia, Pseudozyma, Rhodotorula, Rhodosporidium, Starmerella, Saccharomyces, Saccharomycodes ), Saccharomycopsis, Scheffersomyces, Schizosaccharomyces, Schwanniomyces, Torulopsis, Torulaspora, Trichosporon, Trigonopsis, Yarrowia, Xanthophyllomyces, and Zygosaccharomyces.

[0091] filamentous fungi

[0092] Acremonium, Agaricus, Agrocybe, Akanthomyces, Alternaria, Ampelomyces, Amylosporus, Antrodia, Armillaria, Ashbya, Aspergillus, Atkinsonella, Aureobasidium, Auricularia, Balansia, Balans iopsis, Beauveria, Bispora, Bjerkandera, Boletus, Cantharillus, Catenaria, Cephalosporium, Chaetomium, Chrysonilia, Cladosporium, Claviceps, Clitocybe, Clitopilus, Colletotrichum, Collybia , Coniochaeta, Coprinus, Cordyceps, Coriolus, Cunninghamella, Cyathus, Cyclocybe, Cylindrocarpon, Cylinrocarpum, Cytonaema, Cytospora, Daldinia, Dentipellis, Doratomyces, Echinodothis, Emer icella), Emericellopsis, Entoloma, Epichloe, Epicoccum, Exophiala, Favolaschia, Flammulina, Fomes, Fomitopsis, Fusarium, Ganoderma, Giberella, Gliocladium, Grifola, Gymnoascus,Hericium, Hohenbuehelia, Hormonema, Humicola, Hydropus, Hypomontagnella, Hypomyces, Hypoxylon, Hypsizigus, Inocutis, Inocybe, Inonotus, Isaria, Kuehneromyces, Lactarius, Laetiporus, Laxitextum), Lecanicillium, Lentinula, Lentinus, Lepista, Leptoshaeria, Lignosus, Lycoperdon, Lyophyllum, Martierella, Metarhizium, Monascus, Monilia, Monocillum, Morchella, Mortiere lla), Mucor, Mycelia, Myriogenospora, Neurospora, Nigrospora, Omphalotus, Ophiocordyceps, Oudemansiella, Paecilomyces, Panellus, Panus, Paraconiothyrium, Paraepichloe, Penicillium, Peniophora, Pe riconia), Pestalotiopsis, Phellinus, Phlebia, Pholiota, Phoma, Phomopsis, Piptoporus, Pleurotus, Pochonia, Polyporus, Preussia, Pycnoporus, Ramaria, Rhizoctonia, Rhizopus,Rhodotorula, Rhodotus, Sarcodon, Schizophyllum, Scytalidium, Scytalidium, Scytinostroma, Sparassis, Sphaerodes, Spicaria, Stachybotrys, Steccherinum, Stropharia, Suillus, Thymus alaromyces), Thermoascus, Thermomyces, Tolypocladium, Torula, Trametes, Tremella, Trichoderma, Tricholoma, Tuber, Verticillium, Volvariella, Wolfiporia, Wrightoporia, and Xylaria.

[0093] Preferably, the microbial inoculant comprises one or more filamentous fungal strains belonging to a genus selected from the group consisting of Alternaria, Aspergillus, Chaetomium, Exophiala, Fusarium, Penicillium, Phoma, Talaromyces and Trichoderma, or a filamentous fungal strain selected from the group consisting of Alternaria, Aspergillus, Chaetomium, Exophiala, Fusarium, Penicillium, Phoma, Talaromyces and Trichoderma. The genus selected from the group consisting of Beauveria, Chaetomium, Exophiala, Fusarium, Penicillium, Phoma, Talaromyces and Trichoderma, more preferably a genus selected from the group consisting of Trichoderma and Penicillium or a genus selected from the group consisting of Beauveria, Trichoderma and Penicillium.

[0094] The choice of microorganisms to be included in the microbial inoculant may depend on parameters such as the desired end product, their ability to develop in one or more types of given substrates, their availability and price. By way of illustration and not limitation, plant beneficial microorganisms that may be included in the microbial inoculant may include fungi belonging to the phylum Ascomycota (e.g., fungi of the genus Ampelomyces), fungi of the phylum Basidiomycota and Zygomycota, bacteria of the family Rhizobiaceae, Frankia, Azotobacter, Azospirillum, Acetobacter, Azoarcus, Burkholderia, Herbaspirillum, Pseudomonas (e.g., Pseudomonas fluorescens, P. putida, P. gladioli), Bacillus (Bacillus subtilis), subtilis), Bacillus cereus, Bacillus circulans), and other bacteria such as Serratia marcescens, Flavobacterium spp., Alcaligenes sp., Agrobacterium radiobacter, etc. For example, but not limited to, the biological control microorganisms that may be included in the microbial inoculant may include bacteria of the genus Agrobacterium, Pseudomonas, Streptomyces or Bacillus, and / or fungi of the genus Gliocladium, Trichoderma, Ampelomyces, Candida or Coniothyrium.

[0095] In certain embodiments, the microbial inoculant comprises a bacterium selected from a Streptomyces spp. (e.g., Streptomyces griseoviridis), a Bacillus spp. (e.g., Bacillus subtilis), an Azospirillum spp. (e.g., Azospirillum brasilense), or a Bradyrhizobium spp. (e.g., Bradyrhizobium japonicum), or a filamentous fungus selected from a Trichoderma spp. (e.g., Trichoderma harzianum), or a Penicillium spp. (e.g., Penicillium bilaiae), or any combination thereof.

[0096] In certain embodiments, the microbial inoculant comprises a bacterium selected from an Azotobacter spp. (e.g., Azotobacter chroococcum), a Pseudomonas spp. (e.g., Pseudomonas fluorescens), a Streptomyces spp. (e.g., Streptomyces griseoviridis), a Bacillus spp. (e.g., Bacillus subtilis), an Azospirillum spp. (e.g., Azospirillum brasilense), or a Bradyrhizobium spp. (e.g., Bradyrhizobium japonicum), or a Beauveria spp. spp.) (e.g., Beauveria bassiana), Trichoderma spp. (e.g., Trichoderma harzianum), or Penicillium spp. (e.g., Penicillium bilaiae), or any combination thereof.

[0097] Inoculating the slurry with a microbial inoculant may comprise adding the microbial inoculant to the slurry and optionally mixing the slurry.Mixing may be performed using a mixing device, by pumping, for example, a gas through the slurry, or by shaking.

[0098] Inoculating the wet hydrochar with a microbial inoculant may include optionally mixing the microbial inoculant with a liquid (e.g., water), adding the inoculant and optionally the liquid to the wet hydrochar, and optionally mixing the inoculant throughout the wet hydrochar to ensure exposure to and adherence to various surfaces of the hydrochar.

[0099] The inoculation step is performed under sterile conditions. "Inoculation under sterile conditions" herein means conditions that prevent the slurry or wet hydrochar from being contaminated by microorganisms other than those contained in the microbial inoculant.

[0100] Fermentation

[0101] The method further comprises fermenting the inoculated slurry or wet hydrochar. The fermentation comprises storing the inoculated slurry or wet hydrochar for a sufficient time under conditions that promote growth and expansion of the microbial inoculant and degradation of the hydrochar by the microorganisms.

[0102] The time that is enough for the fermentation of described hydrochar is different because of the composition of biomass, microbial inoculant and fermentation conditions.Described fermentation may not be complete, but at least a portion of the contents of described hydrochar should be fermented.The time that is enough for the fermentation of described hydrochar can be, for example, at least 1 day, at least 5 days, at least 10 days, at least 15 days or at least 20 days.The time that is enough for the fermentation of described hydrochar can be, for example, at most 60 days, at most 50 days, at most 40 days, at most 30 days or at most 20 days.In embodiments, the slurry of described inoculation or wet hydrochar can be fermented for about 2 days to about 14 days.

[0103] Preferably, fermentation conditions are controlled during the fermentation step. For example, temperature can be controlled. In certain embodiments, the fermentation is carried out at a temperature below 85°C, preferably below 75°C, more preferably between 15°C and 70°C, or between 15°C and 65°C, or between 15°C and 60°C. Fermentation is typically carried out at a temperature between 15°C and 65°C, depending on the microorganisms and biomass involved. Other parameters that can be controlled include humidity, oxygen, and CO2 levels. For example, humidity can be maintained above 35%, preferably above 45%, more preferably above 50%, even more preferably above 60%, for example above 65%. CO2 levels can be maintained between 600 and 1200 ppm, preferably between 800 and 1000 ppm.

[0104] The fermentation can be carried out under aerobic (i.e. in the presence of oxygen) or anaerobic (i.e. in the absence of oxygen) conditions, depending on the microorganism involved. For aerobic fermentation, oxygen can be fed to the slurry as air. However, pure oxygen and / or oxygen-enriched air and / or separate feed air and oxygen can also be fed to the slurry. For aerobic solid-state fermentation, the wet hydrochar can be exposed to air.

[0105] In a preferred embodiment, the fermentation is carried out under aseptic conditions. As used herein, "fermentation under aseptic conditions" or "sterile fermentation" means the absence or minimal presence of unwanted microorganisms, e.g., preferably no contamination by microorganisms other than the species of microorganisms contained in the microbial inoculant. Techniques for aseptic fermentation are known to those skilled in the art and include, for example, sterilizing the fermentor prior to inoculation with the microbial inoculant, using a sterile fermentation broth, using sterile (e.g., filtered) air or oxygen, and the like, and any combination thereof.

[0106] The fermentation can be carried out in a single stage or in two or more stages (i.e., multi-stage fermentation), such as a two-step fermentation. In embodiments, the fermentation process can include fermenting the inoculated slurry to form a first fermentation product, then inoculating the second slurry formed in the HTC process with the first fermentation product, and fermenting the second slurry of the inoculation to form a (second) fermentation product. In other embodiments, the fermentation of the (first) slurry inoculated with the microbial inoculant is carried out under aseptic conditions. In other embodiments, the fermentation of the (second) slurry inoculated with the first fermentation product is carried out under non-sterile or aseptic conditions. In other specific embodiments, the fermentation of the (first) slurry inoculated with the microbial inoculant is carried out under aseptic conditions, and the fermentation of the (second) slurry inoculated with the first fermentation product is carried out under non-sterile conditions.

[0107] In certain embodiments, the fermentation process is submerged fermentation. The term "submerged fermentation" generally refers to the process of fermenting microorganisms submerged in a liquid medium that provides nutrients to the microorganisms. As used herein, "submerged fermentation" can refer to hydrothermal char fermentation, wherein a microbial inoculant is inoculated into the slurry or partially dehydrated slurry of the HTC process.

[0108] In certain embodiments, the fermentation is solid-state fermentation. The term "solid-state fermentation" generally refers to a process in which a microorganism is fermented on a solid medium or substrate that provides an anchoring point and nutrients for the microorganism. "Solid-state fermentation" as used herein can refer to the fermentation of hydrochar, wherein a microbial inoculant is inoculated into the wet hydrochar. Statement "fermentation product" as used herein refers to the product produced in a fermentation step according to the present invention. Fermentation product can correspond to a substrate (particularly the hydrochar of fermentation), spores, biomass (such as bacteria, yeast or filamentous fungi), molecule or any mixture thereof that is fermented.

[0109] As used herein, "fermented hydrochar" refers to hydrochar that has been at least partially degraded during the fermentation process. The term fermented hydrochar encompasses "partially fermented hydrochar," which includes a portion of fermented hydrochar and a portion of fermentable hydrochar, as well as "fully fermented hydrochar."

[0110] Recycling of microorganisms and nutrient delivery systems

[0111] The fermentation product can be used as, for example, a microorganism or a nutrient delivery system, or the fermentation product can be subjected to one or more processing steps to obtain a microorganism and a nutrient delivery system.

[0112] Recovery of the microorganisms and nutrient delivery system from the fermentation product may comprise one or more processing steps selected from a solid-liquid separation step, a drying step, grinding, milling, granulation, micronization process, extrusion, and the like.

[0113] In certain embodiments, reclaiming microorganisms and nutrient delivery systems from the fermentation product comprises separating the fermentation product into a solids-rich fraction and a liquid fraction (or subjecting the fermentation product to solid-liquid separation), wherein the microorganisms and nutrient delivery systems are obtained or recovered from the solids-rich fraction. The solid-liquid separation techniques described elsewhere herein for solid-liquid separation of slurries can be used.

[0114] In certain embodiments, recovering the microorganism and nutrient delivery system from the fermentation product comprises drying the fermentation product or a solids-rich fraction of the fermentation product. Depending on the type of microorganism in the microbial inoculant (sensitive microorganisms may require more gentle drying techniques), different drying techniques can be used, including but not limited to freeze drying, spray drying, fluidized bed drying, vacuum tray drying, etc.

[0115] In certain embodiments, the microorganism and nutrient delivery system are recovered from the fermentation product and include extruding the fermentation product, for example, by granulating the fermentation product. The step of "extruding" the fermentation product can be broadly described as a step of forcing the fermentation product through an opening. In an extrusion step that typically involves at least one screw and a die, high pressure is accumulated at the ends of the screw and die. Specifically, it can be considered that the extrusion step combines various unit operations into a system: transportation of materials, particle size reduction, particle size change, shape change, moisture change (increase or decrease), mixing, extraction, washing, cooling and / or heating, steaming, compression and / or expansion.

[0116] After the step of extruding the fermentation product, the extruded fermentation product may be dried.

[0117] In certain embodiments, recovering the microorganisms and nutrient delivery system from the fermentation product comprises grinding or milling the fermentation product or a solids-rich fraction thereof, optionally after a drying step.

[0118] In certain embodiments, recovering the microorganism and nutrient delivery system from the fermentation product comprises a micronization process.

[0119] In certain embodiments, particularly those in which the microbial inoculant comprises a spore-forming microorganism, the fermentation product can be subjected to a spore separation step in which the spores are separated from the fermentation product. Both the separated spores and the residual fermentation product can be used as the microbial and nutrient delivery system described herein. Spore separation can be performed in a variety of ways known to those skilled in the art, including but not limited to screening, filtration, centrifugation, cyclone separation, and any combination thereof.

[0120] system

[0121] Also disclosed herein is a system for producing a microorganism and a nutrient delivery system. Specifically, the system for producing a microorganism and a nutrient delivery system can be used to perform the method for producing a microorganism and a nutrient delivery system according to the present invention.

[0122] The system can include a unit for carrying out the HTC process (e.g., a pressurized reactor equipped with a thermocouple; Ingelia SL HTC plant), a cooling unit (e.g., a heat exchanger), and a fermentation unit (e.g., a fermentor tank, an open container). Suitable equipment and cooling equipment for carrying out the HTC process or fermentation process according to an embodiment of the present invention are known to any person skilled in the art and can be suitably selected by a professional technician. For example, immersion fermentation can be carried out in a (stirring) tank. The non-limiting examples of suitable solid-state fermenters include tray-type bioreactors, stirred tank bioreactors, drum bioreactors, fluidized bed bioreactors, etc., known to professional technicians.

[0123] The system can also comprise one or more of a liquid-solid separator (eg, a centrifuge), a dryer, a grinder, a mill, and an extruder for recovering the microorganism and nutrient delivery system from the fermentation product.

[0124] Microorganisms and nutrient delivery systems

[0125] Another aspect relates to a microorganism and nutrient delivery system obtainable by the method of the present invention. Specifically, a microorganism and nutrient delivery system is provided, comprising:

[0126] - Fermented hydrochar; and

[0127] - Microorganisms attached to the surface or pores of the fermented hydrochar.

[0128] As used herein, a "microorganism and nutrient delivery system" refers to a system or composition comprising microorganisms and nutrients.

[0129] The term "nutrient" generally refers to the material that organisms such as microorganisms and plants are used to survive, grow and reproduce. The term nutrient covers macronutrients and micronutrients, and includes but is not limited to carbon, oxygen, nitrogen, phosphorus, mineral matter (for example calcium, sodium, potassium, magnesium, chloride) etc. The nutrient can be present in the system in any form, for example as ion, salt, compound (for example carbohydrate) etc. Depend on application, the microorganism and nutrient delivery system can comprise the microorganism existing in the system and the nutrient for other organisms (for example plant). Advantageously, the microorganism in the system can convert some nutrients into certain form, makes it become that other organisms (for example plant) have bioavailability.

[0130] The microorganism and nutrient delivery system according to the present invention comprises fermented hydrochar, microorganisms and optionally spores.The microorganism and nutrient delivery system may also comprise (residual) process water.

[0131] The microorganisms adhere to the surface or pores of the fermented hydrochar. Without wishing to be bound by any theory, the adhesion may be stronger and / or the colonization of the pores may be better due to the fermentation step compared to using non-fermented hydrochar as a microbial carrier.

[0132] Due to the aseptic conditions of application at least in the cooling and inoculation steps of the method of the present invention, the feature of microorganism and nutrient delivery system according to the present invention is that it is rarely contaminated by the microorganism species that do not comprise in the unwanted microorganism or the described microorganism inoculant.The feature of microorganism and nutrient delivery system of the present invention is that the described microorganism is basically composed of the microorganism species that comprise in the described microorganism inoculant.In embodiments, at least 50% in the described microorganism and nutrient delivery system, preferably at least 55%, 60%, 65%, 70% or 75%, more preferably at least 80%, 82%, 85%, 86%, 88% or 90%, for example at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% microorganism is composed of the microorganism species that comprise in the described microorganism inoculant.As previously mentioned, the hydrochar of described fermentation can be the hydrochar of partial fermentation or the hydrochar of complete fermentation.The hydrochar of complete fermentation may be preferred because the lack of fermentable substrate makes it less attractive to unwanted and / or pathogenic microorganisms.

[0133] Compared to systems in which non-fermented hydrochar is used as a microbial carrier, the microbial and nutrient delivery system is more stable and has a longer shelf life due to the fermentation of the hydrochar.

[0134] In certain embodiments, e.g., wherein the microorganism and nutrient delivery system is obtainable by a method according to the present invention comprising a spore isolation step, the microorganism consists essentially of spores, preferably spores of the microorganism species comprised in the microbial inoculant.

[0135] The microorganism and nutrient delivery system may also include one or more adjuvants.

[0136] The terms "adjuvant," "auxiliary agent," "additive," or "adjuvant" are used interchangeably herein. The adjuvant can be a natural or synthetic organic or inorganic material, preferably a natural material, that facilitates application of the microorganism and nutrient delivery system to, for example, a plant, plant part, seed, plant growth site, or environmental site. The adjuvant can be one or more of a solvent, carrier, adhesive, surfactant, sticker, tackifier, antifreeze, thickener, buffer, defoamer, antioxidant, preservative, stabilizer, fragrance, colorant, and the like.

[0137] Suitable adjuvant is known in the art and is commercially available.In general, described microorganism and nutrient delivery system can be combined with any solid, semisolid or liquid additive that is usually used for formulation purpose.Carrier should be understood to mean natural or synthetic organic or inorganic substance, it mixes or combines with described microorganism and nutrient delivery system to obtain better applicability, for example, is applied to plant or plant part such as seed.Described carrier can be solid, semisolid or liquid, normally inert, and is applicable to agriculture or gardening.For example, liquid carrier can comprise water, organic solvent, mineral oil and vegetable oil.Suitable liquefied gaseous extender or carrier are gaseous liquids under ambient temperature and atmospheric pressure, for example aerosol propellant, for example butane, propane, nitrogen and carbon dioxide.Adhesive should be understood to mean the additive or adjuvant that improves the adhesion performance of described composition to plant or its part.Suitable surfactant is emulsifier, dispersant or wetting agent with ionic or nonionic property, or the mixture of these surfactants. Colorants such as inorganic pigments (e.g., iron oxide, titanium oxide, Prussian blue) and organic dyes (e.g., alizarin dyes, azo dyes, and metal phthalocyanine dyes) may be used, as well as trace nutrients such as salts of iron, manganese, boron, copper, cobalt, molybdenum, and zinc. Stabilizers (e.g., low-temperature stabilizers), preservatives, antioxidants, light stabilizers, or other agents that improve chemical and / or physical stability may also be present.

[0138] For some applications, the adjuvant added to the microorganism and nutrient delivery system is preferably an agriculturally acceptable adjuvant. The term "agriculturally acceptable" or "agriculturally compatible" has the same meaning as in the art, meaning that it is not harmful to the recipient plant, for example, it does not produce, have or cause any adverse effects when applied to a plant or plant part, or has no adverse effects on plants grown from the plant part.

[0139] In certain embodiments, the microorganism and nutrient delivery system comprises one or more other active ingredients, such as nutrients and / or microorganisms.

[0140] The terms "active ingredient" or "active component" are used interchangeably and generally refer to a material, such as an element, molecule, substance, and / or microorganism, that, when provided in an effective amount, achieves a desired result, such as one or more effects on one or more plant growth characteristics, or one or more effects on bioremediation. Generally, the active ingredients described herein can achieve such results by interacting with and / or modulating plants, parts thereof, seeds in which the plants are grown, the locus of the plants (e.g., soil), or an environmental locus (e.g., contaminated soil).

[0141] For example, microorganism can be added, it forms the microbial flora that is applicable to bioremediation application together with the microorganism of described microorganism and nutrient delivery system, but they and the microorganism that is used for fermentation slurry or wet hydrothermal charcoal of described microorganism and nutrient delivery system are not too compatible or incompatible (for example different fermentation conditions).Other limiting examples that can add to the active component of described microorganism and nutrient delivery system comprise fertilizer (for example chemical fertilizer), pesticide (for example chemical pesticide, chitosan), biostimulant (as microbial biostimulant, plant extract, (plant) hormone, (biological) chemical) or its any combination.

[0142] Microorganism and nutrient delivery system or composition as taught herein can be liquid, semisolid or solid, and can comprise solution or dispersion.Non-limiting examples of the composition taught herein can be (soluble) powder, (soluble) granule, wettable granule, pellet, tablet formulation, dry concentrated suspension, aqueous concentrated suspension, wettable dispersible granule, oil dispersion, suspension concentrate, dispersible concentrate, emulsifiable concentrate, aqueous suspension, fertilizer granule, sprayable thing etc.Term " powder " refers to the dry block solid being made up of many very fine particles, and it can flow freely when shaking or tilting.

[0143] use

[0144] Other aspects relate to the use of the microorganisms and nutrient delivery systems of the present invention.The microorganisms and nutrient delivery systems of the present invention can be used as agricultural biologicals, in particular as biostimulants, more particularly as biofertilizers and / or biocontrol agents.

[0145] Thus, in one aspect, the present invention relates to the use of a microorganism and a nutrient delivery system according to the invention as a biostimulant.

[0146] As used herein, "biostimulant" refers to a substance or microorganism that, when applied to a plant, a part thereof (e.g., roots), a seed for growing the plant, or the locus of the plant (e.g., the soil surrounding the plant or the plant growth medium), stimulates natural processes to improve the growth characteristics of the plant.

[0147] The term "plant growth characteristic" is intended to broadly encompass any characteristic that is related in some way to plant growth. The characteristic may be associated with or observable for an individual plant or a population of plants. Examples of such characteristics include, but are not limited to, wet or dry plant biomass, plant height, plant size, percent emergence, emergence date, canopy cover, flowering status, seed yield, grain yield, fruit yield, number of tillers per plant, stem length, root length, root system architecture, seed weight, senescence, stay-greenness, number of mature plant reproductive elements per plant, visual appearance, and the like.

[0148] Reference to improvement encompasses any qualitative or quantitative change or modification in a plant growth characteristic that is industrially beneficial, particularly in an agricultural context. To the extent that a plant growth characteristic is quantifiable, improvement may be synonymous with an increase or decrease in that quantity, depending on the nature of the plant growth characteristic. By way of example and not limitation, it may be desirable to increase a quantifiable characteristic such as wet or dry plant biomass, plant height, plant size, canopy cover, seed yield, grain yield, fruit yield, number of tillers per plant, stem length, root length, seed weight, and the like.

[0149] For example, biostimulants may be able to increase nutrient uptake and / or nutrient use efficiency of treated plants compared to untreated plants, increase nitrogen fixation or phosphorus uptake of treated plants compared to untreated plants, increase biomass of treated plants compared to untreated plants, increase the number of tillers per plant of treated plants compared to untreated plants, increase growth and / or yield of treated plants compared to untreated plants, and / or help treated plants overcome stress conditions such as nutritional stress or abiotic stress (e.g., drought, high temperature, and saline soil) compared to untreated plants; etc. Other plant growth traits that can be improved by biostimulants may include disease resistance, drought tolerance, heat tolerance, cold tolerance, salt tolerance, metal tolerance, herbicide tolerance, chemical tolerance, improved water use efficiency, improved phosphorus solubility, improved phosphorus mobilization, improved nitrogen use efficiency, improved nitrogen fixation, insect resistance, herbivore resistance, pathogen resistance, increased yield, increased yield under water-limiting conditions, enhanced health, improved vigour, improved growth, improved plant emergence, improved photosynthetic capacity, enhanced nutrition, altered protein content, altered oil content, increased biomass per plant, Increased tiller number, increased stem length, increased root length, improved root architecture, increased seed weight, altered seed carbohydrate composition, altered seed oil composition, increased radicle length, delayed senescence, retention of green color, altered seed protein composition, increased dry weight of mature plant propagation elements, increased fresh weight of mature plant propagation elements, increased number of mature plant propagation elements per plant, increased chlorophyll content, reduced number of wilted leaves per plant, reduced number of severely wilted leaves per plant, increased number of non-wilted leaves per plant, and / or improved visual appearance of the plant.

[0150] In an embodiment, the present invention relates to the use of microorganisms and nutrient delivery systems as biofertilizers.

[0151] As used herein, the term "biofertilizer" refers to a substance containing one or more nutrients (e.g., nitrogen, phosphorus, and / or potassium) and live microorganisms that, when applied to a plant, a part thereof (e.g., roots), a seed for growing the plant, or the locus of the plant (e.g., the soil surrounding the plant or the plant growth medium), colonizes the locus of the plant or plant structure and promotes growth by increasing the availability of nutrients to the plant.

[0152] In embodiments, the present invention relates to the use of microorganisms and nutrient delivery systems as biocontrol agents.

[0153] As used herein, the term "biological control agent" refers to a substance containing one or more microorganisms that are capable of reducing the population of or preventing the effects of potential pathogens. When applied to a plant, a part thereof (e.g., roots), a seed from which the plant is grown, or the locus of the plant (e.g., the soil surrounding the plant or the plant growth medium), the biological control agent significantly reduces the incidence and severity of plant diseases. This mode of action may be due to the biocontrol agent outcompeting the pathogen for a niche, secreting or containing substances that are toxic to the pathogen, parasitizing the pathogen, or some combination of these or other effects.

[0154] The use may entail applying or applying the microorganism and nutrient delivery system according to the present invention to the plant, a part thereof (e.g., roots), a seed for growing the plant, or the locus of the plant (e.g., the soil surrounding the plant or plant growth medium).

[0155] Reference to plants includes any plant. The plants may include wild plants and domesticated varieties. In certain embodiments, the plants may be agricultural plants. The term "agricultural plants" or "crops" includes plants grown by humans for, but not limited to, food, feed, fiber, fuel, horticultural, and / or industrial purposes.

[0156] As used herein, the phrase "part of a plant" or "plant part" refers to any one or more parts of a plant, such as any one or more of a seed, shoot, stem, leaf, root (including tuber), flower, etc. Furthermore, "plant part" is intended to refer generally to any part of a plant that is capable of initiating other plants through sexual or asexual reproduction of the plant, such as, but not limited to, a seed, seedling, root, shoot, cutting, scion, graft, runner, bulb, tuber, corm, keikis, or bud. In certain embodiments, the microorganism and nutrient delivery system are applied to a seed.

[0157] When treated with the microorganisms and nutrient delivery systems according to the present invention, the plant part can be attached to (e.g., grown on) the whole plant, or can be detached from (e.g., not grown on) the whole plant. For example, a seed can be detached from (e.g., not grown on) the whole plant when treated with the microorganisms and nutrient delivery systems of the present invention.

[0158] As used herein, the phrase "plant site" or "plant growing site" refers to the immediate vicinity of a plant (including parts thereof, such as seeds). For example, the plant site can be a circular area around the plant (e.g., around the seed), such as a circular area around the plant (e.g., around the seed) having a diameter of at most 1 meter, such as at most 50 centimeters (cm), at most 40 cm, at most 30 cm, at most 20 cm, at most 10 cm, or at most 5 cm. The growing site can include a growing medium (e.g., soil, hydroponic medium, or aquaponic medium) for growing the plant.

[0159] Phrase " application " generally refers to the configuration, application, delivery or provision of the narrated object (for example microorganism and nutrient delivery system) driven or implemented by people and / or machines to receptor entity (for example plant, its part, for the seed of growing plant or plant site).Microorganism and nutrient delivery system taught herein can be used by any known method, wherein all or part of the plant is processed, for example, by root or seed inoculation.For example, described application can be for the root of described plant, before seed is planted in soil for described plant seed, or for soil or plant growth medium around described plant or plant seed.Can adopt application method such as spraying, coating, covering, contact and / or dipping.In certain embodiments, application can be for surface, for example the surface of the plant of growth medium (for example soil), plant, plant part, seed, the plant of results or plant part (for example root, bulb or tuber of results).In certain embodiments, described application can be for plant, its part or plant site existing on field or farmland.

[0160] In certain embodiments, the microorganisms and nutrient delivery systems taught herein can be applied to a plant site, such as by inoculating soil or a growth medium. Thus, in certain embodiments, the method comprises inoculating soil or a plant growth medium with the microorganisms and nutrient delivery system and growing the plant in the soil or medium.

[0161] As used herein, the terms "growth medium" or "plant growth medium" refer to a matrix or medium used to grow plants. The growth medium can be soil, compost, peat, coconut fiber, wood fiber, a matrix that simulates soil such as a mineral lava or basalt matrix, a textile, or a soilless matrix. For example, the growth medium can be sand, gravel, polysaccharides, topsoil, peat moss, straw, logs, clay, or a combination thereof. The plant growth medium can also include a hydroponic system or an in vitro culture system. The plant growth medium can also be a hydroponic medium or a hydroponic medium. It will be understood by those skilled in the art that different types of growth media can be used to grow different types of plants. For example, liquids, powders, granules, or pellets can be used to inoculate the plant growth medium. For example, aquatic plants can be grown in granules or pellets of the microorganism and nutrient delivery system described herein.

[0162] Inoculation of plant growth media with the microorganisms and nutrient delivery systems taught herein can be performed during and / or after sowing, or before, during, or after the start of the plant growth cycle in the case of hydroponics or in vitro cultivation. The inoculation can be performed once or multiple times during the plant growth cycle.

[0163] In certain embodiments, the sprayable liquid can be applied by spraying the plant, parts thereof, or plant loci, preferably the plant loci, with conventional spraying equipment known in the art (e.g., aircraft, backpack sprayers, tractor-mounted boom sprayers, etc.).

[0164] In certain embodiments, the microorganisms and nutrient delivery systems taught herein can be applied directly to plants, parts thereof, or plant growth sites, or by acting on their surroundings or habitats using conventional treatment methods, such as by dipping, pouring, spraying, coating, atomizing, irrigating, evaporating, dusting, misting, broadcasting, foaming, painting, smearing, watering (drenching), or drip irrigation. For example, the application may include spraying, sprinkling, showering, spurting, spreading in droplet form, splashing; dispersing, spreading, or rinsing the plants, parts thereof, or plant growth sites with the microorganisms and nutrient delivery system.

[0165] Also disclosed herein are uses of the microorganisms and nutrient delivery systems for environmental applications, such as as bioremediation agents.

[0166] Thus, another aspect relates to the use of the microorganisms and nutrient delivery systems taught herein as bioremediation agents.

[0167] "Bioremediation" generally refers to the remediation of contaminated soil by harnessing the ability of certain microorganisms to convert harmful substances into non-toxic compounds. The key requirements for effective bioremediation are biodegradable organic matter, an appropriate active microbial community (population), and bioavailability of the contaminants. Bioremediation may also require nutrients for the microorganisms.

[0168] According to the present invention, a slurry or wet hydrochar from an HTC process is inoculated with an appropriate microorganism or blend of microorganisms and fermented, causing the microorganisms to attach to the fermented hydrochar and colonize its pores. The fermentation product, optionally after processing steps as described elsewhere herein, can be applied to contaminated soil. The hydrochar serves as a nutrient source for the growth and proliferation of the microorganisms in the system and provides protection against predation by protozoa. Furthermore, due to the fermentation of the hydrochar, competition with indigenous soil microorganisms can be reduced.

[0169] In other aspects, the present invention relates to the use of the microorganisms and nutrient delivery system as a microbial inoculant, for example, for anaerobic digesters, septic tank systems, water treatment systems, etc. Depending on the specific application, a skilled person can select suitable microorganisms for the microbial inoculant.

[0170] In certain embodiments, the microorganism and nutrient delivery system can be (repeatedly) used as a microbial inoculant in the methods of the invention.

[0171] Reference Attachment Figure 1 The present invention is explained in more detail. Figure 1 A schematic diagram is provided illustrating an embodiment for implementing a method according to the present invention, without limiting the invention to the specific steps and parameters shown. In the method, biomass (101) is subjected to a hydrothermal carbonization process (10) to form a slurry (103) comprising hydrothermal char and process water. The slurry is cooled (20) to a temperature suitable for the growth of a microbial inoculant. The cooled slurry (105) can be inoculated with a microbial inoculant (109) and then subjected to a fermentation process (40), particularly immersion fermentation.

[0172] Optionally, before the inoculation step, the cooled slurry (105) may first be subjected to a solid-liquid separation (30). The solid-liquid separation reduces the process water content of the slurry to obtain a wet hydrochar (106). The process water (107) separated from the wet hydrochar (106) may be recycled to the HTC process (10). The solid-liquid separation step also allows for easy addition of additional ingredients (110) to the wet hydrochar, and it allows for the addition of microbial inoculum (109) to the wet hydrochar in a convenient manner. The fermentation (40) of the wet hydrochar (106) may be a solid-state fermentation.

[0173] Additional ingredients (110) such as nutrients may be added at various steps of the process, e.g., before the HTC process (10), during solid-liquid separation (30), before fermentation (40), etc., to promote the growth of beneficial microorganisms or to adjust the composition of the microorganisms and nutrient delivery system.

[0174] The fermentation product (111) comprising fermented hydrothermal char can be used, for example, as a microbial and nutrient delivery system.

[0175] Alternatively, the fermentation product (111) can be subjected to one or more processing steps (50), such as a solid-liquid separation step, a drying step, grinding, milling, granulation, etc., and / or formulation (50) (optionally with one or more co-formulators (112)) to provide a formulated microorganism and nutrient delivery system (113).

[0176] Statements. In these statements, the phrase "[the subject matter] according to statement [number], wherein ..." or "[the subject matter] [the subject matter] according to any one of statement [number], wherein ..." also discloses the simple phrase "in certain embodiments ..." and may be used in place of it.

[0177] Statement 1. A method for producing a microorganism and nutrient delivery system (113), the method comprising:

[0178] - providing biomass (101);

[0179] - subjecting the biomass (101) to a hydrothermal carbonization process (HTC) (10) to form a slurry (103) comprising hydrothermal char and HTC process water (in particular HTC process water);

[0180] - cooling (20) the slurry (103) to a temperature suitable for the growth of the microbial inoculant;

[0181] - inoculating the cooled slurry (105) with the microbial inoculant (109);

[0182] - subjecting the inoculated slurry to a fermentation process (40) to form a fermentation product (111); and

[0183] - Obtaining or recovering (50) the microorganism and nutrient delivery system (113) from the fermentation product.

[0184] Statement 2. The method of statement 1 further comprising the step (30) of reducing the process water (particularly HTC process water) content of the slurry (103, 105) to form a wet hydrothermal char (106) prior to inoculation with the microbial inoculant.

[0185] Statement 3. The method of statement 1 or 2, wherein the cooling step (20) and the inoculation step are performed under sterile conditions.

[0186] Statement 4. The method according to statement 2 or 3, wherein the cooling step (20), the inoculation step and the step of reducing the process water (especially HTC process water) content of the slurry (30) are carried out under sterile conditions.

[0187] Statement 5. A method according to any one of statements 2 to 4, wherein the process water (particularly HTC process water) content of the slurry is reduced (30) by a mechanical process, preferably solid-liquid separation such as decantation, centrifugation or filtration.

[0188] Statement 6. The method of any one of statements 1 to 5, wherein the fermentation process (40) is solid-state fermentation.

[0189] Statement 7. The method of any one of statements 1 to 5, wherein the fermentation process (40) is submerged fermentation.

[0190] Statement 8. The method of any one of statements 1 to 7, wherein the fermentation process is a single-stage fermentation.

[0191] Statement 9. The method of statement 8, wherein the fermentation is performed under sterile conditions.

[0192] Statement 10. A method according to any one of statements 1 to 7, wherein the fermentation process is a multi-stage fermentation, such as a two-step fermentation, wherein the fermentation process includes fermenting the inoculated slurry to form a first fermentation product, and then inoculating a second slurry formed in the HTC process with the first fermentation product and fermenting the inoculated second slurry to form the (second) fermentation product.

[0193] Statement 11. The method of statement 10, wherein the fermentation of the (first) slurry inoculated with the microbial inoculant is performed under sterile conditions.

[0194] Statement 12. The method of statement 10 or 11, wherein fermentation of the (second) slurry inoculated with the first fermentation product is performed under non-sterile conditions.

[0195] Statement 13. The method of any one of statements 1 to 12, wherein the fermentation is carried out at a temperature below 85°C, preferably below 75°C, more preferably between 15°C and 70°C or between 15°C and 65°C or between 15°C and 60°C.

[0196] Statement 14. The method of any one of statements 1 to 13, wherein the slurry (103) or the wet hydrochar is cooled (20) to a temperature between about 4°C and about 65°C, preferably between about 20°C and about 50°C, more preferably between about 20°C and about 30°C.

[0197] Statement 15. The method of any one of statements 1 to 14, wherein the cooling (20) is performed using a heat exchanger.

[0198] Statement 16. The method of any one of statements 1 to 15, wherein the heat released in the cooling step (20) is utilized in the hydrothermal carbonization process (10).

[0199] Statement 17. A method according to any one of statements 1 to 16, wherein one or more nutrients (110) such as a carbon source are added to the slurry or wet hydrochar prior to the fermentation process, and / or wherein the pH of the slurry or wet hydrochar is adjusted to a pH suitable for growth of the microbial inoculant prior to the fermentation process.

[0200] Statement 18. The method of any one of statements 1 to 17, wherein the hydrothermal carbonization process (10) is at a temperature between about 180°C and about 250°C, preferably between about 200°C and about 220°C.

[0201] Statement 19. The method of any one of statements 1 to 18, wherein the hydrothermal carbonization process (10) is carried out at a pressure between about 10 bar and about 50 bar, preferably between about 15 bar and about 25 bar.

[0202] Statement 20. A method according to any one of statements 1 to 19, wherein the hydrothermal carbonization process (10) is carried out for a period of at least 30 minutes, preferably between about 30 minutes and about 8 hours, more preferably between about 30 minutes and about 4 hours, and even more preferably between about 2 hours and about 3 hours.

[0203] Statement 21. The method of any one of statements 1 to 20, wherein the hydrothermal carbonization process (10) is carried out at a temperature between about 180°C and about 250°C and a pressure between about 10 bar and 50 bar for at least 30 minutes.

[0204] Statement 22. The method of any one of statements 1 to 21, wherein the hydrothermal carbonization process (10) is carried out at a temperature between about 200°C and about 220°C and a pressure between about 15 bar and about 25 bar for about 2 hours to 3 hours.

[0205] Statement 23. The method of any one of statements 1 to 22, wherein the biomass (101) is a wet biomass having a water content between 10% and 95% by weight, preferably between 10% and 90% by weight.

[0206] Statement 24. The method of any one of statements 1 to 23, wherein the biomass (101) is selected from food waste, agricultural waste, animal by-products, or any combination thereof.

[0207] Statement 25. The method of any one of statements 1 to 24, wherein the biomass (101) comprises animal byproducts such as manure.

[0208] Statement 26. The method of any one of statements 1 to 25, wherein the microbial inoculant (109) comprises one or more bacteria belonging to a genus selected from the group consisting of Achromobacter, Aeromonas, Azospirillum, Bacillus, Bradyrhizobium, Enterobacter, Gluconacetobacter, Klebsiella, Pseudomonas, Rhizobia, Rhizobium, and Streptomyces, or selected from the group consisting of Achromobacter, Aeromonas, Azospirillum, Bacillus, Bradyrhizobium, Enterobacter, Gluconacetobacter, Klebsiella, Pseudomonas, Rhizobia, Rhizobium, and Streptomyces. r), Bacillus, Bradyrhizobium, Enterobacter, Gluconacetobacter, Klebsiella, Pseudomonas, Rhizobia, Rhizobium and Streptomyces, for example a genus selected from the group consisting of Streptomyces, Bacillus, Azospirillum and Bradyrhizobium or a genus selected from the group consisting of Azotobacter, Streptomyces, Bacillus, Azospirillum and Bradyrhizobium.

[0209] Statement 27. The method of any one of statements 1 to 26, wherein the microbial inoculant (109) comprises one or more filamentous fungi belonging to a genus selected from the group consisting of Alternaria, Aspergillus, Chaetomium, Exophiala, Fusarium, Penicillium, Phoma, Talaromyces, and Trichoderma, or a filamentous fungus selected from the group consisting of Alternaria, Aspergillus, Chaetomium, Exophiala, Fusarium, Penicillium, Phoma, Talaromyces, and Trichoderma. gillus, Beauveria, Chaetomium, Exophiala, Fusarium, Penicillium, Phoma, Talaromyces and Trichoderma, for example a genus selected from the group consisting of Trichoderma and Penicillium or a genus selected from the group consisting of Beauveria, Trichoderma and Penicillium.

[0210] Statement 28. The method of any one of statements 1 to 27, wherein the recovering step (50) comprises separating the fermentation product into a solids-rich fraction and a liquid fraction.

[0211] Statement 29. The method of any one of statements 1 to 28, wherein the recovering step (50) comprises drying the fermentation product or the solids-rich fraction of the fermentation product.

[0212] Statement 30. The method of any one of statements 1 to 29, wherein the recovering step (50) comprises the step of separating spores from the fermentation product, wherein the microbial inoculant comprises spore-forming microorganisms.

[0213] Statement 31. The method of any one of statements 1 to 30, wherein the recovery step (50) comprises a processing step selected from the group consisting of grinding, milling, and granulation, or wherein the recovery step (50) comprises one or more of the processes of grinding, milling, granulation, extrusion, and micronization.

[0214] Statement 32. A microorganism and nutrient delivery system obtainable by the method of any one of statements 1 to 31, the system comprising:

[0215] - Fermented hydrocharcoal;

[0216] - Microorganisms attached to the surface or pores of the fermented hydrothermal charcoal.

[0217] Statement 33. A microorganism and nutrient delivery system according to statement 32, wherein at least 50%, preferably at least 55%, 60%, 65%, 70% or 75%, more preferably at least 80%, 82%, 85%, 86%, 88% or 90%, for example at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% of the microorganisms in the microorganism and nutrient delivery system are composed of microbial species contained in the microbial inoculant.

[0218] Statement 34. The microorganism and nutrient delivery system of statement 32 or 33, further comprising one or more adjuvants, such as one or more adjuvants selected from solvents, carriers, binders, surfactants, adhesives, viscosity enhancers, antifreeze agents, thickeners, buffers, defoaming agents, antioxidants, preservatives, stabilizers, fragrances, and colorants.

[0219] Statement 35. Use of a microorganism and a nutrient delivery system according to any one of statements 32 to 34 as a biostimulant.

[0220] Statement 36. Use of a microorganism and a nutrient delivery system according to any one of statements 32 to 34 as a biofertilizer.

[0221] Statement 37. Use of a microorganism and nutrient delivery system according to any one of statements 32 to 34 as a biocontrol agent.

[0222] Statement 38. Use of a microorganism and nutrient delivery system according to any one of statements 32 to 34 as a bioremediation agent.

[0223] Statement 39. Use of the microorganism and nutrient delivery system of any one of statements 32 to 34 as a microbial inoculant, for example, for an anaerobic digester, a septic system, or a water treatment system.

[0224] Statement 40. Use of the microorganism and nutrient delivery system of any one of statements 32 to 34 as a microbial inoculant in the method of any one of statements 1 to 31.

[0225] Although the present invention has been described in conjunction with specific embodiments thereof, it is apparent that many alternatives, modifications and variations will be apparent to those skilled in the art based on the above description. It is therefore intended that all such alternatives, modifications and variations below be included within the spirit and scope of the appended claims.

[0226] The following non-limiting examples further support the aspects and embodiments of the invention disclosed herein.

[0227] Example

[0228] Example 1: Microbial Growth on Hydrothermal Charcoal and Process Water in Agar

[0229] Materials and methods:

[0230] Hydrochar was produced at the Ingelia SLHTC plant (València, Spain). The feedstock used to produce the hydrochar was pig manure (HC-C). Hydrothermal carbonization was performed at 210°C and 20-25 bar for 3 to 4 hours.

[0231] The slurry obtained after hydrothermal carbonization was separated into (wet) hydrothermal char and process water fractions using gravity filtration.

[0232] As reference material, golden peat moss from Baltic origin (Novabalt) was used.

[0233] Three types of agar plates were prepared and sterilized by autoclaving (121°C for 15 minutes):

[0234] PW agar: Dilute 1.5% w / v agar in process water (adjust to pH 7 using 1M sodium hydroxide)

[0235] HC agar: Dilute 2% w / v hydrocharcoal HC-C and 1.5% w / v agar in reverse osmosis water.

[0236] HC+PW agar: Dilute 2% w / v hydrocharcoal HC-C and 1.5% w / v agar in process water (adjust to pH 7 with 1M sodium hydroxide)

[0237] ● Peat agar: Dilute 2% w / v peat and 1.5% w / v agar in reverse osmosis water.

[0238] Bacterial strains Streptomyces griseoviridis (LMG19321), Bacillus subtilis (LMG 23370), Azospirillum brasilense (LMG28319) and Bradyrhizobium japonicum (LMG 4252), as well as fungal strains Trichoderma harzianum (MUCL 22194) and Penicillium bilaiae (MUCL31187) were obtained from the Belgian Coordinated Collection of Microorganisms (BCCM). All strains were revived and grown on yeast mannitol agar (YMA) medium at 28°C for bacterial strains and 25°C for fungal strains. Agar for microbiology (900040) and yeast mannitol agar NutriSelect Plus (900050) were purchased from Merck Life Sciences BV (Belgium).

[0239] The six different strains were inoculated with an inoculating loop by streaking pure cultures from YMA plates onto four custom agar plates in a laminar air flow cabinet. For bacterial and fungal strains, the plates were inoculated under normal atmosphere at 28°C and 25°C, respectively. After four days, growth on the culture medium was visually scored.

[0240] result:

[0241] Table 1: Growth of selected microorganisms on PW agar, HC agar, HC+PW agar, and peat agar after 4 days. Scoring system: 0: no colonies visible; +: some colonies visible, minimal growth; ++: good growth, many colonies; +++: excessive growth, diffuse colonies; C: contamination.

[0242] microorganism PW agar HC agar HC+PW agar Peat agar Streptomyces glaucum 0 +++ + C Bacillus subtilis + + ++ 0 Azospirillum brasiliensis + ++ + C Bradyrhizobia soybean 0 + ++ C Trichoderma harzianum + ++ +++ 0 Penicillium bilairum 0 ++ +++ ++

[0243] The results in Table 1 show that all six selected microbial strains were able to grow well on HC agar and HC + PW agar, indicating that the hydrochar (optionally combined with process water) provided the necessary nutrients for uninhibited microbial growth. No contamination was observed on PW agar, HC agar, or HC + PW agar plates.

[0244] Only Penicillium bilaiae was able to grow on peat agar. Most peat agar plates showed contamination with spores that were not destroyed during the standard sterilization process at 121°C. The lack of nutrients and the risk of contamination indicate that peat performs poorly as a growth medium for the selected microorganisms.

[0245] Example 2: Microbial Growth on Hydrochar and Process Water (No Agar)

[0246] Materials and methods:

[0247] Hydrochar was produced at the Ingelia SLHTC plant (València, Spain). The feedstock used to produce the hydrochar was cow manure (HC-A), pig manure (HC-C), or lignocellulosic biomass (HC-BM). Hydrothermal carbonization was performed at 210°C and 20-25 bar for 3 to 4 hours. The slurry obtained after hydrothermal carbonization was separated into (wet) hydrochar and process water fractions using gravity filtration.

[0248] As reference material, golden peat moss from Baltic origin (Novabalt) was used.

[0249] The same microorganisms and microorganism culture conditions as in Example 1 were used.

[0250] Three types of hydrochar (containing approximately 50% by weight of moisture (process water)) and peat (moistened with reverse osmosis water) were sterilized for 15 minutes at 121° C. The materials were distributed in empty (sterile) Petri dishes (approximately 1 g of dry matter each) in a laminar air flow cabinet.

[0251] The material was inoculated from microbial cultures grown on YMA agar plates using an inoculating loop. The plates were wrapped with parafilm to prevent moisture loss. For bacterial and fungal strains, the material was incubated at 28°C and 25°C, respectively, and subjected to solid-state fermentation processes.

[0252] After 7 days, growth was assessed visually and scored. For HC-C samples, growth was quantified as follows: the dried material was resuspended in sterile physiological water (0.8% NaCl) and 10 -2 to 10 -10 The number of colony forming units (CFU) in the dilution groups was determined by plating on YMA plates.

[0253] result:

[0254] Table 2: Growth of selected microorganisms on hydrochar (HC) from cow dung (HC-A), pig dung (HC-C) or lignocellulosic biomass (HC-B) or peat after 7 days. Scoring system: 0: no colonies visible; +: some growth visible; ++: good growth, clearly visible; +++: excessive growth, diffuse colonies; NVC: no visible growth.

[0255] microorganism HC-A HC-C HC-BM peat Streptomyces glaucum ++ ++ ++ ++ Bacillus subtilis NVC NVC NVC NVC Azospirillum brasiliensis NVC NVC NVC NVC Bradyrhizobia soybean + ++ ++ + Trichoderma harzianum + ++ ++ 0 Penicillium bilairum ++ ++ +++ 0

[0256] With the exception of Bacillus subtilis and Azospirillum brasilense, which produced no visible signs of growth of the microorganisms, the selected microorganisms could grow on the three different types of hydrochar without providing any other nutrients (Table 2).

[0257] Table 3: Quantification of selected microbial growth after 7 days on hydrothermal charcoal from swine manure (HC-C).

[0258] microorganism Average value (CFU / g) Standard Deviation Streptomyces glaucum 3,88E+08 1,06E+08 Bacillus subtilis 1,18E+08 7,10E+07 Azospirillum brasiliensis 1,81E+07 1,58E+07 Bradyrhizobia soybean 1,03E+08 5,18E+07 Trichoderma harzianum 1,40E+08 1,09E+08 Penicillium bilairum 4,23E+07 2,81E+07

[0259] CFU counts confirmed that each of the selected microbial strains could be grown to industrially relevant concentrations on HC-C (Table 3), even Bacillus subtilis and Azospirillum brasilense, which showed no visible signs of growth. No contamination was observed on the CFU count agar plates.

[0260] Example 3: Microbial growth on hydrochar and process water compared to peat

[0261] Materials and methods:

[0262] Hydrochar was produced from pig manure (HC-C) as described in Examples 1 and 2. The hydrochar slurry was separated into (wet) hydrochar and process water fractions by filtration using a chamber filter press.

[0263] As reference material, golden peat moss from Baltic origin (Novabalt) was used.

[0264] The bacterial strain Bradyrhizobium japonicum (LMG 4252) and the fungal strain Trichoderma harzianum (MUCL 22194) from the Belgian Coordinated Collection of Microorganisms (BCCM) were used. All strains were revived and grown in yeast mannitol broth (YMB) medium at 25° C. in shake flasks to obtain liquid inocula. The concentration of colony-forming units in the liquid inoculum was quantified by CFU counting.

[0265] In a laminar air flow cabinet, 1 mL of the liquid inoculum was diluted to 50 mL in sterile saline (0.8% NaCl). The diluted inoculum was used to inoculate hydrothermal charcoal (HC-C; 100 g dry matter) and peat (100 g dry matter) to ensure good distribution of the inoculum throughout the material. All materials were adjusted to a moisture content of 40% using reverse osmosis water. The materials were aerobically incubated in a sterile environment to perform the solid-state fermentation process.

[0266] After 7 days, microbial growth was quantified as follows: the fermented hydrochar and peat were resuspended in sterile physiological water (0.8% NaCl) and 10 -4 to 10 -10 The number of colony forming units (CFU) in the dilution groups was determined by plating on YMA plates.

[0267] result:

[0268] Table 4: Quantification of growth of B. japonicum and T. harzianum after 7 days on hydrothermal charcoal (HC-C) or peat from pig manure.

[0269] microorganism substrate Average value (CFU / g) Standard Deviation Bradyrhizobia soybean HC-C 1,32E+08 3.06E+07 Bradyrhizobia soybean peat NVC Trichoderma harzianum HC-C 1,36E+09 6,47E+08 Trichoderma harzianum peat 9,42E+05 3,3E+05

[0270] Comparative analysis of microbial growth (measured in CFU / g) showed that the concentration of microbial activity was significantly higher on fermented HC-C compared to fermented peat (Tables 4 and Figure 2 Bradyrhizobium japonicum did not grow on the peat. These results suggest that, in contrast to peat, HC-C has the potential to serve as a nutrient source for a wider range of microorganisms.

[0271] Example 4: Shelf life of fermented hydrothermal charcoal

[0272] Materials and methods:

[0273] Hydrochar was produced from pig manure (HC-C) as described in Examples 1 and 2. The hydrochar slurry was separated into (wet) hydrochar and process water fractions by filtration using a chamber filter press.

[0274] Liquid inocula of the bacterial strain Azospirillum brasilense (LMG28319) and the fungal strain Trichoderma harzianum (BCCM: MUCL 22194) were prepared as described in Example 3.

[0275] In a laminar air flow cabinet, 1 mL of the liquid inoculum was diluted to 50 mL in sterile saline solution (0.8% NaCl). The diluted liquid inoculum was mixed with the wet hydrochar (HC-C; 100 g dry matter) in duplicate. The moisture content of all samples was adjusted to 40% with reverse osmosis water. The samples were aerobically incubated in a sterile environment for 7 days to perform the solid-state fermentation process (fermented hydrochar). After drying, the material was distributed in multiple aluminum foil-lined packages, which were separated and stored at 4°C or 25°C to assess shelf life.

[0276] As a reference, a commercially available reference of the fungal strain Trichoderma harzianum formulated on mineral clay was included.

[0277] After 0, 1, 7, 14, 28, 42 and 56 days of storage, CFU counts were measured as follows: the fermented hydrochar and reference were resuspended in sterile physiological water (0.8% NaCl) and 10 -1 to 10 -7 The number of colony forming units (CFU) in the dilution groups was determined by plating on YMA plates.

[0278] result:

[0279] Table 5: Quantification of growth of Trichoderma harzianum on fermented hydrothermal charcoal (HC-C) and formulated with mineral clay (reference) at different storage temperatures.

[0280]

[0281] Table 5 shows that T. harzianum fermented on hydrothermal charcoal had a longer shelf life compared to the commercially available reference of formulations containing mineral clay at different storage temperatures, resulting in higher CFU counts after storage.

[0282] Table 6: Quantification of the growth of Azospirillum brasilense on fermented hydrothermal charcoal (HC-C) at different storage temperatures.

[0283]

[0284] Table 6 shows that the A. brasilense population in the fermented hydrothermal charcoal exhibited a longer shelf life compared to A. brasilense formulated with PBS, resulting in higher CFU counts after storage at both 4°C and 25°C; with counts reaching 5.5E+07 CFU / mL and 1.77E+05 CFU / mL after 60 days of storage at 4°C and 28°C, respectively. and Bonilla, (2015), African Journal of Biotechnology 14:2547-2553. Figure 2 A and 2C).

[0285] Example 5: Resistance of fermented hydrothermal charcoal to desiccation and oxidative stress

[0286] Materials and methods:

[0287] Hydrochar was produced from pig manure (HC-C) as described in Examples 1 and 2. The hydrochar slurry was separated into (wet) hydrochar and process water fractions by filtration using a chamber filter press.

[0288] Liquid inocula of the bacterial strain Bacillus subtilis (BCCM: LMG23370) and the fungal strain Trichoderma harzianum (BCCM: MUCL 22194) were prepared as described in Example 3.

[0289] 1 mL of the liquid inoculum was diluted to 50 mL in a sterile saline solution (0.8% NaCl). The diluted liquid inoculum was mixed with the wet hydrochar (HC-C; 100 g dry matter) in duplicate. The moisture content of all materials was adjusted to 40% with reverse osmosis water. One sample was immediately placed in a desiccator and allowed to air dry at 25°C (microorganisms prepared on hydrochar). Another sample was aerobically incubated for 7 days in a sterile environment to perform a solid-state fermentation process (fermented hydrochar). After drying (microorganisms prepared on hydrochar) or solid-state fermentation (fermented hydrochar), the materials were distributed on multiple air-permeable and water-permeable packages, which were stored at 25°C to evaluate the shelf life under desiccation and oxidative stress.

[0290] After 0, 1, 7, 14, 28 and 56 days of storage, CFU counts were measured as follows: the fermented hydrochar and the microorganisms formulated with hydrochar were resuspended in sterile physiological water (0.8% NaCl) and 10 -4 to 10 -10 The number of colony forming units (CFU) in the dilution groups was determined by plating on YMA plates.

[0291] result:

[0292] Higher CFU counts were obtained in fermented hydrothermal charcoal during storage and after environmental stress.

[0293] Example 6: Anti-pollution properties of fermented hydrothermal charcoal

[0294] Materials and methods:

[0295] Hydrochar was produced from pig manure (HC-C) as described in Examples 1 and 2. The hydrochar slurry was separated into (wet) hydrochar and process water fractions by filtration using a chamber filter press.

[0296] A liquid inoculum of the fungal strain Trichoderma harzianum (BCCM: MUCL22194) was prepared as described in Example 3.

[0297] In a laminar air flow cabinet, 1 mL of the liquid inoculum was diluted to 50 mL in sterile saline solution (0.8% NaCl). The diluted liquid inoculum was mixed with the wet hydrochar (HC-C; 100 g dry matter). The moisture content of the material was adjusted to 40% with reverse osmosis water and incubated aerobically in a sterile environment for 7 days to perform a solid-state fermentation process (fermented hydrochar).

[0298] The other (control) sample was wet hydrothermal charcoal (HC-C; 100 g dry matter) that was not inoculated or fermented with microorganisms and adjusted to a moisture content of 40% with reverse osmosis water (control).

[0299] The non-sterile samples, fermented hydrothermal charcoal and control, were both exposed to the outside air for 2 hours to allow for contamination by microorganisms. After two hours, both materials were incubated again in a sterile environment at 25°C for 7 days.

[0300] Microbial growth was visually observed and quantified as follows: the fermented hydrochar and wet hydrochar (control) were resuspended in sterile physiological water (0.8% NaCl) and 10 -1 to 10 -7The number of colony forming units (CFU) in the dilution groups was determined by plating on nutrient agar (NA) plates.

[0301] result:

[0302] The growth of contaminating microorganisms was significantly reduced on the fermented hydrochar compared to the control hydrochar ( Figure 3 Our analysis showed that the only microbial species in the fermented hydrochars was Trichoderma harzianum, whether exposed to the outside air (non-sterile) or not (sterile) before fermentation (Tables 7 and Figure 3 ). These observations suggest that the fermentation process of the desired microorganisms is effective in creating an environment that is less favorable for the growth of undesirable microorganisms.

[0303] Table 7: Quantification of the growth of T. harzianum on control and fermented hydrothermal charcoal from pig manure (HC-C) after 7 days.

[0304]

[0305] Example 7: Immersed Fermentation on Hydrothermal Charcoal

[0306] Materials and methods:

[0307] Hydrothermal charcoal was produced from pig manure as described in Examples 1 and 2 (HC-C).

[0308] Liquid inocula of the bacterial strain Azospirillum brasilense (LMG28319) and the fungal strain Trichoderma harzianum (BCCM: MUCL 22194) were prepared as described in Example 3.

[0309] Four different liquid fermentation media were prepared and sterilized by autoclaving (121 °C for 15 min):

[0310] 50 g of HC-C (dried at 105°C) was diluted to 1 L (HC-C) with saline solution (0.8% NaCl)

[0311] ● Dilute 50g of process water to 1L with brine solution (0.8% NaCl) (PW)

[0312] 50g HC-C (dried at 105°C) and 50g process water were diluted to 1L (HC-C + PW) with brine solution (0.8% NaCl)

[0313] 1 L saline solution (0.8% NaCl) - (control)

[0314] 100 mL of fermentation medium was distributed in triplicate in 250 mL shake flasks. The shake flasks were inoculated with 1 mL of liquid inoculum and incubated in a shaker at 25°C and 150 rpm for 3 days.

[0315] A. brasilense growth was quantified by CFU counts (diluted in saline and plated on YMA medium) for each shake flask. For T. harzianum, growth under each liquid fermentation condition was quantified by measuring the dry mass of mycelium (which is typically produced in liquid medium fermentations). The mycelial mat was filtered onto filter paper (e.g., Whatman No. 1) and dried overnight at 50°C.

[0316] result:

[0317] Table 8: Quantification of the growth of A. brasilense in the different liquid fermentation media tested.

[0318] Liquid fermentation medium Average value (CFU / g) Standard Deviation comparison 7,40E+05 1,22E+05 HC-C 2,28E+07 1,28E+07 PW 4,30E+07 1,43E+07 HC-C+PW 1,49E+08 3,87E+07

[0319] Table 8 shows that the highest population of A. brasilense occurred in the liquid fermentation containing HC-C and PW as nutrient sources for the microorganisms.

[0320] Table 9: Dry mass of mycelium of T. harzianum grown in the different liquid fermentation media tested.

[0321] Liquid fermentation medium Dry mass (g) comparison 0,023 HC-C 0,093 PW 0,459 HC-C+PW 0,878

[0322] Table 9 illustrates that the most abundant populations of T. harzianum were observed in liquid fermentations incorporating HC-C and PW as sources of microbial nutrients.

[0323] Example 8: Microbial Growth on Hydrochar and Process Water (Without Agar)

[0324] Materials and methods:

[0325] Hydrothermal charcoal was produced from pig manure as described in Examples 1 and 2 (HC-C).

[0326] Bacterial strains Azotobacter chroococcum (LMG 3852) and Pseudomonas fluorescens (LMG 1244) and the fungal strain Beauveria bassiana (IHEM 3558) were obtained from the Belgian Coordinated Collection of Microorganisms (BCCM).

[0327] HC-C (containing about 50% by weight of moisture (process water)) was distributed into microcartridge containers (50 g of dry matter per container). These microcartridges were sterilized at 121° C. for 15 minutes.

[0328] In a laminar air flow cabinet, an inoculation loop taken from a microbial culture grown on a YMA agar plate was diluted in 50 mL of process water (PW) and then introduced into the material. The microboxes with the inoculated HC-C were incubated at 28°C and 25°C for bacterial and fungal strains, respectively, and the solid-state fermentation process was carried out.

[0329] After 7 days, growth was visually assessed and scored. For HC-C samples, growth was quantified as follows: the dried material was resuspended in sterile physiological water (0.8% NaCl) and 10 -1 to 10 -7 The number of colony forming units (CFU) in the dilution groups was determined by plating on YMA plates.

[0330] result:

[0331] Table 10: Quantification of the growth of selected microorganisms on hydrothermal charcoal from pig manure (HC-C) after 7 days.

[0332] microorganism Average value (CFU / g) Standard Deviation Brown spherical nitrogen-fixing bacteria 1,10E+07 1,91E+06 Pseudomonas fluorescens 9,52E+08 5,52E+07 Beauveria bassiana 3,33E+08 7,7E+07

[0333] The CFU quantification confirmed the successful cultivation of Azotobacter chroococcum, Pseudomonas fluorescens, and Beauveria bassiana on HC-C at concentrations that were industrially relevant (Table 10). Notably, no contamination was observed on the CFU count agar plates.

[0334] Example 9: Importance of Maintaining a Sterile Workflow

[0335] Materials and methods:

[0336] Hydrochar was produced from pig manure (HC-C) as described in Examples 1 and 2. The hydrochar slurry was separated into (wet) hydrochar and process water fractions by filtration using a chamber filter press.

[0337] Liquid cultures of the fungal strain Trichoderma harzianum (BCCM: MUCL22194) were prepared as described in Example 3.

[0338] In a laminar flow cabinet, 1 mL of the liquid inoculum was diluted to 50 mL in sterile saline solution (0.8% NaCl). The diluted liquid inoculum was mixed with hydro-hot charcoal (HC-C; 100 g dry matter). The moisture content of the material was adjusted to 40% with reverse osmosis water. Another (control) sample had the same composition, but the steps were performed under non-sterile conditions outside the laminar flow cabinet.

[0339] Both samples were incubated aerobically in a sterile environment for 7 days to perform a solid-state fermentation process (fermented hydrochar).

[0340] Microbial growth was visually observed and quantified as follows: Both samples were resuspended in sterile physiological water (0.8% NaCl) and 10 -1 to 10 -7 The number of colony forming units (CFU) in the dilution groups was determined by plating on YMA plates.

[0341] result:

[0342] Table 11: Trichoderma harzianum growth measurements after 7 days on hydrothermal charcoal from swine manure (HC-C) fermentation, where the evaluation was performed under sterile or non-sterile conditions.

[0343]

[0344]

[0345] Trichoderma harzianum was detected in samples processed outside the laminar flow cabinet, although the population was low due to the growth of unwanted contaminating microorganisms ( Figure 4 ).

Claims

1. A method of producing a microorganism and nutrient delivery system (113), the method comprising: - providing biomass (101); - subjecting the biomass (101) to a hydrothermal carbonization process (HTC) (10) to form a slurry (103) comprising hydrothermal char and HTC process water; - cooling (20) the slurry (103) to a temperature suitable for the growth of the microbial inoculant; - inoculating the cooled slurry (105) with the microbial inoculant (109); - subjecting the inoculated slurry to a fermentation process (40) to form a fermentation product (111); and - obtaining or recovering (50) said microorganism and nutrient delivery system (113) from said fermentation product (111), The cooling step and the inoculation step are performed under sterile conditions.

2. The method according to claim 1, further comprising a step (30) of reducing the HTC process water content of the slurry (103, 105) to form wet hydrothermal char (106) before inoculating with the microbial inoculant, wherein the step (30) of reducing the HTC process water content of the slurry (103, 105) is performed under sterile conditions.

3. The method according to claim 2, wherein the process water content of the slurry is reduced (30) by a mechanical process, preferably solid-liquid separation such as decantation, centrifugation or filtration.

4. The method according to any one of claims 1 to 3, wherein the fermentation is carried out at a temperature below 85°C, preferably below 75°C, more preferably between 15°C and 70°C or between 15°C and 65°C or between 15°C and 60°C.

5. The method according to any one of claims 1 to 4, wherein the fermentation process (40) is submerged fermentation.

6. The method according to any one of claims 2 to 4, wherein the fermentation process (40) is a solid-state fermentation.

7. The method according to any one of claims 1 to 6, wherein the fermentation process is a single-stage fermentation.

8. The method according to claim 7, wherein the fermentation is carried out under sterile conditions.

9. The method according to any one of claims 1 to 6, wherein the fermentation process is a multi-stage fermentation such as a two-step fermentation, wherein the fermentation process comprises fermenting the inoculated slurry to form a first fermentation product, and then inoculating a second slurry formed in the HTC process with the first fermentation product and fermenting the inoculated second slurry to form the (second) fermentation product.

10. The method according to claim 9, wherein the fermentation of the (first) slurry inoculated with the microbial inoculant is performed under sterile conditions.

11. The method according to any one of claims 1 to 10, wherein the hydrothermal carbonization process (10) is carried out at a temperature between about 180°C and about 250°C and a pressure between about 10 bar and 50 bar for at least 30 minutes.

12. The method according to any one of claims 1 to 11, wherein the heat released during the cooling step (20) is used for the hydrothermal carbonization process (10).

13. The method according to any one of claims 1 to 12, wherein the biomass (101) is a wet biomass having a water content between 10% and 95% by weight, preferably between 10% and 90% by weight.

14. The method according to any one of claims 1 to 13, wherein the biomass (101) is selected from food waste, agricultural waste, animal by-products or any combination thereof, preferably animal by-products such as manure.

15. The method according to any one of claims 1 to 14, wherein the recovery step (50) comprises one or more of the following: - separating the fermentation product into a solids-rich fraction and a liquid fraction; - drying the fermentation product or a solids-rich fraction of the fermentation product; and - a processing step selected from the group consisting of grinding, milling, granulation, extrusion and micronization processes.

16. The method according to any one of claims 1 to 15, wherein the recovering step (50) comprises the step of separating spores from the fermentation product, wherein the microbial inoculant comprises spore-forming microorganisms.

17. The method according to any one of claims 1 to 16, wherein the microbial inoculant (109) comprises one or more bacteria belonging to a genus selected from the group consisting of Azotobacter, Streptomyces, Bacillus, Azospirillum and Bradyrhizobium and / or one or more filamentous fungi belonging to a genus selected from the group consisting of Beauveria, Trichoderma and Penicillium.

18. A microorganism and nutrient delivery system obtainable by the method according to any one of claims 1 to 17, the system comprising: - Fermented hydrocharcoal; - microorganisms attached to the surface or pores of the fermented hydrochar; and - optionally one or more auxiliary agents selected from solvents, carriers, binders, surfactants, stickers, tackifiers, antifreeze agents, thickeners, buffers, defoamers, antioxidants, preservatives, stabilizers, fragrances and colorants.

19. Use of the microorganism and nutrient delivery system according to claim 18 as a biostimulant, preferably as a biofertilizer or biocontrol agent.

20. Use of the microorganism and nutrient delivery system according to claim 18 as a bioremediation agent.

21. Use of the microorganism and nutrient delivery system according to claim 18 as a microbial inoculant, such as a microbial inoculant for an anaerobic digester, a septic system or a water treatment system or in a method according to any one of claims 1 to 17.

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