Microorganisms and Nutrient Delivery Systems

Abstract

The present invention relates to a method for producing microorganisms and nutrient delivery systems comprising hydrothermally carbonizing suitable biomass to form a slurry comprising hydrochar and HTC process water, cooling the slurry, inoculating the cooled slurry with a microbial inoculant, fermenting the inoculated slurry, and obtaining or recovering the microorganisms and nutrient delivery systems from the fermentation product, wherein the cooling and inoculating steps are performed under sterile conditions. The present invention further relates to the microorganisms and nutrient delivery systems obtainable by the method and uses thereof, including agricultural and environmental applications.

Description

[Technical Field]

[0001] Field The present invention relates to valorization of the products of a hydrothermal carbonization process. In particular, a production process for microorganisms and nutrient delivery systems is provided that involves the integration of a hydrothermal carbonization process and a fermentation process. The present invention further relates to the microorganisms and nutrient delivery systems and their uses, including agricultural and environmental applications. [Background technology]

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

[0003] Agricultural fertilizers are widely used to promote crop growth and yield and prevent soil depletion. Generally, fertilizers deliver nutrients to the soil, allowing crops to grow and develop better in that soil. A wide range of fertilizer formulations is known in the field of agricultural science. The most commonly used fertilizers are raw animal manure and mineral fertilizers produced by chemical processes, such as nitrogen fertilizers produced by the Haber-Bosch process. While both fertilizers are rapidly available sources of nutrients, they have the disadvantage of carrying a significant risk of nutrient runoff, which not only results in economic losses but also disrupts aquatic ecosystems (e.g., water pollution, eutrophication, and loss of biodiversity). Raw manure has another significant drawback in 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 associated with the use and development of biofertilizers are reliability, inappropriate formulations, high levels of contamination, poor quality, and the poor shelf life and consistency of the inoculants under field conditions.

[0005] Appropriate formulations can ensure the survival of microorganisms during storage and application. Formulations can be broadly classified as those using solid materials as carriers or liquid formulations. The disadvantage of liquid formulations is that the metabolic activity of beneficial microorganisms declines rapidly after production, which can pose a high risk of contamination. Peat is a frequently used solid carrier material used for both seed coating and soil application. To prevent contamination, peat must be sterilized before use, which is an energy-intensive process. Furthermore, because it is a fossil resource, its use in agricultural applications is gradually being phased out.

[0006] Hydrochar has been proposed as a promising vector for beneficial microorganisms (Thunshirn et al., 2022. Critical Reviews in Environmental Science and Technology 52:4147-4171). Hydrochar is the solid product of the hydrothermal carbonization (HTC) process of wet biomass, e.g., the digestate from the anaerobic digestion process. 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 prevent its use as a carrier for biofertilizer formulations. Furthermore, a by-product of the HTC process is the generation of large amounts of process water that must be managed and treated.

[0007] There remains a need in the art for additional and / or improved biostimulants, such as biofertilizers, that are stable, have an improved shelf life, are environmentally sustainable, and whose production process has minimal impact on the environment. Summary of the Invention [Problem to be solved by the invention]

[0008] overview The present invention is based, at least in part, on the inventors' discovery that process water produced in a hydrothermal carbonization (HTC) process functions surprisingly well as a growth medium for microorganisms. The hydrochar, together with the (residual) process water, can support and promote the growth and amplification of microorganisms and can therefore be used as a substrate for fermentation. [Means for solving the problem]

[0009] Accordingly, one aspect of the present invention provides a method or process for producing a microorganism and a nutrient delivery system, the method comprising: -Preparing biomass; - subjecting the biomass to a hydrothermal carbonization process to form a slurry comprising hydrochar and HTC process water; - cooling the slurry to a temperature suitable for the growth of microbial inoculants; - inoculating the cooled slurry with a microbial inoculant; subjecting the inoculated slurry to a fermentation process to form a fermentation product; and - Obtaining or recovering the microorganisms and nutrient delivery systems from the fermentation product Including, The inoculation and cooling steps are carried out under sterile conditions.

[0010] By combining the HTC process with a fermentation process, the process water of the HTC process is valorized, i.e., valorized as a growth medium for microorganisms. Furthermore, microorganisms can convert water-soluble nutrients (e.g., biochemical oxygen demand (BOD) nutrients, nitrate / ammonium) in the process water into biomass, thereby improving nutrient recovery from the biomass substrate. The integration of both the HTC process and the fermentation process is advantageous in that it does not require a separate microbial cultivation step, reducing operational costs. Furthermore, the HTC process, conducted under conditions suitable for killing or inactivating microorganisms, eliminates the need for a sterilization step prior to the fermentation process. The method is also energy efficient in that at least a portion of the heat required for the HTC process can be recovered from a cooling step prior to the fermentation process.

[0011] Further aspects are directed to microorganisms and / or nutrient delivery systems obtainable by the methods of the present invention. The microorganisms and nutrient delivery systems provided herein are -Fermented hydrochar; and -Microorganisms attached to the surface or pores of fermented hydrochar Includes:

[0012] Due to the sterile conditions during at least the cooling and inoculation steps of the method, the microbial and nutrient delivery system is characterized by minimal contamination with undesirable microorganisms, such that the microorganisms in the system consist essentially of the microbial species contained in the microbial innoculant (the desired microorganisms).

[0013] The fermented hydrochar provides nucleation sites (carrier / vector function) for microorganisms. Furthermore, the porous structure of the hydrochar protects the 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, attachment and / or pore colonization may be better for microbial growth and amplification on the hydrochar compared to using the hydrochar as a carrier / vector, whereby the microorganisms are dried on the surface and / or mixed with the hydrochar. Also advantageously, due to at least partial fermentation of the hydrochar by beneficial microorganisms, the hydrochar is more stable (less fermentable material), resulting in a product with improved shelf life. Also advantageously, due to at least partial fermentation of the hydrochar, the growth of undesirable and / or pathogenic microorganisms and plant toxicity may be reduced. Additionally, certain nutrients in hydrochar that are less bioavailable (e.g., phosphors) can be solubilized by beneficial microorganisms, resulting in a product with enhanced nutrient supply.

[0014] Further aspects are directed to the use of the microorganisms and nutrient delivery systems of the invention as biostimulants, in particular as biofertilizers, and / or as biocontrol agents, bioremediation agents or as microbial inoculants, for example for anaerobic digesters, for sewage treatment systems, for water treatment systems or in methods according to the invention.

[0015] These and further aspects and preferred embodiments of the present invention are set out in the following sections and in the appended claims, the subject matter of which is specifically incorporated into this specification.

[0016] BRIEF DESCRIPTION OF THE DRAWINGS The following description of figures of specific embodiments of the invention are merely exemplary in nature and are in no way intended to limit the present teachings, their application, or uses. [Brief explanation of the drawings]

[0017] [Figure 1] 1 shows a schematic diagram of an embodiment of a process according to the present invention. [Figure 2] Mycelial growth of Trichoderma harzianum after 7 days on fermented peat (A) or fermented hydrochar from pig manure (fermented HC) (B). [Figure 3] Comparison of the growth of microbial species on nutrient agar (NA) plates of control (non-fermented) (A, B) and fermented (C, D) hydrochars exposed to non-sterile conditions (B, D) or left sterilized (A, C). [Figure 4] Comparison of Trichoderma harzianum growth on yeast mannitol agar (YMA) plates inoculated with fermented hydrochar from pig manure (HC-C) under sterile (A) or non-sterile (B) conditions. DETAILED DESCRIPTION OF THE INVENTION

[0018] Description of the embodiment As used herein, the singular forms "a," "an," and "the" include both singular and plural referents unless the context clearly dictates otherwise.

[0019] As used herein, the terms "comprising," "comprises," and "comprised of" are synonymous with "including," "includes," "containing," or "contains," and are inclusive or open-ended and do not exclude additional, unrecited members, elements, and method steps. This term also encompasses "consisting of," "consists in," "consisting of," and "consists of," as well as the terms "consisting essentially of," "consisting essentially in," and "consisting essentially of," which enjoy well-established meanings in patent terminology.

[0020] The recitation of numerical ranges by endpoints includes all integers, and, where appropriate, fractions subsumed within each range, and the recited endpoints. This applies to numerical ranges whether introduced by the phrase "from to" or "between" or otherwise. Any numerical range recited herein is intended to include all subranges subsumed therein.

[0021] The terms "about" or "approximately," as used herein when referring to a measurable value such as a parameter, amount, duration, etc., are meant to encompass variation of the specified value and variation from the specified value, to the extent that such variation is appropriate to occur in the disclosed invention, such as variation of no more than ±10%, preferably no more than ±5%, more preferably no more than ±1%, and even more preferably no more than ±0.1% from the specified value. It is to be understood that the value to which the modifier "about" or "approximately" refers is itself specifically, preferably disclosed.

[0022] Furthermore, the terms first, second, third, etc. in the specification and claims, unless otherwise specified, are used to distinguish between like elements and do not necessarily describe a sequential or chronological order. The terms so used are interchangeable under appropriate circumstances, and it is understood that the embodiments of the invention described herein may operate in orders other than those described or illustrated herein.

[0023] The term "one or more" or "at least one," e.g., one or more members or at least one member of a group of members, will itself be clear by further example, but the term specifically encompasses reference to any one of the members or any two or more of the members, e.g., any >3, >4, >5, >6, or >7, 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.

[0024] As used herein, the term "and / or," when used in a list of two or more items, means that any one of the listed items may be used by itself, or any combination of two or more of the listed items may be used. For example, if a list is stated to include 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.

[0025] The discussion of the background of the invention herein is included to explain the context of the invention and should not be construed as an admission that any of the material referred to was published, known, or part of the common general knowledge in any country on the priority date of any of the claims.

[0026] Throughout this disclosure, various publications, patents, and published patent specifications are referenced by an identifying citation. All documents cited herein are incorporated by reference in their entirety. In particular, the teachings or sections of such documents that are specifically mentioned herein are incorporated by reference.

[0027] 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 this invention belongs. By way of further guidance, definitions of terms are included to better understand the teachings of the present invention. When a particular term is defined in connection with a particular aspect of the present invention or a particular embodiment of the present invention, it is meant that such connotation or meaning also applies throughout this specification, i.e., in the context of other aspects or embodiments of the present invention, unless otherwise defined.

[0028] In the following sections, various aspects or embodiments of the present invention are defined in more detail. Each aspect or embodiment so defined can be combined with any other aspect or embodiment, unless expressly indicated otherwise. In particular, any feature indicated as being preferred or advantageous can be combined with any other feature indicated as being preferred or advantageous.

[0029] References throughout this specification to "one 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 do not necessarily all refer to the same embodiment, but may. Furthermore, particular features, structures, or characteristics can be combined in any suitable manner in one or more embodiments, as would be apparent to one of ordinary skill in the art from this disclosure. Furthermore, some embodiments described herein include some features but not other features included in other embodiments, meaning that combinations of features from different embodiments form different embodiments within the scope of the present invention and as would be understood by one of ordinary skill in the art. For example, in the appended claims, any of the claimed embodiments can be used in any combination.

[0030] Similarly, in describing exemplary embodiments of the invention, it will be understood that various features of the invention may be grouped together in a single embodiment, figure, or description thereof for the purpose of streamlining the disclosure and aiding in the understanding of one or more of the various inventive aspects.

[0031] In one aspect, the present invention provides a method for producing a microorganism and a nutrient delivery system, the method comprising: -Preparing biomass; - subjecting the biomass to a hydrothermal carbonization process to form a slurry comprising hydrochar and HTC process water; - cooling the slurry to a temperature suitable for the growth of microbial inoculants; - inoculating the cooled slurry with a microbial inoculant; subjecting the inoculated slurry to a fermentation process to form a fermentation product; and - Obtaining or recovering the microorganisms and nutrient delivery systems from the fermentation product Including, The inoculation and cooling steps are carried out under sterile conditions.

[0032] Biomass preparation As used herein, the term "biomass" refers to organic material that is biodegradable.

[0033] 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 domestic waste, and mixtures thereof. Non-limiting examples of biomass suitable for use in the methods of the present invention include food residues, agricultural residues (e.g., straw, bark, wood chips...), animal by-products (e.g., manure, feathers, wool, exoskeletons...), organic fractions of domestic waste, organic fractions of industrial waste and by-products, sewage sludge, digestate from anaerobic digestion, etc.

[0034] In certain embodiments, the biomass is an animal by-product, such as manure. Advantageously, animal by-products can provide high concentrations of nutrients (nitrogen, phosphorus, potassium, micronutrients, etc.).

[0035] The method of the present invention is particularly useful for biomass with a high moisture content because the HTC process uses water as a reaction medium. Compared to pyrolysis, there is no need to dry the wet biomass before or during the process, resulting in significant energy savings. In certain embodiments, the biomass is wet biomass, such as biomass having a moisture content of 5% to 95% by weight, preferably 10% to 95% by weight, more preferably 10% to 90% by weight, e.g., 10% to 80% by weight. Non-limiting examples of suitable wet biomass include manure and digestate from anaerobic digestion, which inherently contain water. If the biomass is too dry (e.g., moisture content less than 10% or 5% by weight), water may be added before use in the method of the present invention as a substrate for the HTC process. To make the HTC process sustainable from an energy standpoint, the moisture content of the substrate is preferably less than 95% by weight, more preferably less than 90% by weight, e.g., less than 80% by weight.

[0036] Biomass, also referred to herein as feedstock or substrate, can be either a single input (e.g., animal manure) or a mixture of two or more feedstock types.

[0037] Biomass may be used directly in the HTC process or may be subjected to one or more pretreatment steps before being used as a substrate for the HTC process. The purpose of such pretreatment may be to enable a more effective HTC process, such as blending different feedstocks, liquefying the biomass, removing (separating) undesirable materials such as large items and / or inert, non-biodegradable materials (e.g., plastic, glass), reducing the particle size of the biomass (e.g., de-clumping, crushing, grinding, milling, ...), adding additives (e.g., acids, bases, salts), and / or adjusting the properties / composition of the final product. A wide variety of pretreatment processes are available, and the choice depends, among other things, on the type of biomass.

[0038] hydrothermal carbonization 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 hydrochar and aqueous and gaseous by-products. Without wishing to be bound by any theory, a series of hydrolysis, condensation, decarboxylation, dehydration, aromatization, and / or polymerization reactions may occur during HTC.

[0039] The reaction temperature may be in the range of about 160°C to about 300°C, preferably about 180°C to about 250°C, more preferably about 200°C to about 220°C, and may be applied at a corresponding pressure that ensures that the water remains in a liquid state, such as about 10 bar to about 88 bar, preferably about 10 bar to 50, 40, or 30 bar, more preferably about 15 bar to about 25 bar. The treatment time may vary from a few minutes to several hours or days, and preferably the HTC process is carried out for at least 30 minutes, such as 30 minutes to 20, 19, 18, 17, 16, 15, 14, or 13 hours, preferably about 30 minutes to about 12, 11, 10, or 9 hours, or about 30 minutes to about 8, 7, 6, or 5 hours, more preferably about 30 minutes to about 4 hours, such as 2 hours to about 3 hours. The selected conditions of temperature, pressure, and time can depend on the biomass substrate and the desired yield and / or properties of the hydrochar product. In certain embodiments, the HTC process is carried out at a temperature of about 180°C to about 250°C and a pressure of about 10 bar to about 50 bar for at least 30 minutes. In certain embodiments, the HTC process is carried out at a temperature of about 200°C to about 220°C and a pressure of about 15 bar to about 25 bar for about 2 hours to about 3 hours.

[0040] As used herein, the term "hydrochar" 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 production yield depend on the biomass substrate and HTC process parameters.

[0041] As used herein, the terms "process water" or "HTC process water" refer to the liquid or aqueous by-product of the HTC process. Process water is rich in dissolved organic components and inorganic salts. As with hydrochar, the yield and composition of process water are highly dependent on the biomass being treated and the parameters of the HTC process.

[0042] The hydrochar, together with the process water, forms a solid-liquid "slurry." The heat treatment renders the slurry containing the hydrochar and process water biologically sterilized.

[0043] As used herein, the phrase "subjecting biomass to a hydrothermal carbonization process to form a slurry comprising hydrochar and process water" refers to the conversion of biomass into a slurry comprising hydrochar and process water by the HTC process.

[0044] cooling The slurry is typically at a temperature above 160° C., for example, about 200° C. to about 220° C. To allow for inoculation with the desired microorganisms for fermentation, the slurry or wet hydrochar must be cooled to a temperature suitable for growth of the microbial inoculum, such as a temperature of about 4° C. to about 65° C. In embodiments, the slurry or wet hydrochar is cooled to a temperature of about 20° C. to about 50° C., preferably about 20° C. to about 30° C., depending on the microbial inoculum.

[0045] The cooling step is carried out under sterile conditions, by which is meant herein conditions that prevent microbial contamination of the slurry.

[0046] Cooling or aseptic cooling of the slurry or wet hydrochar can be achieved by a heat exchanger.

[0047] Advantageously, the heat released during the cooling step can be used in the HTC process. Cooling can be carried out in one or more stages. For example, the slurry may be partially cooled to a temperature of about 80°C to about 120°C, and the partially cooled slurry may then be further cooled to a temperature suitable for growth of the microbial inoculant, such as a temperature of about 4°C to about 65°C. In embodiments including a solid-liquid separation step, it is preferred to first cool the slurry before subjecting it to solid-liquid separation. Alternatively, the slurry may be subjected to solid-liquid separation to form a wet hydrochar and the wet hydrochar may be cooled, or the slurry may be partially cooled (preferably to a temperature below 100°C) and subjected to solid-liquid separation, and the wet hydrochar may be further cooled to a temperature suitable for growth of the microbial inoculant.

[0048] solid-liquid separation In certain embodiments, the process water content of the slurry is reduced or the slurry is at least partially dewatered or dried, preferably by solid-liquid separation techniques, thereby forming wet hydrochar. As used herein, "wet hydrochar" refers to 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% or less than 70% by weight, and more preferably less than 60% by weight. In embodiments, the wet hydrochar has a process water content of about 40% to about 60% by weight, e.g., about 50% to about 55% by weight. Solid-liquid separation techniques are known to those skilled in the art and include, but are not limited to, gravity decanting, centrifugation (various disc stack, basket, decanter centrifuges, ...), and filtration (filter press, tangential flow filtration, belt filter, screw press, ...).

[0049] In a preferred embodiment, the step of reducing the HTC process water content of the slurry or the dewatering process is carried out under sterile conditions.

[0050] When the slurry is subjected to solid-liquid separation, the wet hydrochar may be washed with (sterile) water. Thus, in embodiments, the method may further comprise a step of washing the wet hydrochar. This may be particularly advantageous when the process water contains water-soluble compounds that inhibit beneficial microorganisms.

[0051] The solid-liquid separation process also allows for the 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 beneficial microorganisms. Additionally, it also allows for the addition of microbial inoculants to the wet hydrochar in a convenient manner.

[0052] Inoculation of microbial inoculant After cooling, the slurry or wet hydrochar is inoculated with a microbial inoculant.

[0053] "Microbial inoculant" refers to a composition or concentrate of specific microorganisms. As used herein, it refers to a composition or concentrate of specific microorganisms that are added to a slurry or wet hydrochar to ferment the hydrochar. Microbial inoculants can contain one or a combination of various types of live microorganisms. Microbial inoculants can be used to ferment 10 6 ~10 12 cfu / g or cfu / ml, preferably 10 8 ~10 12 It may contain cfu / g or cfu / ml. The composition or concentrate may be in solid, liquid, or other form (e.g., a suspension), or a mixture thereof.

[0054] As used herein, "colony forming unit" or "CFU" refers to a unit of measure of viable microorganisms in a sample. A CFU is an individual viable cell capable of forming a visible colony on a solid medium, where the individual cell is derived by cell division from a single parent cell. The terms "CFU," "CFU / ml," and "CFU / g" also encompass reference to "spores," "spores / ml," or "spores / g," respectively, when the microorganism itself is suitable for administration in the form of spores.

[0055] As used throughout this specification, the term "microorganism" or "microbe" refers to any strain, species, or taxon 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 microbe or microorganism is a bacterial strain. In some embodiments, the microbe or microorganism is a fungal strain, such as a yeast strain or a filamentous fungal strain. In some embodiments, the microbe or microorganism encompasses an individual cell (e.g., a unicellular microorganism) or two or more cells (e.g., a multicellular microorganism).

[0056] As used herein, the terms "bacterium," "bacteria," or "bacterial" generally refer to any prokaryotic organism and may 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 (e.g., but not limited to, the evolving field of whole genome sequencing), and such nomenclature reassignments are understood to be within the scope of any claimed genus.

[0057] The terms "fungi," "fungus," or "fungal" refer broadly to a wide variety of nucleated (eukaryotic) spore-bearing organisms that lack chlorophyll (i.e., fungi are not photosynthetic and are heterotrophic). These organisms are classified in the kingdom Fungi, separate from other eukaryotic kingdoms. 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 of the organism's life cycle and encompasses fungal cells of any ploidy, such as haploid, diploid, and polyploid fungal cells; and encompasses vegetative cells as well as fungal spores.

[0058] The term "microalgae" as used herein refers to microscopic algae. "Microalgae" includes, but is not limited to, (i) several eukaryotic phyla, including Rhodophyta (red algae), Chlorophyta (green algae), Dinoflagellata, and Haptophyta; (ii) several classes from the eukaryotic phylum Heterokontophyta, including, but not limited to, Bacillariophycea (diatoms), Eustigmatophycea, Phaeophyceae (brown algae), Xanthophyceae (yellow-green algae), and Chrysophyceae (golden algae); and (iii) organisms within the prokaryotic phylum Cyanobacteria (blue-green algae). The term "microalgae" includes, for example, Achnanthes, Amphora, Anabaena, Ankistrodesmis, Arachnoidiscus, Aster, Botryococcus, Chaetoceros, Chlamydomonas, Chlorella, Chlorococcum, Chorethron, Cocconeis, Coscinodiscus, Crypthecodinium, and the like. ecodinium, 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, Phaeodactylum, Platymonas The genera include those selected from the group consisting of Onas, Pleurochrysis, Porphyra, Pseudoanabaena, Pyramimonas, Scenedesmus, Schyzochitrium, Stichococcus, Synechococcus, Synechocystis, Tetraselmis, Thalassiosira, and Trichodesmium.

[0059] The term "strain" (e.g., the phrases "fungal strain" and "bacterial strain"), as the fundamental operating unit of microbial classification, such as fungal or bacterial classification, is frequently used to refer to a population composed of the descendants of a single isolate in pure culture, usually consisting of a series of cultures ultimately derived from that original single fungal or bacterial colony. When a species encompasses two or more distinct isolates, the term "strain" can be used to refer to an isolate or group of isolates that can be distinguished from other isolates of the same genus and species by phenotypic or genotypic characteristics, or both.

[0060] In embodiments, the microbial innoculant may comprise one or more bacterial strains, yeast strains, and / or filamentous fungi strains, preferably bacterial strains and / or filamentous fungi strains. The microorganism may be genetically modified or not. For example, one or several microorganisms of the following genera can be used in the present invention:

[0061] bacteria Acetobacter, Achromobacter, Acinetobacter, Actinomyces, Actinoplanes, Actinomadura, Aerococcus, Aeromonas, Alcaligenes, Alcanivorax, Alloiococcus, Alteromo Alteromonas, Amycolatopsis, Anabaena, Arthrobacter, Arthrospira, Atopobium, Azoarcus, Azobacter, Azorhizobium, Azospirillum, Azotobacter, Bacillus, Beijeri nckia, Bifidobacterium, Bradyrhizobium, Brevibacterium, Brevundimonas, Carnobacterium, Catenisphaera, Cellulomonas, Chryseobacterium, Citrobacter, Clostridium , Corynebacterium, Cyanobacteria, Dermatophilus, Desulfotomaculum, Dietzia, Enterobacter, Enterococcus, Escherichia, Frankia, Flavobacterium, Geobacillus,Gluconacetobacter, Gluconobacter, Gordonia, Herbaspirillum, Humicola, Janthinobacterium, Lactobacillus, Lactococcus, Leuconostoc, Klebsiella, Marinobacter er), Mycobacterium, Micromonospora, Microtetraspora, Moraxella, Mycobacterium, Mycococcus, Micrococcus, Nocardia, Oenococcus, Paenibacillus, Pediococcus , Phormidium, Phyllobacterium, Propionibacterium, Pseudomonas, Raoultella, Rastonia, Rhizobia, Rhizobium, Rhodococcus, Saccharopolyspora, Serratia, Shigella, Sinorhizobium, Sphingomonas, Staphylococcus, Streptococcus, Streptomyces, Symbiobacterium, Synechococcus, Synechocystis, Tetragenococcus, Thermoactinomyces,Thermomonospora, Vagococcuswhich, Vibrio, Weissella, Xanthomonas.

[0062] Preferably, the microbial inoculant is 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. The bacterial strains include one or more bacterial strains belonging to a genera selected from the group consisting of Streptomyces, Bacillus, Azospirillum, and Bradyrhizobium, or a genera selected from the group consisting of Azotobacter, Streptomyces, Bacillus, Azospirillum, and Bradyrhizobium.

[0063] yeast Arxula, Aureobasidum, Blastobotrys, Brettanomyces (its sexual form, Dekkera), Candida, Citeromyces, Cryptococcus, Cystofilobasidium, Debaryomyces, Endomycopsis, Phyllobaziella Filobasidiella, Galactomyces, Geotrichum, Glaciozyma, Guehomyces, Hansenula, Hanseniaspora (its asexual counterpart Kloeckera), Hyphopichia, Kluyveromyces, Kodamaea, Komagatael la), Lachancea, Lipomyces, Metschnikowia, Meyerozyma, Moniella, Murakia, Ogataea, Pichia, Phaffia, Pseudozyma, Rhodotorula, Rhodosporidium, Starmerella, Saccharomyces Saccharomyces, Saccharomycodes, Saccharomycopsis, Scheffersomyces, Schizosaccharomyces, Schwanniomyces, Torulopsis, Torulaspora, Trichosporon, Trigonopsis, Yarrowia,Xanthophyllomyces, and Zygosaccharomyces.

[0064] filamentous fungi Acremonium, Agaricus, Agrocybe, Akanthomyces, Alternaria, Ampelomyces, Amylosporus, Antrodia, Armillaria, Ashbya, Aspergillus, Atkinsonella, Aureobasidium asidium, Auricularia, Balansia, Balansiopsis, Beauveria, Bispora, Bjerkandera, Boletus, Cantharellus, Catenaria, Cephalosporium, Chaetomium, Chrysonilia, Cladosporium Cladosporium, Claviceps, Clitocybe, Clitopilus, Colletotrichum, Collybia, Coniochaeta, Coprinus, Cordyceps, Coriolus, Cunninghamella, Cyathus, Cyclocybe, Cylindrocarpon indrocarpon, Cylinrocarpum, Cytonaema, Cytospora, Daldinia, Dentipellis, Doratomyces, Echinodothis, Emericella, Emericellopsis, Entoloma, Epichloe, Epicoccum,Exophiala, Favolaschia, Flammulina, Fomes, Fomitopsis, Fusarium, Ganoderma, Giberella, Gliocladium, Grifola, Gymnoascus, Hericium, Hohenbuehelia, Hormonema onema, Humicola, Hydropus, Hypomontagnella, Hypomyces, Hypoxylon, Hypsizigus, Inocutis, Inocybe, Inonotus, Isaria, Kuehneromyces, Lactarius, Laetiporus, Laxitextum Laxitextum, Lecanicillium, Lentinula, Lentinus, Lepista, Leptoshaeria, Lignosus, Lycoperdon, Lyophyllum, Martierella, Metarhizium, Monascus, Monilia, Monocillium , Morchella, Mortierella, Mucor, Mycelia, Myriogenospora, Neurospora, Nigrospora, Omphalotus, Ophiocordyceps, Oudemansiella, Paecilomyces, Panellus, Panus,Paraconiothyrium, Paraepichloe, Penicillium, Peniophora, Periconia, 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, Talaromyces, Thermoascus, Thermomyces ermomyces, Tolypocladium, Torula, Trametes, Tremella, Trichoderma, Tricholoma, Tuber, Verticillium, Volvariella, Wolfiporia, Wrightoporia, Xylaria.

[0065] Preferably, the microbial inoculant comprises one or more filamentous fungal strains belonging to the group consisting of Alternaria, Aspergillus, Chaetomium, Exophiala, Fusarium, Penicillium, Phoma, Talaromyces and Trichoderma, or to a genera selected from the group consisting of Alternaria, Aspergillus, Beauveria, Chaetomium, Exophiala, Fusarium, Penicillium, Phoma, Talaromyces and Trichoderma, more preferably to the group consisting of Trichoderma and Penicillium, or to a genera selected from the group consisting of Beauveria, Thrichoderma and Penicillium.

[0066] The choice of microorganisms to be included in the microbial inoculant may depend, among other parameters, on the desired end product, their ability to develop in one or more types of given substrate, their availability, and price. By way of example, and without limitation, plant beneficial microorganisms that may be included in the microbial inoculant include fungi belonging to the phyla Ascomycota (e.g., fungi of the genus Ampelomyces), 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 (e.g., Bacillus subtilis), and the like. Additional bacteria may include bacteria of the genera B. subtilis, B. cereus, B. circulans, Serratia marcescens, Flavobacterium spp., Alcaligenes sp., Agrobacterium radiobacter, etc. For example, but not limited to, biocontrol microorganisms that may be included in the microbial inoculant may include bacteria of the genera Agrobacterium, Pseudomonas, Streptomyces, or Bacillus, and / or fungi of the genera Gliocladium, Trichoderma, Ampelomyces, Candida, or Coniothyrium.

[0067] In certain embodiments, the microbial inoculant comprises a bacterium selected from Streptomyces spp. (e.g., Streptomyces griseoviridis), Bacillus spp. (e.g., Bacillus subtilis), Azospirillum spp. (e.g., Azospirillum brasilense), or Bradyrhizobium spp. (e.g., Bradyrhizobium japonicum), or a filamentous fungus selected from Trichoderma spp. (e.g., Trichoderma harzianum) or Penicillium spp. (e.g., Penicillium bilaiae), or any combination thereof.

[0068] In certain embodiments, the microbial inoculant is a bacterium selected from Azotobacter spp. (e.g., Azotobacter chroococcum), Pseudomonas spp. (e.g., Pseudomonas fluorescens), Streptomyces spp. (e.g., Streptomyces griseoviridis), Bacillus spp. (e.g., Bacillus subtilis), Azospirillum spp. (e.g., Azospirillum brasilense), or Bradyrhizobium spp. (e.g., Bradyrhizobium japonicum), or a Beauveria spp. (e.g., Beauveria bassiana), Trichoderma spp. (e.g., Trichoderma harzianum), or Penicillium spp. (e.g., Penicillium bilaiae), or any combination thereof.

[0069] Inoculating the slurry with the microbial inoculant can include adding the microbial inoculant to the slurry and optionally mixing the slurry. Mixing can be performed using a mixing device, for example, by pumping a gas through the slurry, or by shaking.

[0070] 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 adhesion to various surfaces of the hydrochar.

[0071] The inoculation step is carried out under sterile conditions. "Inoculation under sterile conditions" as used herein means conditions that prevent contamination of the slurry or wet hydrochar with microorganisms other than those contained in the microbial innoculant.

[0072] fermentation The method further includes fermenting the inoculated slurry or wet hydrochar, which includes storing the inoculated slurry or wet hydrochar for a sufficient time under conditions that promote growth and amplification of the microbial inoculant and degradation of the hydrochar by the microorganisms.

[0073] The time sufficient for fermentation of the hydrochar varies depending on, among other things, the composition of the biomass, the microbial inoculant, and the fermentation conditions. Fermentation does not have to be complete, but at least a portion of the hydrochar content should be fermented. A time sufficient for fermentation of the 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. A time sufficient for fermentation of the hydrochar can be, for example, up to 60 days, up to 50 days, up to 40 days, up to 30 days, or up to 20 days. In embodiments, the inoculated slurry or wet hydrochar can be fermented for about 2 days to about 14 days.

[0074] Preferably, fermentation conditions are controlled during the fermentation process. For example, temperature may be controlled. In certain embodiments, fermentation is carried out at temperatures 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 temperatures between 15°C and 65°C, depending on the microorganisms and biomass involved. Other parameters that may be controlled include humidity measurement, oxygen, and CO2 levels. For example, humidity may be maintained above 35%, preferably above 45%, more preferably above 50%, even more preferably above 60%, for example above 65%. CO2 levels may be maintained at 600-1200 ppm, preferably 800-1000 ppm.

[0075] 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, for example, on the microorganisms involved. For aerobic fermentation, oxygen may be supplied to the slurry as air. However, it is also possible to supply pure oxygen to the slurry and / or oxygen-enriched air and / or air and oxygen in separate feeds. For aerobic solid-state fermentation, the wet hydrochar may be exposed to air.

[0076] In a preferred embodiment, fermentation is carried out under sterile conditions. As used herein, "fermentation under sterile conditions" or "sterile fermentation" means that there are no or minimal undesirable microorganisms, e.g., there is no contamination by microorganisms other than the microbial species preferably contained in the microbial innoculant. Techniques for sterile fermentation are known to those skilled in the art and include, for example, sterilizing the fermenter before inoculating the microbial innoculant, using a sterile fermentation broth, using sterile (e.g., filtered) air or oxygen, and the like, and any combination thereof.

[0077] Fermentation may be carried out in one single stage, or in two or more stages (i.e., multi-stage fermentation), for example, two-stage fermentation. In embodiments, the fermentation process may include fermenting an inoculated slurry to form a first fermentation product, followed by inoculating a second slurry formed in the HTC process with the first fermentation product and fermenting the inoculated second slurry to form a (second) fermentation product. In further embodiments, the fermentation of the (first) slurry inoculated with the microbial innoculant is carried out under sterile conditions. In further embodiments, the fermentation of the (second) slurry inoculated with the first fermentation product is carried out under non-sterile or sterile conditions. In further particular embodiments, the fermentation of the (first) slurry inoculated with the microbial innoculant is carried out under sterile conditions and the fermentation of the (second) slurry inoculated with the first fermentation product is carried out under non-sterile conditions.

[0078] In certain embodiments, the fermentation process is submerged fermentation. The term "submerged fermentation" generally refers to a process in which microorganisms are fermented while submerged in a liquid medium that provides nutrients to the microorganisms. As used herein, "submerged fermentation" can refer to the fermentation of hydrochar in which a microbial innoculant is inoculated into a slurry or partially dewatered slurry from an HTC process.

[0079] In certain embodiments, the fermentation is solid-state fermentation. The term "solid-state fermentation" generally refers to a process in which microorganisms are fermented on a solid medium or substrate that provides the microorganisms with a foothold and nutrients. As used herein, "solid-state fermentation" may refer to the fermentation of hydrochar, in which a microbial innoculant is inoculated into wet hydrochar. The expression "fermentation product" as used herein refers to a product resulting during the fermentation process according to the present invention. The fermentation product may correspond to the fermented substrate, in particular the fermented hydrochar, spores, biomass, such as bacteria, yeast, or filamentous fungi, molecules, or any mixture thereof.

[0080] As used herein, "fermented hydrochar" refers to hydrochar that has been at least partially decomposed in a fermentation process. The term fermented hydrochar encompasses "partially fermented hydrochar," which includes a fermented hydrochar portion and a fermentable hydrochar portion, as well as "fully fermented hydrochar."

[0081] Recovery of Microorganisms and Nutrient Delivery Systems The fermentation product may be used as such, as a microorganism or nutrient delivery system, or the fermentation product may be subjected to one or more processing steps to obtain the microorganism and nutrient delivery system.

[0082] Recovering the microorganisms and nutrient delivery systems from the fermentation product may involve one or more processing steps selected from solid-liquid separation steps, drying steps, grinding, comminution, pelleting, microgranulation processes, extrusion, and the like.

[0083] In certain embodiments, recovering the microorganisms and nutrient delivery systems from the fermentation product comprises separating the fermentation product into a solid-rich fraction and a liquid fraction (or subjecting the fermentation product to solid-liquid separation), and the microorganisms and nutrient delivery systems are obtained or recovered from the solid-liquid fraction. For solid-liquid separation of the slurry, solid-liquid separation techniques described elsewhere herein can be used.

[0084] In certain embodiments, recovering the microorganisms and nutrient delivery 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 innoculant, various drying techniques can be used, including but not limited to freeze drying, spray drying, fluidized bed drying, vacuum tray drying, etc. (Sensitive microorganisms may require gentler drying techniques).

[0085] In certain embodiments, recovering the microorganisms and nutrients from the fermentation product includes extruding the fermentation product, e.g., pelleting the fermentation product. The process of "extruding" the fermentation product can be broadly described as forcing the fermentation product through an orifice. During the extrusion process, which generally involves at least one screw and a die, high pressure can build up at the end of the screw and die. In particular, the extrusion process can be considered to combine various unit operations into one system: material transport, 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.

[0086] After the step of extruding the fermentation product, the extruded fermentation product can be dried. In certain embodiments, recovering the microorganisms and nutrient delivery from the fermentation product comprises grinding or milling the fermentation product or a solids-rich fraction thereof, optionally after a drying step.

[0087] In certain embodiments, recovering the microorganisms and nutrient delivery from the fermentation product comprises a microgranulation process.

[0088] In certain embodiments, particularly those in which the microbial innoculant comprises spore-forming microorganisms, the fermentation product can be subjected to a spore separation process, in which the spores are separated from the fermentation product. Both the separated spores and the residual fermentation product can be used as the microorganisms and nutrient delivery systems described herein. Spore separation can be carried out in a variety of ways known to those skilled in the art, including, but not limited to, sieving, filtering, centrifugation, cyclone separation, and any combination thereof.

[0089] system Also disclosed herein are systems for producing microorganisms and nutrient delivery systems. In particular, the systems for producing microorganisms and nutrient delivery systems can be used to carry out methods for producing microorganisms and nutrient delivery systems according to the present invention.

[0090] The system may include a unit for carrying out the HTC process (e.g., a pressurized reactor equipped with a thermocouple; Ingelia SLHTC plant), a cooling unit (e.g., a heat exchanger), and a fermentation unit (e.g., a fermenter, an open vessel). Suitable equipment for carrying out the HTC process or the fermentation process according to embodiments of the present invention, as well as cooling equipment, are known to those skilled in the art and can be appropriately selected by those skilled in the art. For example, submerged fermentation may be carried out in a (stirred) tank. Non-limiting examples of suitable solid-state fermenters include tray bioreactors, stirred tank bioreactors, rotating drum bioreactors, fluidized bed bioreactors, etc., known to those skilled in the art.

[0091] The system may further include one or more of a liquid-solid separator (e.g., a centrifuge), a dryer, a grinder, a mill, and an extruder to recover the microorganisms and nutrient delivery system from the fermentation product.

[0092] Microorganisms and Nutrient Delivery Systems Further aspects are directed to microorganisms and nutrient delivery systems obtainable by the methods of the invention. In particular, there is provided a microorganism and nutrient delivery system comprising: -Fermented hydrochar; and -Microorganisms attached to the surface or pores of fermented hydrochar.

[0093] As used herein, "microorganism and nutrient delivery system" means a system or composition that includes microorganisms and nutrients.

[0094] The term "nutrient" broadly refers to substances used by organisms such as microorganisms and plants for survival, growth, and reproduction. The term nutrient encompasses macronutrients and micronutrients, including, but not limited to, carbon, oxygen, nitrogen, phosphorus, minerals (e.g., calcium, sodium, potassium, magnesium, chloride), and the like. Nutrients can be present in a system in any form, such as, for example, as ions, as salts, or as chemical compounds (e.g., carbohydrates). Depending on the application, the microorganisms and nutrient delivery system can include nutrients for the microorganisms present in the system as well as other organisms, such as plants. Advantageously, the microorganisms in the system can convert certain nutrients into a form that makes them bioavailable to other organisms (e.g., plants).

[0095] 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 further comprise (residual) process water.

[0096] Microorganisms adhere to the surface or pores of the fermented hydrochar, and without wishing to be bound by any theory, the attachment may be stronger and / or the colonization of the pores may be better due to the fermentation process compared to using non-fermented hydrochar as a microbial carrier.

[0097] Due to the aseptic conditions applied during at least the cooling and inoculation steps of the method of the present invention, the microorganisms and nutrient delivery systems of the present invention are characterized by their resistance to contamination by undesirable microorganisms or microbial species not contained in the microbial innoculant. The microorganisms and nutrient delivery systems of the present invention are characterized by the fact that the microorganisms consist essentially of the microbial species contained in the microbial innoculant. In embodiments, at least 50%, preferably at least 55%, 60%, 65%, 70%, or 75%, more preferably at least 80%, 82%, 85%, 86%, 88%, or 90%, e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, of the microorganisms in the microorganisms and nutrient delivery system consist of the microbial species contained in the microbial innoculant. As mentioned above, the fermented hydrochar may be partially fermented or fully fermented. Fully fermented hydrochar may be preferred, as the absence of fermentable substrate makes the hydrochar less attractive to undesirable and / or pathogenic microorganisms.

[0098] Due to the fermentation of the hydrochar, the microorganism and nutrient delivery system is more stable and has an improved shelf life compared to systems in which non-fermented hydrochar is used as the microbial carrier.

[0099] In certain embodiments, for example, the microorganisms and nutrient delivery systems are obtainable by a method according to the invention that includes a spore isolation step, and the microorganisms consist essentially of spores, preferably spores of the microbial species contained in the microbial inoculant.

[0100] The microorganism and nutrient delivery system may further include one or more supplements. The terms "auxiliary agent," "supplement," "additive," or "adjuvant" may be used interchangeably herein. An auxiliary agent may be a natural or synthetic organic or inorganic material, preferably a natural material, that facilitates administration of the microorganism and nutrient delivery system to, for example, a plant, a plant part, a seed, a plant-growing locus, or an environmental locus. An auxiliary agent may be one or more of a solvent, carrier, binder, surfactant, adhesive, tackifier, antifreeze agent, thickener, buffer, antifoaming agent, antioxidant, preservative, stabilizer, fragrance, colorant, etc.

[0101] Suitable adjuvants are known in the art and commercially available. Generally, the microorganisms and nutrient delivery system can be combined with any solid, semi-solid, or liquid additive commonly used for formulation purposes. The carrier should be understood to mean a natural or synthetic organic or inorganic substance that is mixed or combined with the microorganisms and nutrient delivery system for better applicability, such as application to plants or plant parts such as seeds. The carrier may be solid, semi-solid, or liquid, and is generally inert and suitable for use in agriculture or horticulture. For example, liquid carriers may include water, organic solvents, mineral oils, and vegetable oils. Suitable liquefied gas extenders or carriers are liquids that are gaseous at ambient temperature and atmospheric pressure, such as aerosol propellants such as butane, propane, nitrogen, and carbon dioxide. The adhesive should be understood to mean an additive or adjuvant that improves the adhesive properties of the composition to plants or their parts. Suitable surfactants are emulsifiers, dispersants, or wetting agents with ionic or non-ionic properties, or mixtures of these surfactants. Colorants such as inorganic pigments, e.g., iron oxide, titanium oxide, Prussian blue, and organic dyes, such as alizarin dyes, azo dyes, and metal phthalocyanine dyes, as well as trace nutrients, such as salts of iron, manganese, boron, copper, cobalt, molybdenum, and zinc, can be used. Stabilizers, such as low-temperature stabilizers, preservatives, antioxidants, light stabilizers, or other agents that improve chemical and / or physical stability, may also be present.

[0102] In certain applications, the adjuvants added to the microorganism and nutrient delivery system are preferably agriculturally acceptable adjuvants. The terms "agriculturally acceptable" or "agriculturally compatible" are consistent in the art and mean not harmful to recipient plants, such as not producing, having, or causing adverse effects when applied to a plant or plant part, or to plants grown from that plant part.

[0103] In certain embodiments, the microorganism and nutrient delivery system includes one or more additional active ingredients, such as nutrients and / or microorganisms.

[0104] The terms "active ingredient" or "active component" can be used interchangeably and refer broadly to materials, such as elements, molecules, substances, and / or microorganisms, that, when provided in an effective amount, achieve a desired result, such as achieving one or more effects on one or more plant growth characteristics or achieving one or more effects for bioremediation. Typically, active ingredients contemplated herein may achieve such a result by interacting with and / or modulating a plant, a part thereof, a seed for growing a plant, the plant's locus (e.g., soil), or an environmental locus (e.g., contaminated soil).

[0105] For example, microorganisms may be added that, together with the microorganisms and the microorganisms of the nutrient delivery system, form a microbial consortium suitable for bioremediation applications, but that are less compatible or incompatible (e.g., different fermentation conditions) with the microorganisms for fermenting the slurry or wet hydrochar and the microorganisms of the nutrient delivery system. Further non-limiting examples of active ingredients that can be added to the microorganisms and the nutrient delivery system include fertilizers (e.g., chemical fertilizers), pesticides (e.g., chemical pesticides, chitosan), biostimulants (e.g., microbial biostimulants, plant extracts, (phyto)hormones, (bio)chemicals), or any combination thereof.

[0106] The microorganism and nutrient delivery systems or compositions taught herein can be liquid, semi-solid, or solid, and can include solutions or dispersions. Non-limiting examples of compositions taught herein can be (soluble) powders, (soluble) granules, wettable granules, pellets, tablet formulations, dry flowables, aqueous flowables, wettable dispersible granules, oil dispersions, suspension concentrates, dispersible concentrates, emulsifiable concentrates, aqueous suspensions, fertilizer granules, sprayable materials, etc. The term "powder" refers to a dry bulk solid composed of many very fine particles that can flow freely when shaken or tilted.

[0107] use Further aspects are directed to applications of the microorganisms and nutrient delivery systems of the present invention, which are useful as agricultural biological materials, particularly as biostimulants, and more particularly as biofertilizers and / or biocontrol agents.

[0108] Thus, in one aspect, the present invention relates to the use of microorganisms and nutrient delivery systems according to the present invention as biostimulants.

[0109] 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 a plant, or the locus of a plant (e.g., the soil or plant growth medium surrounding the plant), stimulates natural processes to improve the growth characteristics of the plant.

[0110] The term "plant growth characteristics" is intended to broadly encompass any characteristic related in any 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, plant wet or dry biomass, plant height, plant size, % emergence, date of emergence, canopy cover, flowering status, seed yield, grain yield, fruit yield, number of tillers per plant, shoot length, root length, root architecture, seed weight, senescence, stay green, number of mature plant reproductive elements per plant, visual appearance, etc.

[0111] Reference to improvement encompasses any qualitative or quantitative change or modification of a plant's growth characteristics that is industrially useful, particularly in the agricultural context. To the extent that a plant's growth characteristics are quantifiable, improvement can be synonymous with an increase or decrease in their amount, depending on the nature of the plant's growth characteristics. By way of example, and not limitation, an increase in quantifiable characteristics such as plant wet or dry biomass, plant height, plant size, canopy cover, seed yield, grain yield, fruit yield, number of tillers per plant, shoot length, root length, seed weight, etc. may be desired.

[0112] By way of example, a biostimulant may be able to increase nutrient uptake and / or nutrient utilization efficiency in treated plants compared to untreated plants; increase the nitrogen fixation capacity or phosphorus uptake in treated plants compared to untreated plants; increase the amount of biomass in treated plants compared to untreated plants; increase the number of tillers per plant in treated plants compared to untreated plants; increase the growth and / or yield of treated plants compared to untreated plants; and / or help treated plants overcome stress conditions such as nutritional or abiotic stress (e.g., drought, heat, and saline soils) compared to untreated plants, etc. Additional plant growth characteristics that can be improved by biostimulants include disease resistance, drought tolerance, heat tolerance, cold tolerance, salt tolerance, metal tolerance, herbicide tolerance, chemical tolerance, improved water use efficiency, improved phosphorus solubilization, improved phosphorus mobilization, improved nitrogen utilization, improved nitrogen fixation, pest resistance, herbivore resistance, pathogen resistance, increased yield, increased yield under water-limited conditions, enhanced health, improved vigor, improved growth, improved plant emergence, improved photosynthetic capacity, nutritional enhancement, changes in protein content, changes in oil content, increased biomass, increased number of tillers per plant, and increased shoot length. These may include increased root size, increased root length, improved root architecture, increased seed weight, changes in seed carbohydrate composition, changes in seed oil composition, increased radical length, delayed senescence, retention of green color, changes in seed protein composition, increased dry weight of mature plant reproductive elements, increased fresh weight of mature plant reproductive elements, increased number of mature plant reproductive elements per plant, increased chlorophyll content, decreased number of wilted leaves per plant, decreased number of severely wilted leaves per plant, increased number of non-wilted leaves per plant, and / or improved visual appearance of the plant.

[0113] In an embodiment, the present invention relates to the use of microorganisms and nutrient delivery systems as biofertilizers.

[0114] As used herein, the term "biofertilizer" refers to a substance containing one or more nutrients (e.g., nitrogen, phosphorus, and / or potassium) and living microorganisms that, when applied to a plant, a part thereof (e.g., roots), a seed for growing a plant, or the locus of a plant (e.g., the soil or plant growth medium surrounding the plant), colonizes the plant locus or plant structure and promotes growth by increasing the availability of nutrients to the plant.

[0115] In an embodiment, the present invention relates to the use of microorganisms and nutrient delivery systems as biocontrol agents.

[0116] As used herein, the term "biological control agent" refers to a substance containing microorganisms capable of reducing the population of or avoiding the effects of potentially pathogenic agents. When applied to a plant, its parts (e.g., roots), seeds for growing plants, or the location of the plant (e.g., the soil surrounding the plant or plant growth medium), the biological control agent significantly reduces the incidence and severity of plant disease. The mode of action may be due to the biological control agent secreting or containing a substance that is toxic to the pathogen, due to the biological control agent's parasitism of the pathogen, or some combination of these or other effects, so that the biological control agent competes with the pathogen in the ecological niche.

[0117] Use may involve administering or applying the microorganisms and nutrient delivery systems according to the invention to a plant, a part thereof (e.g., a root), a seed for growing a plant, or the locus of the plant (e.g., the soil surrounding the plant or plant growth medium).

[0118] Reference to a plant includes any plant. Plants can include wild plants and domesticated varieties. In certain embodiments, the plant can be an agricultural plant. The term "agricultural plant" or "crop" includes plants cultivated by humans for purposes including, but not limited to, food, feed, fiber, fuel, horticulture, and / or industry.

[0119] 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 collectively to any part of a plant from which other plants can be derived either through sexual or asexual reproduction of the plant, for example, but not limited to, a seed, seedling, root, shoot, cutting, scion, scion, shoot, bulb, tuber, corm, tuber, or sprout. In certain embodiments, the microorganism and nutrient delivery system are applied to a seed.

[0120] Plant parts, when treated with the microorganisms and nutrient delivery systems of the present invention, may be attached to (e.g., growing on) or separated from (e.g., not growing on) the whole plant. For example, seeds, when treated with the microorganisms and nutrient delivery systems of the present invention, may be separated from (e.g., not growing on) the whole plant.

[0121] As used herein, the phrase "plant location" or "plant growth location" refers to an area adjacent to a plant (including parts thereof, such as seeds). For example, a plant location can be a circular area around a plant, e.g., a seed, such as a circular area having a diameter of up to 1 meter, e.g., up to 50 centimeters (cm), up to 40 cm, up to 30 cm, up to 20 cm, up to 10 cm, or up to 5 cm, around a plant, e.g., a seed. A growth location can include a growth medium (e.g., soil, hydroponic medium, or hydroculture medium) for cultivating a plant.

[0122] The phrase "administering" generally refers to the placing, application, delivery, or provision, whether humanly and / or mechanically driven or performed, of the recited object, such as a microorganism and a nutrient delivery system, to a recipient entity, such as a plant, a part thereof, a seed for growing a plant, or the locus of a plant. The microorganism and nutrient delivery system taught herein can be administered by any known method in which all or a part of a plant is treated, such as by root or seed inoculation. For example, administration can be to the roots of a plant, to the seeds of a plant before sowing in soil, or to the soil or plant growth medium surrounding the plant or plant seed. Application methods such as spraying, coating, covering, contacting, and / or immersion can be employed. In certain embodiments, application can be to a surface, such as the surface of a growth medium (such as soil), a plant, a plant part, a seed, a harvested plant, or a plant part (e.g., a harvested root, bulb, or tuber). In certain embodiments, administration can be to a plant, a part thereof, or the locus of a plant present in a field or agricultural land.

[0123] In certain embodiments, the microorganisms and nutrient delivery systems taught herein can be administered to the locus of the plant, such as by inoculating soil or growth medium. Thus, in certain embodiments, the method includes inoculating soil or plant growth medium with the microorganisms and nutrient delivery system and growing a plant in the soil or medium.

[0124] As used herein, the term "growth medium" or "plant growth medium" refers to a substrate or medium for culturing plants. The growth medium can be soil, compost, peat, coco coir, wood fiber, mineral lava, or basalt substrate, a soil-simulating substrate, textile, or a soil-free substrate. For example, the growth medium can be sand, gravel, polysaccharides, mulch, peat moss, straw, logs, clay, or a combination thereof. The plant growth medium can also include a water culture system or an in vitro culture system. The plant growth medium can be a hydroponic or water culture medium. Those skilled in the art will understand that different types of growth media can be used to grow different types of plants. Inoculation of the plant growth medium can be performed using, for example, liquids, powders, granules, or pellets. For example, aquatic plants can be grown with granules or pellets of the microorganism and nutrient delivery system described herein.

[0125] Inoculation of plant growth media with the microorganisms and nutrient delivery systems taught herein can occur before, during, and / or after sowing, or before, during, and / or after the start of the plant's growth cycle, in the case of aqueous culture or in vitro culture. Inoculation can occur one or more times during the plant's growth cycle.

[0126] In certain embodiments, the sprayable liquid can be applied by spraying the plant, part thereof, or the location of the plant, preferably the location of the plant, with conventional spraying equipment known in the art, such as an aircraft, backpack sprayer, tractor-mounted boom sprayer, etc.

[0127] In certain embodiments, the microorganisms and nutrient delivery systems taught herein can be applied to plants, parts thereof, or the locus of plant growth directly or by using conventional treatment methods, such as dipping, drenching, spraying, coating, atomizing, irrigating, evaporating, dusting, misting, broadcasting, foaming, painting, spreading, drenching, or drip irrigation, to affect their surroundings or habitat. For example, application can include spraying, sprinkling, showering, misting, spreading, droplet spreading, sputtering, dispersing, diffusing, or dousing the microorganisms and nutrient delivery systems onto the plant, parts thereof, or the locus of plant growth.

[0128] Further disclosed herein is the use of the microorganisms and nutrient delivery systems in environmental applications, for example, as bioremediation agents.

[0129] Thus, a further aspect is directed to the use of the microorganisms and nutrient delivery systems taught herein as bioremediation agents.

[0130] "Bioremediation" generally refers to the remediation of contaminated soils that utilizes the ability of certain microorganisms to convert hazardous substances into non-toxic compounds. The main requirements for effective bioremediation are a biodegradable organic substrate, a suitable active microbial consortium, and the bioavailability of the contaminant. Bioremediation may also require nutrients for the microorganisms.

[0131] According to the present invention, the slurry or wet hydrochar from the HTC process is inoculated with a suitable microorganism or blend of microorganisms, and the inoculated slurry or wet hydrochar is fermented, allowing the microorganisms to attach to the fermented hydrochar and colonize its pores. The fermentation product can be applied to contaminated soil, optionally after several treatment steps taught elsewhere herein. The hydrochar acts as a nutrient source for the growth and reproduction of microorganisms in the system and provides protection against predation by protozoa. Additionally, fermentation of the hydrochar can reduce competition with native microorganisms in the soil.

[0132] In a further aspect, the present invention relates to the use of the microorganisms and nutrient delivery systems as microbial innoculants, for example for anaerobic digesters, sewage treatment systems, water treatment systems, etc. Depending on the specific application, one skilled in the art will be able to select appropriate microorganisms for the microbial innoculant.

[0133] In certain embodiments, the microorganisms and nutrient delivery systems may be (re)used as microbial innoculants in the methods of the present invention.

[0134] The present invention will now be described in more detail with reference to the accompanying Figure 1, which provides a schematic diagram illustrating an embodiment for implementing a process according to the present invention, without limiting the invention to the particular steps and parameters presented. In the method, biomass (101) is subjected to a hydrothermal carbonization process (10) to form a slurry (103) comprising hydrochar and process water. The slurry is cooled (20) to a temperature suitable for the growth of microbial inoculants. The cooled slurry (105) may be inoculated with microbial inoculants (109) and subsequently subjected to a fermentation process (40), particularly submerged fermentation.

[0135] Optionally, the cooled slurry (105) may first be subjected to solid-liquid separation (30) prior to the inoculation step. Solid-liquid separation reduces the process water content of the slurry to obtain wet hydrochar (106). The process water (107) separated from the wet hydrochar (106) can be recycled to the HTC process (10). The solid-liquid separation step also allows for the easy addition of additional components (110) to the wet hydrochar and allows for the addition of microbial inoculants (109) to the wet hydrochar in a convenient manner. Fermentation (40) of the wet hydrochar (106) can be solid-state fermentation.

[0136] Additional components (110), such as nutrients to promote the growth of beneficial microorganisms or to adjust the composition of the microorganisms and nutrient delivery system, may be added at various steps in the process, such as before the HTC process (10), during solid-liquid separation (30), before fermentation (40), etc.

[0137] The fermentation products (111), including the fermented hydrochar, can be used as microbial and nutrient delivery systems, and the like.

[0138] Alternatively, the fermentation product (111) may be subjected to one or more processing steps (50), such as from solid-liquid separation, drying, grinding, milling, pelleting, etc., and / or optionally blended (50) with one or more co-formulants (112) to provide a blended microorganism and nutrient delivery system (113).

[0139] DESCRIPTIONS. These descriptions also disclose the phrase "...the [subject matter] described in description [number]" or "...the [subject matter] described in any one of description [number]," which can be replaced with the simple phrase "in one particular embodiment...."

[0140] Description 1. A method for producing a microorganism and nutrient delivery system (113), the method comprising: -Preparing biomass (101); - subjecting the biomass (101) to a hydrothermal carbonization process (10) to form a slurry (103) comprising hydrochar and process water, in particular HTC process water; - cooling the slurry (103) to a temperature suitable for the growth of microbial inoculants (20); - inoculating the cooled slurry (105) with a microbial inoculant (109); - subjecting the inoculated slurry to a fermentation process (40) to form a fermentation product (111); and - Obtaining or recovering (50) the microorganisms and nutrient delivery systems (113) from the fermentation product; A method comprising:

[0141] Statement 2. The method of statement 1, further comprising reducing (30) the process water, particularly HTC process water, content of the slurry (103, 105) prior to inoculating with the microbial inoculant to form a wet hydrochar (106).

[0142] Statement 3. The method of statements 1 or 2, wherein the cooling step (20) and the inoculation step are performed under sterile conditions.

[0143] Statement 4. The method of statements 2 or 3, wherein the cooling step (20), the inoculation step, and reducing the process water, particularly the HTC process water content of the slurry (30) are performed under sterile conditions.

[0144] Statement 5. The method of any one of statements 2-4, wherein the process water, particularly the HTC process water content of the slurry is reduced by a mechanical process, preferably solid-liquid separation such as decantation, centrifugation, or filtration (30).

[0145] Statement 6. The method of any one of statements 1 to 5, wherein the fermentation process (40) is solid-state fermentation.

[0146] Statement 7. The method of any one of statements 1 to 5, wherein the fermentation process (40) is a submerged fermentation.

[0147] Statement 8. The method of any one of statements 1-7, wherein the fermentation process is a single-stage fermentation. Statement 9. The method of statement 8, wherein the fermentation is carried out under sterile conditions.

[0148] Statement 10. The method of any one of statements 1-7, wherein the fermentation process is a multi-stage fermentation, e.g., a two-stage fermentation, and the fermentation process comprises fermenting an inoculated slurry to form a first fermentation product, followed by inoculating a second slurry formed in an HTC process with the first fermentation product, and fermenting the inoculated second slurry to form a (second) fermentation product.

[0149] Statement 11. The method of statement 10, wherein fermentation of the (first) slurry inoculated with the microbial inoculant is carried out under sterile conditions.

[0150] Statement 12. The method of statements 10 or 11, wherein the fermentation of the (second) slurry inoculated with the first fermentation product is carried out under non-sterile conditions.

[0151] 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.

[0152] Statement 14. The method of any one of statements 1-13, wherein the slurry (103) or wet hydrochar is cooled (20) to a temperature of about 4°C to about 65°C, preferably about 20°C to about 50°C, and more preferably about 20°C to about 30°C.

[0153] Statement 15. The method of any one of statements 1 to 14, wherein the cooling (20) is performed by a heat exchanger.

[0154] Statement 16. The method of any one of statements 1-15, wherein the heat released during the cooling step (20) is used in the hydrothermal carbonization process (10).

[0155] Statement 17. The method of any one of statements 1-16, 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 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.

[0156] Statement 18. The method of any one of statements 1 to 17, wherein the hydrothermal carbonization process (10) is carried out at a temperature of about 180°C to about 250°C, preferably about 200°C to about 220°C.

[0157] Statement 19. The method of any one of statements 1 to 18, wherein the hydrothermal carbonization process (10) is carried out at a pressure of about 10 bar to about 50 bar, preferably about 15 bar to about 25 bar.

[0158] Statement 20. The method of any one of statements 1-19, wherein the hydrothermal carbonization process (10) is carried out for at least 30 minutes, preferably from about 30 minutes to about 8 hours, more preferably from about 30 minutes to about 4 hours, and even more preferably from about 2 hours to about 3 hours.

[0159] Statement 21. The method of any one of statements 1-20, wherein the hydrothermal carbonization process (10) is carried out at a temperature of about 180°C to about 250°C and a pressure of about 10 bar to 50 bar for at least 30 minutes.

[0160] Statement 22. The method of any one of statements 1 to 21, wherein the hydrothermal carbonization process (10) is carried out at a temperature of about 200°C to about 220°C, a pressure of about 15 bar to about 25 bar, and for about 2 hours to 3 hours.

[0161] Statement 23. The method of any one of statements 1 to 22, wherein the biomass (101) is a wet biomass having a moisture content of 10% to 95% by weight, preferably 10% to 90% by weight.

[0162] Statement 24. The method of any one of statements 1-23, wherein the biomass (101) is selected from food residues, agricultural residues, animal by-products, or any combination thereof.

[0163] Statement 25. The method of any one of statements 1-24, wherein the biomass (101) comprises animal by-products, such as manure.

[0164] Statement 26. The microbial inoculant (109) is a member of the group consisting of Achromobacter, Aeromonas, Azospirillum, Bacillus, Bradyrhizobium, Enterobacter, Gluconacetobacter, Klebsiella, Pseudomonas, Rhizobia, Rhizobium, and Streptomyces, or a member of the group consisting of Achromobacter, Aeromonas, Azospirillum, Azotobacter, Bacillus, Bradyrhizobium, and Enterobacter. 26. The method of any one of statements 1-25, comprising one or more bacteria belonging to a genus selected from the group consisting of Gluconacetobacter, Klebsiella, Pseudomonas, Rhizobia, Rhizobium, and Streptomyces, such as the group consisting of Streptomyces, Bacillus, Azospirillum, and Bradyrhizobium, or a genus selected from the group consisting of Azotobacter, Streptomyces, Bacillus, Azospirillum, and Bradyrhizobium.

[0165] Statement 27. The method of any one of statements 1-26, wherein the microbial inoculant (109) comprises one or more filamentous fungi belonging to the group consisting of Alternaria, Aspergillus, Chaetomium, Exophiala, Fusarium, Penicillium, Phoma, Talaromyces, and Trichoderma, or a genus selected from the group consisting of Alternaria, Aspergillus, Beauveria, Chaetomium, Exophiala, Fusarium, Penicillium, Phoma, Talaromyces, and Trichoderma, such as the group consisting of Trichoderma and Penicillium, or a genus selected from the group consisting of Beauveria, Thrichoderma, and Penicillium.

[0166] Statement 28. The method of any one of statements 1-27, wherein the recovering step (50) comprises separating the fermentation product into a solids-rich fraction and a liquid fraction.

[0167] Statement 29. The method of any one of statements 1-28, wherein the recovering step (50) comprises drying the fermentation product or a solids-rich fraction of the fermentation product.

[0168] Statement 30. The method of any one of statements 1-29, wherein the recovering step (50) comprises separating spores from the fermentation product, and the microbial inoculant comprises spore-forming microorganisms.

[0169] Statement 31. The method of any one of statements 1-30, wherein the recovering step (50) comprises a process selected from the group consisting of grinding, pulverizing, and pelletizing, or wherein the recovering step (50) comprises one or more of a grinding, pulverizing, pelletizing, extrusion, and microgranulation process.

[0170] Statement 32. A microorganism and nutrient delivery system obtainable by the method according to any one of statements 1 to 31, -Fermented hydrochar; and -Microorganisms attached to the surface or pores of fermented hydrochar microbial and nutrient delivery systems, including

[0171] Statement 33. The microorganism and nutrient delivery system of 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%, such as at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, of the microorganisms in the microorganism and nutrient delivery system consist of microbial species contained in the microbial inoculant.

[0172] Statement 34. The microorganism and nutrient delivery system of statements 32 or 33, further comprising one or more adjuvants, such as one or more adjuvants selected from solvents, carriers, binders, surfactants, adhesives, tackifiers, antifreeze agents, thickeners, buffers, antifoaming agents, antioxidants, preservatives, stabilizers, fragrances, and colorants.

[0173] Statement 35. Use of the microorganism and nutrient delivery system of any one of statements 32-34 as a biostimulant.

[0174] Statement 36. Use of the microorganism and nutrient delivery system of any one of statements 32-34 as a biofertilizer.

[0175] Statement 37. Use of the microorganism and nutrient delivery system of any one of statements 32-34 as a biocontrol agent.

[0176] Statement 38. Use of the microorganism and nutrient delivery system of any one of statements 32-34 as a bioremediation agent.

[0177] Statement 39. Use of the microorganism and nutrient delivery system of any one of statements 32-34 as a microbial inoculant, such as a microbial inoculant for an anaerobic digester, a sewage treatment system, or a water treatment system.

[0178] Statement 40. Use of the microorganism and nutrient delivery system of any one of statements 32-34 as a microbial inoculant in a method according to any one of statements 1-31.

[0179] While the present invention has been described in conjunction with specific embodiments thereof, it is evident that many alternatives, modifications, and variations will be apparent to those skilled in the art in light of the foregoing description. Accordingly, it is intended to embrace within the spirit and scope of the appended claims all such alternatives, modifications, and variations as fall within the scope of the appended claims.

[0180] The presently disclosed aspects and embodiments of the present invention are further supported by the following non-limiting examples. [Example]

[0181] Example Example 1: Microbial growth in hydrochar and process water in agar material and method: The hydrochar was produced at the Ingelia SLHTC plant (Valencia, Spain). The feedstock used for the production of the hydrochar was pig manure (HC-C). Hydrothermal carbonization was carried out at 210 °C and 20–25 bar for 3–4 h.

[0182] Gravity filtration was used to separate the slurry obtained after hydrothermal carbonization into (wet) hydrochar and process water fractions.

[0183] As a reference material, Blonde peat moss (Novabalt) from the Baltic Sea was used. Three types of agar plates were prepared and sterilized by autoclaving (121°C for 15 minutes): PW-Agar: 1.5% w / v agar diluted in process water (adjusted to pH 7 using 1M sodium hydroxide) · HC-Agar: 2% w / v Hydrochar HC-C, 1.5% w / v agar diluted in reverse osmosis water.

[0184] HC+PW-Agar: 2% w / v Hydrochar HC-C, 1.5% w / v agar diluted in process water (adjusted to pH 7 using 1M sodium hydroxide) · Peat-agar: 2% w / v peat, 1.5% w / v agar diluted in reverse osmosis water.

[0185] The bacterial strains Streptomyces griseoviridis (LMG19321), Bacillus subtilis (LMG23370), Azospirillum brasilense (LMG28319), and Bradyrhizobium japonicum (LMG4252) and the fungal strains Trichoderma harzianum (MUCL22194) and Penicillium bilaiae (MUCL31187) were obtained from the Belgium Coordinated Collections of Microorganisms (BCCM). All strains were reactivated and grown on yeast mannitol agar (YMA) medium at 28°C for bacterial strains and 25°C for fungal strains. Microbiology agar (900040) and yeast mannitol agar NutriSelect Plus (900050) were obtained from Merck Life Sciences BV (Belgium).

[0186] Six different strains were inoculated onto four custom agar plates in a laminar airflow cabinet by streaking pure cultures from YMA plates with an inoculating loop. Plates were inoculated under normal atmosphere at 28°C and 25°C for bacterial and fungal strains, respectively. After 4 days, the media were visually scored for growth.

[0187] result:

[0188] [Table 1]

[0189] 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 hydrochar, possibly combined with process water, provides the nutrients necessary for uninhibited microbial growth. No contamination was observed on PW-agar, HC-agar, and HC+PW-agar plates.

[0190] Only Penicillium bilaiae was able to grow on peat-agar. Most peat-agar plates showed contamination with spores that were not destroyed during standard sterilization processes at 121°C. The lack of nutrients and risk of contamination indicate poor performance of peat as a growth substrate for selected microorganisms.

[0191] Example 2: Microbial Growth in Hydrochar and Process Water (without Agar) material and method: Hydrochar was produced at the Ingelia SLHTC plant (Valencia, Spain). The feedstocks used for hydrochar production were cow manure (HC-A), pig manure (HC-C), or lignocellulosic biomass (HC-BM). Hydrothermal carbonization was carried out at 210 °C and 20–25 bar for 3–4 h. Gravity filtration was used to separate the slurry obtained after hydrothermal carbonization into (wet) hydrochar and process water fractions.

[0192] As a reference material, Blonde peat moss (Novabalt) from the Baltic Sea was used. The same microorganisms and microbial culture conditions as in Example 1 were used.

[0193] Three types of hydrochar (containing approximately 50% moisture by weight (process water)) and peat (moistened with reverse osmosis water) were sterilized for 15 minutes at 121° C. The materials were distributed (approximately 1 gram of dry matter each) into empty (sterile) Petri dishes in a laminar air flow cabinet.

[0194] Materials were inoculated using an inoculating loop from microbial cultures grown on YMA agar plates. Plates were taped with parafilm to prevent moisture loss. Materials were incubated at 28°C and 25°C for bacterial and fungal strains, respectively, for solid-state fermentation.

[0195] After 7 days, growth was visually assessed and scored. For HC-C samples, growth was quantified by resuspending the dried material in sterile saline (0.8% NaCl) and then 10 -2 ~10 -10 A dilution series of 100 mg of 10 ...

[0196] result:

[0197] [Table 2]

[0198] It was possible to grow the selected microorganisms on the three different types of hydrochar without providing any other nutrients, except for Bacillus subtilis and Azospirillum brasilense, which did not produce any visual signs of microbial growth (Table 2).

[0199] [Table 3]

[0200] CFU counting confirmed that each of the selected microbial strains could be cultivated in HC-C to industrially relevant concentrations (Table 3), even Bacillus subtilis and Azospirillum brasilense, which showed no visual signs of growth. No contamination was observed on the CFU count agar plates.

[0201] Example 3: Microbial growth in hydrochar and process water versus peat material and method: Hydrochar was produced from hog manure (HC-C) as described in Examples 1 and 2. Chamber filter press filtration was used to separate the post-hydrothermal carbonization slurry into (wet) hydrochar and process water fractions.

[0202] As a reference material, Blonde peat moss (Novabalt) from the Baltic Sea was used. The bacterial strain Bradyrhizobium japonicum (LMG4252) and the fungal strain Trichoderma harzianum (MUCL22194) from the Belgian Collection of Microorganisms (BCCM) were used. All strains were reactivated 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 inocula was quantified by CFU counting.

[0203] In a laminar airflow cabinet, 1 mL of liquid inoculum was diluted to 50 mL in sterile saline (0.8% NaCl). Wet hydrochar (HC-C; 100 g dry matter) and peat (100 g dry matter) were inoculated with the diluted inoculum to ensure good distribution of the inoculum throughout the material. All materials were adjusted to 40% moisture content with reverse osmosis water. The materials were incubated aerobically in a sterile environment to carry out the solid-state fermentation process.

[0204] After 7 days, microbial growth was quantified by resuspending the fermented hydrochar and peat in sterile physiological water (0.8% NaCl) and measuring 10 -4 ~10 -10 A dilution series of 100 mg of 10 ...

[0205] result:

[0206] [Table 4]

[0207] Comparative analysis of microbial growth, measured in CFU / g, revealed significantly higher concentrations of microbial activity on fermented HC-C compared to fermented peat (Table 4 and Figure 2). For Bradyrhizobium japonicum, there was no growth on peat. These results indicate the potential of HC-C as a nutrient source for a wider range of microorganisms, in contrast to peat.

[0208] Example 4: Shelf life of fermented hydrochar material and method: Hydrochar is produced from hog manure (HC-C) as described in Examples 1 and 2. Chamber filter press filtration is used to separate the post-hydrothermal carbonization slurry into (wet) hydrochar and process water fractions.

[0209] Liquid inocula of the bacterial strain Azospirillum brasilense (LMG28319) and the fungal strain Trichoderma harzianum (BCCM:MUCL22194) were prepared as described in Example 3.

[0210] In a laminar airflow cabinet, 1 mL of liquid inoculum was diluted to 50 mL in sterile saline (0.8% NaCl). The diluted liquid inoculum was mixed in duplicate with wet hydrochar (HC-C; 100 g dry matter). All samples were adjusted to 40% moisture content with reverse osmosis water. The samples were incubated aerobically in a sterile environment for 7 days to undergo the solid-state fermentation process (fermented hydrochar). After drying, the material was distributed into several aluminum foil-lined packages and stored in portions at 4°C or 25°C to assess shelf life.

[0211] A commercially available reference of the fungal strain Trichoderma harzianum formulated against mineral clay was included as a reference.

[0212] After 0, 1, 7, 14, 28, 42, and 56 days of storage, CFU counts were determined by resuspending the fermented hydrochars and references in sterile physiological water (0.8% NaCl) and counting 10 -1 ~10 -7 A dilution series of 100 mg / ml was prepared in triplicate. The number of colony forming units (CFU) in the dilution series was determined by plating on YMA plates.

[0213] result:

[0214] [Table 5]

[0215] Table 5 shows that T. harzianum fermented with hydrochar had improved shelf life and increased CFU counts after storage compared to the commercial reference formulation containing mineral clay at different storage temperatures.

[0216] [Table 6]

[0217] Table 6 shows that the A. brasilense population in the fermented hydrochar exhibited improved shelf life, resulting in higher CFU counts after storage at both 4°C and 25°C compared to formulating A. brasilense with PBS, with 5.5E+07 CFU / mL and 1.77E+05 CFU / mL counted after 60 days of storage at 4°C and 28°C, respectively (Figures 2A and 2C in Cortes-Patiano and Bonilla (2015) African Journal of Biotechnology 14:2547-2553).

[0218] Example 5: Resistance of fermented hydrochar to desiccation and oxidative stress material and method: Hydrochar is produced from hog manure (HC-C) as described in Examples 1 and 2. Chamber filter press filtration is used to separate the post-hydrothermal carbonization slurry into (wet) hydrochar and process water fractions.

[0219] Liquid inocula of the bacterial strain Bacillus subtilis (BCCM: LMG23370) and the fungal strain Trichoderma harzianum (BCCM: MUCL22194) are prepared as described in Example 3.

[0220] One mL of liquid inoculum was diluted to 50 mL in sterile saline (0.8% NaCl). The diluted liquid inoculum was mixed in duplicate with wet hydrochar (HC-C; 100 g dry matter). All materials were adjusted to 40% moisture content with reverse osmosis water. One sample was immediately placed in a desiccator and air-dried at 25°C (microorganisms formulated with hydrochar). The other was incubated aerobically in a sterile environment for 7 days to undergo a solid-state fermentation process (fermented hydrochar). After drying (microorganisms formulated with hydrochar) or solid-state fermentation (fermented hydrochar), the material was distributed into several air- and water-permeable packages stored at 25°C to evaluate shelf life under desiccation and oxidative stress.

[0221] After storage for 0, 1, 7, 14, 28, and 56 days, CFU counts were determined by resuspending the fermented hydrochar and the microorganisms combined with the hydrochar in sterile physiological water (0.8% NaCl) and counting 10 -4 ~10 -10 A dilution series of 1000 mg / ml is prepared in triplicate. The number of colony forming units (CFU) in the dilution series is determined by plating on YMA plates.

[0222] result: Higher CFU counts are obtained in fermented hydrochar after environmental stress during storage.

[0223] Example 6: Fouling resistance of fermented hydrochar material and method: Hydrochar was produced from hog manure (HC-C) as described in Examples 1 and 2. Chamber filter press filtration was used to separate the post-hydrothermal carbonization slurry into (wet) hydrochar and process water fractions.

[0224] A liquid inoculum of the fungal strain Trichoderma harzianum (BCCM:MUCL22194) was prepared as described in Example 3.

[0225] In a laminar airflow cabinet, 1 mL of liquid inoculum was diluted to 50 mL with sterile saline (0.8% NaCl). The diluted liquid inoculum was mixed with wet hydrochar (HC-C; 100 g dry matter). The material was adjusted to 40% moisture content with reverse osmosis water and incubated aerobically in a sterile environment for 7 days to carry out the solid-state fermentation process (fermented hydrochar).

[0226] The other (control) sample was wet hydrochar (HC-C; 100 g dry matter) adjusted to 40% moisture content with reverse osmosis water without being inoculated and fermented with microorganisms (control).

[0227] Both the fermented hydrochar and the control non-sterile samples were exposed to ambient air for 2 hours to allow for microbial contamination. After 2 hours, both materials were again incubated in a sterile environment at 25°C for 7 days.

[0228] Microbial growth was visually observed and quantified by resuspending the fermented hydrochar and wet hydrochar (control) in sterile physiological water (0.8% NaCl) for 10 min. -1 ~10 -7 A dilution series of 100 mg of 10 ...

[0229] result: Compared to the control hydrochar, there was a significant reduction in the growth of contaminating microorganisms in the fermented hydrochar (Figure 3). Our analysis revealed that Trichoderma harzianum was the only microbial species in the fermented hydrochar, both when exposed to ambient air (non-sterile) and when not (sterile) prior to fermentation (Table 7 and Figure 3). These findings suggest the effectiveness of the fermentation process with desirable microorganisms in creating an environment that is not conducive to the growth of undesirable microorganisms.

[0230] [Table 7]

[0231] Example 7: Submerged fermentation of hydrochar material and method: Hydrochar was produced from pig manure (HC-C) as described in Examples 1 and 2.

[0232] Liquid inocula of the bacterial strain Azospirillum brasilense (LMG28319) and the fungal strain Trichoderma harzianum (BCCM:MUCL22194) were prepared as described in Example 3.

[0233] Four different liquid fermentation media were prepared and sterilized by autoclaving (121°C for 15 minutes): 50 g of HC-C (dried at 105°C) diluted to 1 L with saline (0.8% NaCl) (HC-C) · 50g process water (PW) diluted to 1L with saline (0.8% NaCl) 50g of HC-C (dried at 105°C) and 50g of process water (HC-C+PW) diluted to 1L with saline (0.8% NaCl) 1L of normal saline (0.8% NaCl) (control) 100 mL of fermentation medium was distributed in triplicate into 250 mL shake flasks. The shake flasks were inoculated with 1 mL of liquid inoculum and incubated in a shaking incubator at 25° C. and 150 rpm for 3 days.

[0234] Growth of A. brasilense was quantified by performing CFU counts on each shake flask (diluted in saline and plated on YMA medium). Growth of T. harzianum in each liquid fermentation condition was quantified by measuring the dry mass of its 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.

[0235] result:

[0236] [Table 8]

[0237] Table 8 shows that the most abundant population of A. brasilense was in liquid fermentation containing HC-C and PW as microbial nutrient sources.

[0238] [Table 9]

[0239] Table 9 shows that the most abundant population of T. harzianum was observed in liquid fermentation incorporating HC-C and PW as microbial nutrient sources.

[0240] Example 8: Microbial Growth in Hydrochar and Process Water (without Agar) material and method: Hydrochar was produced from pig manure (HC-C) as described in Examples 1 and 2.

[0241] The bacterial strains Azotobacter chrooccum (LMG3852) and Pseudomonas fluorescens (LMG1244), and the fungal strain Beauveria bassiana (IHEM3558) were obtained from the Belgian Comprehensive Collection of Microorganisms (BCCM).

[0242] HC-C (containing approximately 50% by weight of moisture (process water)) was distributed into microbox containers (50 g dry matter each), which were sterilized at 121°C for 15 minutes.

[0243] In a laminar airflow cabinet, an inoculation loop taken from a microbial culture growing on a YMA agar plate was diluted in 50 mL of process water (PW) and subsequently introduced into the material. The microboxes containing the inoculated HC-C were incubated at 28 °C and 25 °C for bacterial and fungal strains, respectively, for solid-state fermentation.

[0244] After 7 days, growth was visually assessed and scored. For HC-C samples, growth was quantified by resuspending the dried material in sterile saline (0.8% NaCl) and then 10 -1 ~10 -7 A dilution series of 100 mg of 10 ...

[0245] result:

[0246] [Table 10]

[0247] CFU quantification confirmed the successful cultivation of Azotobacter chroococcum, Pseudomonas fluorescens, and Beauveria bassiana in HC-C at concentrations reaching industrial relevance (Table 10). Notably, no cases of contamination were observed on the CFU count agar plates.

[0248] Example 9: The importance of maintaining a sterile workflow material and method: Hydrochar was produced from hog manure (HC-C) as described in Examples 1 and 2. Chamber filter press filtration was used to separate the post-hydrothermal carbonization slurry into (wet) hydrochar and process water fractions.

[0249] A liquid inoculum of the fungal strain Trichoderma harzianum (BCCM:MUCL22194) was prepared as described in Example 3.

[0250] In a laminar airflow cabinet, 1 mL of liquid inoculum was diluted to 50 mL in sterile saline (0.8% NaCl). The diluted liquid inoculum was mixed with wet hydrochar (HC-C; 100 g dry matter). The material was adjusted to 40% moisture content with reverse osmosis water. Another (control) sample contained the same composition, but the process was carried out outside the laminar airflow cabinet under non-sterile conditions.

[0251] Both samples were aerobically incubated in a sterile environment for 7 days to undergo the solid-state fermentation process (fermented hydrochar).

[0252] Microbial growth was visually observed and quantified by resuspending both samples in sterile saline (0.8% NaCl) for 10 min. -1 ~10 -7 A dilution series of 100 mg of 10 ...

[0253] result:

[0254] [Table 11]

[0255] T. harzianum was detectable in samples processed outside the laminar airflow cabinet, but in lower populations due to the growth of undesirable contaminating microorganisms (Fig. 4).

Claims

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

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

3. 3. The method of claim 2, wherein the process water content of the slurry is reduced by a mechanical process (30), 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 a 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 of claim 7, wherein the fermentation is carried out under sterile conditions.

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

10. 10. The method of claim 9, wherein the fermentation of the (first) slurry inoculated with the microbial innoculant is carried out under sterile conditions.

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

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

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

15. The recovery step (50) - separating the fermentation product into a solid-rich fraction and a liquid fraction; drying the fermentation product or a solid-rich fraction of the fermentation product; and - a processing step selected from the group consisting of grinding, milling, pelletizing, extrusion, and microgranulation processes The method of any one of claims 1 to 14, comprising any one or more of:

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

17. 17. The method of any one of claims 1 to 16, wherein the microbial inoculant (109) comprises one or more bacteria belonging to a genera selected from the group consisting of Azotobacter, Streptomyces, Bacillus, Azospirillum, and Bradyrhizobium, and / or one or more filamentous fungi belonging to a genera 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, said system comprising: - fermented hydrochar; - microorganisms attached to the surface or pores of the fermented hydrochar; and optionally one or more adjuvants selected from solvents, carriers, binders, surfactants, adhesives, tackifiers, antifreeze agents, thickeners, buffers, antifoaming agents, antioxidants, preservatives, stabilizers, fragrances, and colorants; microbial and nutrient delivery systems, including

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

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

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

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