Edible composition containing filamentous fungi and bioreactor system for its cultivation

Abstract

A method for producing an edible fungal biomat formulation as the sole protein source and / or protein ingredient in food products is provided, as well as a single-use or repeatable self-contained biofilm-biomat reactor comprising a container having at least one compartment and a raw material, a fungal inoculant, a gas-permeable membrane, and optionally a liquid nutrient medium disposed within the compartment.

Description

Technical Field

[0001] The present application relates to edible fungi, and provides a method for producing edible fungi used in food products, liquid and solid formulations of edible fungi, and related uses and methods, food products containing edible fungi, and methods and uses thereof.

Background Art

[0002] The United Nations predicted in August 2017 that the world's population would reach 7.5 billion, which would reach 8 billion in 2023 and 10 billion in 2056. In a related report, the Food and Agriculture Organization of the United Nations (FAO) estimated that if the world's population reaches 9.1 billion by 2050, the world's food production needs to increase by 70%, and double in the developing world. This increase in food production needs to occur despite rising energy costs, decreasing underground aquifer resources, loss of agricultural land due to urban sprawl, and increasingly severe weather due to climate change (e.g., rising temperatures, increasing droughts, increasing floods, etc.). This is particularly a problem in regions such as Africa where the protein intake was already inadequate in 2009, and countries such as China, India, Pakistan, and Indonesia where there is a risk of inadequate protein intake. In addition, world demand is expected to increase by 60% for meat and 50% for dairy products by 2040.

[0003] However, not all protein sources are created equal. Animal-derived foods (meat, eggs, dairy products) provide "complete" proteins as they contain all the essential amino acids, namely methionine, leucine, isoleucine, phenylalanine, valine, threonine, histidine, tryptophan, and lysine. Plant-derived foods contain some essential amino acids but generally lack a complete combination. For example, the proteins found in starchy roots lack the essential amino acid lysine, and as a result, it must be obtained from another food in the diet. Legumes and pulses contain high levels of lysine but lack the essential amino acid methionine. It is possible to assemble complete proteins by combining plant-based foods, but ensuring a nutritionally balanced diet is much easier with complete proteins.

Summary of the Invention

Problems to be Solved by the Invention

[0004] One non-animal source of complete protein is obtained from edible filamentous fungi such as Fusarium venenatum (formerly classified as Fusarium graminearum). However, protein production from these sources has hitherto required significant investment in energy resources and production facilities, such as capital-intensive bioreactors and centrifuges. There remains a need for culture, harvesting, and food production methods that require low energy, consume few natural resources, and are low in cost. The present invention addresses these problems.

[0005] Furthermore, one area for reducing logistics supplies related to natural disasters, logistically isolated environments or responses to military and / or space / extra-terrestrial missions is the life support loop that provides mission-critical products such as nutritious and appetizing foods, fuels, metabolite expression platforms, building materials and / or microbial factories, in particular the closure of waste streams. In many cases, these types of environments may not have access to or have limited access to sterilization facilities and / or require a sealed sterile system to completely contain waste streams and / or food, fuel and produced substances. For example, research by the European Space Agency (Expeditions 25-28, Growth and Survival of Colored Fungi in Space (CFS-A)) has demonstrated that fungi can grow inside a space station and can decompose food and other organic materials under humid conditions. Here, the containment of the fungal system is excellent at preventing accidental contamination of supplies and surfaces. In addition to the need to decompose food and waste in the developing field of space travel, these needs also exist when dealing with natural disasters, at the site of military operations, working in the wilderness, public health and situations in third world countries where refrigeration is unreliable, confined spaces, logistically difficult arenas and certain agricultural operations / production processes. There is a need to have an in-built sterile system that operates efficiently with minimal space, energy, and maintenance.

Means for Solving the Problems

[0006] A robust and efficient portable in-situ biofilm - biomatt reactor system that can convert a variety of waste streams into numerous valuable products addresses these problems. The present disclosure describes a simple sterile bioreactor platform that requires no agitation, no active aeration, and no energy source (other than temperature control) during fermentation, produces minimal to no waste residue, requires little water, and generates a high-density, easily harvestable, textured biomatt. Additionally, this in-situ biofilm - biomatt reactor system is portable and / or scalable for larger, more concentrated missions and / or populations.

[0007] Summary The present disclosure provides a formulation of edible filamentous fungi. The edible filamentous fungi grow on a liquid medium under surface fermentation conditions to produce a filamentous fungal biomatt. In one embodiment, a method for the surface fermentation production of an edible fungal biomatt is provided, the method comprising inoculating a liquid synthetic growth medium containing a carbon source with plankton-like and / or conidial fungal cells, incubating the inoculated growth medium at room temperature, and harvesting the cohesive biomatt produced by the fungi. In some embodiments, the inoculated growth medium is incubated in an open tray or in an open tray contained in at least a semi-sterile environment.

[0008] In another embodiment, the method for the surface fermentation production of an edible fungal biomatt enables the harvesting of a portion of the biomatt while maintaining the growth potential of the remaining biomatt.

[0009] In a further embodiment, the filamentous fungus is Fusarium oxysporum MK7 strain (deposited with the American Type Culture Collection, 1081 University Boulevard, Manassas, Virginia, USA under ATCC PTA - 10698), which has a complete protein content of 46 - 51%, contains all essential amino acids at high levels, specifically 42 - 43% essential amino acids and 4 - 21% BCAA, which is higher than that of eggs. Additionally, the Fusarium oxysporum MK7 strain has 8 - 10% minerals and ash, contains high levels of calcium (1.3 mg / 100 g serving), iron (5.5 mg / 100 g serving), 1 - 2% nucleic acids, and 6 - 11% lipids, 85% of which are unsaturated.

[0010] In another embodiment, the filamentous fungus is Fusarium venenatum or Fusarium fujikuroi.

[0011] In yet another embodiment, the filamentous fungus is selected from the group consisting of Agaricus bisporus (cream and white), Boletus edulis (porcini), Cantarellus cibarius (chanterelle), Calvatia gigantea (giant puffball), Cyclocybe aegerita (velvet pioppini), Ganoderma lucidum (reishi), Grifola frondosa (maitake), Morchella species (morel), Hypsizygus tessellatus (clam shell), Hypsizygus ulmarius (elm oyster), Laetiporus species [chicken of the woods], Lentinula edodes (shiitake), Pleurotus eryngii (king oyster), Calvatia gigantean (giant puffball), Pleurotus ostreatus (pearl oyster), Pleurotus ostreatus var. columbinus (blue oyster) and other Pleurotus sp. (e.g., P. citrinopileatus, tuberregium), Hypsizygus ulmarius (elm oyster), Pholiota microspora (forest pholiota), Sparassis crispa (cauliflower), and Tuber species (truffle).

[0012] In additional embodiments, the carbon source is a saccharide (e.g., sucrose, maltose, glucose, fructose, oligosaccharide, etc.), sugar alcohol (e.g., glycerol, polyol, etc.), starch (e.g., corn starch, etc.), starch derivative, starch hydrolyzate, hydrolyzed hydrogenated starch, lignocellulose pulp or raw material (e.g., sugar beet pulp, agricultural pulp, wood pulp, distillers dried grains, brewery waste, etc.), corn steep liquor, acid whey, sweet whey, whey, wheat steep liquor, industrial liquor, food refinery product / waste stream, and / or combinations thereof.

[0013] In yet another embodiment, a method for the surface fermentation production of an edible filamentous fungal biomass starting from the fruiting body or spores of a filamentous fungus is provided. For biomass starting from the fruiting body, the method includes surface sterilizing the fungal fruiting body, reducing the size of the sterilized fungal fruiting body, surface sterilizing the reduced fungal fruiting body, inoculating cells from the reduced and sterilized fungal fruiting body into a synthetic liquid growth medium containing a carbon source, incubating the inoculated growth medium at room temperature, and harvesting the cohesive filamentous biomass produced by the fungus. For biomass starting from the spores of a filamentous fungus, the method includes inoculating sterile spores into a synthetic liquid growth medium containing a carbon source, incubating the inoculated growth medium at room temperature, and harvesting the cohesive filamentous biomass produced by the fungal spores.

[0014] In some embodiments, the fruiting bodies or spores of filamentous fungi are selected from the group consisting of mushrooms (cream and white), porcini mushrooms, chanterelle mushrooms, giant puffball mushrooms, velvet pioppini mushrooms, reishi mushrooms, maitake mushrooms, morel mushrooms, clamshell mushrooms, elm oyster mushrooms, chicken of the woods mushrooms, shiitake mushrooms, king oyster mushrooms, pearl oyster and blue oyster mushrooms, enoki mushrooms, cauliflower mushrooms, and truffle mushrooms. Aseptic spores of filamentous fungi were obtained from commercial vendors such as Myco Direct (Huntley, Illinois).

[0015] In yet another embodiment, the filamentous biomats generated from the plankton-like cells, microconidia cells, reduced-sized fruiting bodies, or spores of filamentous fungi comprise aggregates of mycelia and / or hyphae having a length of less than 5 mm. In still yet another embodiment, the reduced-sized filamentous biomats comprise aggregates longer than 5 mm.

[0016] In a further embodiment, the pH of the growth medium inoculated with fruiting body cells has a pH of about 4.0 - 4.1.

[0017] In another further embodiment, the carbon source of the synthetic growth medium for the growth of fruiting bodies and / or cells comprises glycerol, starch, corn steep liquor, acid whey, or combinations thereof, and / or the incubation period is about 2 - 10 days or more.

[0018] Another embodiment relates to a formulation of edible fungal filamentous biomats comprising edible fungal filamentous biomats particles isolated from edible fungal filamentous biomats grown by surface fermentation on a synthetic liquid medium.

[0019] A further embodiment relates to a formulation in the form of a liquid, solid, or gel.

[0020] Still more embodiments relate to formulations that are pastes, powders, porous / ventilated blocks, and / or solid blocks.

[0021] Yet another embodiment relates to a food product comprising a formulation of an edible fungal mycelial biomass, with or without other ingredients.

[0022] Additional embodiments relate to food products made from the formulations, such as meat substitute foods, drinks, beverages, yogurt, desserts, confections, or candies.

[0023] Another embodiment relates to a food product that is a frozen dessert, such as a mousse or an ice cream-like food that does not melt at room temperature, made from the formulation.

[0024] Further embodiments relate to the use of the formulation as a component for increasing and / or mimicking the texture of meat (e.g., burgers, sausages, hot dogs, chicken or turkey nuggets, and / or fillets) in a food product, and / or for increasing the protein content of the food product. Still further embodiments relate to the use of the liquid dispersion formulation as a substitute milk and / or for increasing the protein content of milk, dairy products, and / or substitute dairy products.

[0025] Yet another embodiment relates to the isolation of oil from an edible filamentous fungal biomass.

[0026] The present disclosure also provides an integrated biofilm - biomatt reactor. In one embodiment, the integrated biofilm - biomatt reactor comprises a container, as well as raw materials, fungal inoculum, a gas - permeable membrane, and optionally a liquid nutrient medium contained within the container. In some embodiments, the reactor is a single - use reactor, while in other embodiments, the reactor can be reused.

[0027] Generally, in various embodiments, the container can be sealed and may include a lid for the container in addition to the seal. In some embodiments, the container is a tray with a lid. In other embodiments, the container is a Petri dish with a lid or other type of container with a lid. In still other embodiments, the container is a bag. In still other embodiments, the container is a pipe whose upper part is made of a gas-permeable membrane (2) (see Figure 23). In some embodiments, the container consists of a plurality of growth compartments. In some embodiments, the container has a manifold design and / or a baffle system. In some embodiments, the container is made in whole or in part from one or more consumable raw materials.

[0028] In some embodiments, the raw material is inoculated with ascomycete fungal strains such as, by way of example, Fusarium oxysporum MK7 strain (deposited with the American Type Culture Collection, 1081 University Boulevard, Manassas, Virginia, USA as ATCC PTA-10698), Fusarium venenatum, and Fusarium avenaceum, Gibberella fujikuroi, Rhizopus species, Aspergillus species, and / or combinations thereof.

[0029] In other embodiments, the raw material is inoculated with basidiomycete fungal strains such as mushrooms (cream and white), Boletus edulis (porcini), Cantharellus cibarius (chanterelle), Gyroporus cyanescens (giant puffball), Cyclocybe aegerita (velvet pioppini), Ganoderma lucidum (reishi), Grifola frondosa (maitake), Morchella species (morel), Clitocybe nebularis (clouded agaric), Pleurotus ostreatus (oyster mushroom), Laetiporus species (chicken of the woods), Pleurotus eryngii (king oyster mushroom), Hypsizygus tessellatus (pearl oyster and blue oyster), Pholiota nameko (forest pholiota), Sparassis crispa (cauliflower), and / or Tuber species (truffle).

[0030] In some embodiments, the raw materials are wastes such as naturally occurring urine and / or feces, as well as food waste and by-products, industrial waste and / or by-products, agricultural waste and by-products, plant materials, and / or combinations thereof. In other embodiments, the raw materials can be synthetic or manufactured substitutes such as synthetic urine. With respect to raw materials that are plant materials or that contain plant materials, the plant materials are typically lignocellulose. Lignocellulosic materials include agricultural crop residues (e.g., wheat straw, barley straw, rice straw, pea straw, oats, small grain straw, corn stover, corn fiber (e.g., corn fiber gum (CFG), distillers dried grain (DDG), corn gluten meal (CGM), switchgrass, hay alfalfa, sugarcane bagasse), non-agricultural biomass (e.g., algal biomass, cyanobacterial biomass, urban tree residues), vegetables (e.g., carrots, broccoli, garlic, potatoes, beets, cauliflower), forest products and industrial residues (e.g., softwood primary / secondary mill residues, hardwood softwood primary / secondary mill residues, recycled paper pulp sludge, anaerobic digestion residues), lignocellulose-containing wastes (e.g., newsprint, waste paper, brewing grains, used rubber tires (URT), municipal organic waste, household waste, medical organic waste, sugars, starches, waste oil, olive oil, olive oil processing waste, bat guano, and wastes generated during the production of biofuels (e.g., processed algal biomass, glycerol), and combinations thereof. Typically, the gas-permeable membrane is in direct contact with the surface of one or more raw materials, an appropriate liquid medium, and an inoculation material present within the container and is sealed to that surface. In some embodiments, an appropriate culture medium may be present.

[0031] In some embodiments, the gas-permeable membrane consists of a polymeric material such as polypropylene, polyethylene, polytetrafluoroethylene, polycarbonate, polyamide, polypyrrolone, poly(amidoamine) dendrimer composite, cellulose acetate, butadiene-acrylonitrile, Teflon AF2400, and nylon. In some embodiments, the pore size of the gas-permeable membrane ranges from 0.05 to 1.5 μm, such as 0.2 μm, 0.45 μm, and 1.0 μm. In some embodiments, the gas-permeable membrane is in the form of a sterile cloth-like material, and in other cases, the membrane is in the form of a paper-like material. In some embodiments, the surface is smooth in texture, and in other cases, the surface is rough in texture. In some embodiments, the gas diffusion passageways are essentially straight, and in other cases, the passageways are tortuous.

[0032] In some embodiments, the reactor produces a biofilm-biomat that serves as a food source such as a protein source and / or an oil and fat source. In other embodiments, the biofilm-biomat serves as a leather analog and / or a bioplastic. In still other embodiments, the biofilm-biomat serves as a resource for biofuel precursors or as biofuel itself. In still other embodiments, the biofilm-biomat serves to produce organic products such as organic acids, antibiotics, enzymes, hormones, lipids, mycotoxins, vitamins, pigments, and recombinant heterologous proteins.

Brief Description of the Drawings

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Mode for Carrying Out the Invention

[0056] Edible filamentous fungi can be used alone or incorporated into food products as a protein source. For example, the protein content is 27.25% in pearl oyster mushroom, 24.65% in blue oyster mushroom, 15.05% in shiitake mushroom (Stamets (2005) Int J Medicinal Mushrooms 7:103 - 110), 27.3% in giant puffball (Agrahar - Murugkar and Subbulakshmi (2005) Food Chem 89:599 - 603), and 32.61% in cauliflower mushroom (Kimura (2013) BioMed Res. Int. Article ID 982317).

[0057] Nevertheless, fruiting bodies of filamentous fungi of the phylum Basidiomycota, such as mushrooms (cream and white), Boletus edulis (porcini), Cantharellus cibarius (chanterelle), Ganoderma lucidum (reishi), species of the genus Morchella (morel), Clitocybe nuda (blewit), Pleurotus ostreatus and Pleurotus eryngii (oyster mushrooms), Pleurotus eryngii (king oyster mushroom), Flammulina velutipes (enoki), Sparassis crispa (cauliflower mushroom), Hypsizygus ulmarius (elm oyster mushroom), Cyclocybe aegerita (velvet pioppini), Grifola frondosa (maitake), Lentinula edodes (shiitake), species of the genus Laetiporus (sulphur shelf), Polyporus squamosus (giant polypore), and species of the genus Tuber (truffle), are commonly used in food products, but there are few products mainly containing vegetative mycelia of filamentous fungi of the phylum Basidiomycota or Ascomycota. This is partly because the mycelium usually lives underground or is hardly separable from the substance on which it grows.

[0058] Furthermore, under certain conditions, filamentous fungi can form fungal biomats by surface fermentation under anaerobic, microaerophilic, or aerobic conditions or combinations thereof. Here, the filamentous fungal biomats mainly contain mycelia, fragments of mycelia, hyphae, fragments of hyphae in the form of fungal species and / or strains and / or their progeny, and to a lesser extent contain conidia, microconidia, macroconidia, or any and all combinations thereof, and may also contain pycnidia and chlamydospores in some cases.

[0059] Typically, filamentous biomats mainly consist of mycelium, i.e., a complex network of intertwined vegetative mycelium filaments. The average length of non-cut filaments in the biomats is generally at least 0.1 mm, for example 0.1 mm - 0.5 mm, 0.5 mm - 50 cm, 0.6 mm - 40 cm, 0.7 mm - 30 cm, 0.8 mm - 25 cm, 1.0 mm - 20 cm, 1.4 mm - 15 cm, 1.6 mm - 10 cm, 1.7 mm - 8 cm, 1.8 mm - 6 cm, 2.5 mm - 4 cm, and 5 mm - 2 cm, 2 cm - 25 cm, 4 cm - 30 cm, 5 cm - 40 cm, 6 cm - 50 cm, 8 cm - 60 cm, 10 cm - 100 cm.

[0060] The growth of filamentous fungal biomats can be achieved by surface fermentation. This involves inoculating filamentous fungal cells into a liquid medium containing a carbon source and a nitrogen source. Suitable carbon sources are saccharides (e.g., sucrose, maltose, glucose, fructose, rare sugars in Japan, etc.), sugar alcohols (e.g., glycerol, polyols, etc.), starches (e.g., corn starch, etc.), starch derivatives (e.g., maltodextrin, cyclodextrin, glucose syrup, hydrolyzates and modified starches), starch hydrolyzates, hydrolyzed hydrogenated starch (HSH, e.g., hydrogenated glucose syrup, maltitol syrup, sorbitol syrup, etc.), lignocellulose pulp or raw materials (e.g., sugar beet pulp, agricultural pulp, wood pulp, distillers dried grains, brewery waste, etc.), corn steep liquor, acid whey, sweet whey, whey, wheat extract, carbohydrates, food waste, olive oil processing waste, hydrolyzates derived from lignocellulosic materials, and / or combinations thereof. The filamentous fungal cells generate a biomatt located on the surface of the growth medium. That is, they essentially float on top of the growth medium.

[0061] In many cases, especially with Ascomycota fungi, the growth medium was inoculated with an inoculum containing plankton-like filamentous fungal cells. High-quality inoculum consists of plankton-like cells that do not clump or aggregate together and are preferably isolated from the exponential growth phase and defined as single cells that can contain microconidia. Ideally, the cells of the inoculum float on the surface of the growth medium like cells with a high lipid content, resulting in an increased growth rate. Cells or cell clumps that sink within the growth medium have a negative impact on the cells floating on the surface and the biomats they form. Specifically, biomats resulting from growth media containing a significant number of aggregated and sunken cells usually discolor and tend not to grow a uniformly high-density mat.

[0062] For inoculation with Basidiomycota spores, approximately 2 cc of sterile spores suspended in deionized water from a spore syringe (e.g., MycoDirect, Huntley, IL) were used to inoculate approximately 75 mL of growth medium in a small Pyrex tray. Alternatively, 1 cc of spores suspended in deionized water from a spore syringe was planted into a container with malt extract agar medium + CF (30 g dried malt extract, 20 g agar, 1000 mL water + 0.01% chloramphenicol) using standard aseptic conditions. The container was sealed with parafilm and incubated at room temperature until the mycelium completely covered the surface of the agar. Sections of mycelium from agar specimens approximately 2 cm wide cut in a wedge shape were then cut into the smallest possible size squares and transferred to a tube with the growth medium. The liquid culture tube was sealed and incubated at room temperature, and shaken for approximately 1 minute at least 5 times a day by hand or mechanical means (i.e., continuous shaking or continuous stirred tank reactor) to break the mycelium as much as possible. The liquid culture was incubated for usually more than 3 days until it appeared turbid. The liquid culture was then used to inoculate 10% or 15% of the total growth medium volume in the tray with growth medium.

[0063] Basidiomycete fruiting bodies were also used to create an inoculum for initiating filamentous biomass. In some cases, the inoculum was produced by (a) surface sterilizing the fruiting bodies with, for example, a 5% bleach solution, (b) rinsing in sterile medium, (c) grinding under sterile conditions into aggregates of less than 5 mm in length or greater than 5 mm depending on the end use, and (d) surface sterilizing the ground mushroom biomass with, for example, a 5% bleach solution and rinsing again in sterile medium. 5 grams of ground and surface-sterilized fruiting body biomass was used directly as the inoculum. In other cases, pure cultures derived from the fruiting bodies were used. Here, approximately 3 mm 3 of the fruiting body was placed on an agar medium containing 0.01% chloramphenicol and incubated at room temperature. After 2 - 5 days of growth, the mycelium was transferred onto fresh agar + chloramphenicol medium and grown for an additional 3 - 7 days. The culture purity was confirmed by extracting and purifying the DNA (FastDNA Spin Kit, MP Biomedicals), sequencing the 16S rRNA sequence and / or the ITS region, and performing phylogenetic classification of the sequences using Blast (NCBI database). After confirmation, the mycelium was used to inoculate 50 mL of sterile liquid medium, shaken / rotated at 185 rpm for approximately 5 days, and then used as an inoculum at a ratio of approximately 7.5% inoculum to 92.5% liquid medium.

[0064] Several different media can be used, but some media such as Hansen's medium (per liter = 1.0 g peptone, 0.3 g KH2PO4·7H2O, 2.0 g MgSO4·7H2O, 5.0 g glucose, C:N ratio 26.9) are not well-suited for the growth of filamentous fungal biomass. Sufficient cohesive biomass was not produced in this medium. Exceptionally successful media include MK7A, MK7-1, MK7-3 (all described in WO 2017 / 151684), as well as the media shown below.

[0065] Malt medium 001 (C:N ratio 19.1)

Table A

[0066] MK-7 SF medium (C:N ratio 7.5)

Table B

Table C

[0067] Malted medium 001 supplemented with NH4NO3 (C:N ratio 7.5)

Table D

[0068] The osmotic pressure values were taken using a recently calibrated osmometer (Model 3250 SN: 17060594) that can measure up to 5000 mOsm by aseptically removing 250 μl of the medium. Three measurements were taken, and the following results were obtained. Hansen’s = 39, 39, 38; Malt 001 = 169, 168, 169; MK-7 SF = 1389, 1386, 1387; Malt 001 + NH4NO3 = 288, 287, 286.

[0069] In addition, the medium used in our method can define the protein content of the resulting biomats. For example, the natural protein content of the fruiting bodies of blue oyster mushrooms has been reported to be 24.65% (Stamets (2005) Int J Medicinal Mushrooms 7:103-110), but the blue oyster biomats grown on Malt 001 medium according to our method have a higher moisture-corrected protein content of 29.82%, with a 5.71% increase in protein content. Even more strikingly, the protein content of the fruiting bodies of giant puffball mushrooms has been reported to be 27.3% (Agrahar-Murugkar and Subbulakshmi (2005) Food Chem 89:599-603), yet the giant puffball biomats grown on Malt 001 medium by our method have a moisture-corrected protein content of 32.04%, while the MK7-1 SF medium yields a moisture-corrected protein content of 46.33% and the Malt 001+NH4NO3 medium yields a moisture-corrected protein content of 46.88%, with the protein content being essentially 19.85% higher than the values reported by Agrahar-Murugkar and Subbulakshmi.

[0070] Harvesting of the biomats is usually after 2 - 3 days of growth, but in some cases, a longer growth period is desirable, for example when a thicker or higher density biomats are desired / required. For example, a growth period of 3.5 - 4 days, 3 - 5 months, 4 - 6 days, 5 - 7 days, 6 - 9 days, 7 - 10 days, 19 - 21 days, or even up to 2 months may be desirable. Due to the cohesive structure of the filamentous biomats grown under the surface fermentation conditions described in International Application PCT / US2017 / 020050 and herein, these filamentous biomats have sufficient tensile strength to be lifted essentially intact from the surface of the medium at the end of the growth period. Table 1 shows some examples of the measured tensile strength. Table 1 - Average Tensile Strength of Some Filamentous Fungal Biomats

Table 1

[0071] Surface fermentation can be carried out under various conditions including static medium conditions (described in International Application PCT / US2017 / 020050), semi-static medium conditions, and continuous medium flow conditions.

[0072] Growth under semi-static medium conditions means replacing at least a portion of the medium before harvesting the filamentous fungal biomass. Under these conditions, linear dry biomass production is possible from 4 to 18 days (r 2 = 0.995), after which the biomass weight stabilizes at about 2.5 Kg dry / m 2 .

[0073] The biomass can also be produced under continuous medium flow conditions where the growth of the biomass is restricted to the surface of the growth medium and the medium under the mat is continuously or semi-continuously refreshed.

[0074] However, in some cases, it is desirable to harvest the growing biomass semi-continuously. In this case, some portion of the biomass is removed and then the remaining portion is physically moved to the open area of the medium created by the removal of a portion of the biomass. This can be achieved by physically grasping the biomass and pulling it until it touches the edge of the surface fermentation vessel or by other mechanical means. The resulting open area can then be utilized for the growth of new biomass without a separate or additional inoculation step since the medium already contains viable fungal cells. This process can be repeated periodically, which may be particularly useful when refreshing the medium or reintroducing limited nutrients.

[0075] The biomass can also be harvested continuously. Continuous removal can be facilitated by several mechanisms. One such example is a roller wheel attached to the mature end of the biomass (see Figure 7). The roller wheel rotates slowly, harvesting the mature biomass while creating an open medium for the growth of new biomass at the other end of the surface fermentation vessel. The typical harvesting rate is 1.56 cm / day, but this can be changed according to specific needs or the desires of the user.

[0076] Growth under membrane encapsulation / sealed - seal bioreactor conditions involves encapsulating a liquid growth medium within a suitable system / container without a gas headspace. Suitable systems / containers are, for example, trays, Petri dishes, or containers with a relatively large surface - area - to - depth ratio. A gas - permeable membrane is placed directly on the surface of the liquid medium and sealed tightly to the system / container. Suitable membranes include, for example, polypropylene membranes (e.g., KC100 Kimguard, Kimberly - Clark, Roswell, GA), polyester membranes, polycarbonate membranes, silicone membranes, polyamide membranes, cellulose membranes, and ceramic membranes. Gas exchange between the growing biomass and the surrounding atmosphere occurs solely through the semi - permeable membrane.

[0077] In some cases, UVB light (290 - 320 nm) can cause pigment production by filamentous fungi such as Fusarium oxysporum MK7 strain, resulting in pigment - deposited biomass. In addition to color changes that may be useful for creating various food effects, UVB treatment converts ergosterol present in the fungal cell membrane to vitamin D2 and increases the production of carotenoids such as beta - carotene and astaxanthin. As a result, irradiating filamentous fungi such as Fusarium oxysporum MK7 strain with UVB can be used to increase vitamin D2 and carotenoids in the resulting biomass.

[0078] In some cases, the generated filamentous fungal biomass consists of a layer of cells with a uniform appearance, with one surface of the filamentous biomass in contact with air and one surface in contact with the synthetic medium. In other cases, there are at least two different layers, namely, an aerial mycelium layer on the upper surface and a high-density multicellular bottom layer in contact with the synthetic medium. Often there are three distinct layers, namely, (a) an aerial mycelium layer on the upper surface, (b) a high-density bottom layer, and (c) a transition layer between the top and bottom layers. The transition layer may only loosely adhere to the high-density bottom layer, allowing for easy separation of the bottom layer from the rest of the biomass. The filament density of the transition layer ranges from slightly lower than that of the high-density bottom layer in the zone where the two layers meet to a density equivalent to that of the aerial mycelium near the top of the biomass.

[0079] Inactivation of filamentous fungal biomass The inactivation process starts with biomass harvested at least 2 days after cultivation. The biomass can be rinsed to remove excess growth medium, although it is not necessary to rinse the biomass. In some cases, removal of the growth medium or excess growth medium is preferred. Similarly, the biomass can be squeezed to remove excess growth medium, although this is also not necessary. However, it may be preferred in some applications.

[0080] In some cases, such as when using the biomass as an independent protein source or as a protein component in food products, elimination of cell viability and the possibility of further biomass growth is desirable. This can be achieved by heating, irradiation, and / or steam treatment.

[0081] In the case of the heating process, the filamentous fungal biomass can be treated according to, for example, WO 95 / 23843 or UK Patent No. 1,440,642, or incubated at a temperature that destroys most of the organism's RNA without adversely affecting the protein composition of the organism.

[0082] In the case of irradiation, the filamentous fungal biomass is 60 Co (or rarely 137Expose to ionizing energy such as that generated by radioactive isotopes of Cs), X-rays generated by machines operating at less than a nominal energy of 5 MeV, and accelerated electrons generated by machines operating at less than a nominal energy of 10 MeV.

[0083] Steam treatment can also remove any metabolites produced from the biomatt construct if any specific metabolites are produced, so steam treatment is a preferred method for inactivating some filamentous fungal biomatts such as those produced by Fusarium oxysporum MK7 strain and F. venenatum. At this time, the biomatt is arranged such that the liquid secreted by the biomatt and the condensed steam can easily drip from the biomatt. Suitable biomatt holding systems include porous plastic meshes and porous trays. Other biomatt holding systems include, but are not limited to, systems for fixing the biomatt in a vertical position, such as a system with a clamp mechanism suspended from the clamp and mesh system that fixes at least one end of the biomatt while the remaining end of the biomatt is fixed by the clamp that fixes at least two sides of the biomatt.

[0084] The biomats are placed in a steamer such that heated steam, such as steam at a temperature higher than 85°C, for example 95°C, contacts the biomats. When multiple trays are placed in a single steamer, for example one tray on top of another, it is preferable to protect the biomats at a lower position from the drips of the biomats at a higher position. The protection should be in the form of diverting the liquid secreted by the biomats and the condensed steam generated at a higher level in the steamer so that they do not contact the biomats at a lower level in the steamer, as contact of the steam with the biomats deactivates the viability of the biomats. In one embodiment, a cone is placed between the upper and lower trays to achieve this result. In other embodiments, the separation of the upper and lower trays also includes at least one other geometric shape such as a cylinder, cube and / or cuboid, pyramid, sphere, torus, and / or other Platonic solids. In yet another embodiment, the trays are separated using at least one cylinder, cube and / or cuboid, pyramid, sphere, torus, other Platonic solids, or a combination thereof.

[0085] The biomats are steam-treated at least until the viability of the biomats is reduced to the point where further growth of the biomats and / or replication of the cells within the biomats becomes negligible. The viability of the biomats is a function of the initial substrate, the growth of the biomats, the steam / heat transfer characteristics, the position of the biomats in the steamer, and the orientation of the biomats with respect to the generated steam. By way of example, a Fusarium oxysporum MK7 strain biomats grown on a glycerol or acid whey substrate is non-viable after 5 minutes of steam treatment, and in some cases less than 5 minutes. The steam-treated mats can be rinsed and / or squeezed to remove the mat excretions and the condensed steam.

[0086] The inactivated edible filamentous fungus biomats can be used directly as a protein source, for example, in the manufacture of food products that are comparable to, for example, just a few examples such as tofu, bacon, and jerky.

[0087] The inactivated edible filamentous fungal biomass can also be reduced in size for use as a protein source in food products. The size reduction can be effected by mechanical means, such as cutting, shredding, dicing, milling, grinding, blending, etc. or by sonication, and is carried out prior to mixing with other ingredients or liquids. The particles of reduced size can be of uniform size or be liable to vary. Usually, the length of the particles of reduced size is from 0.05 to 500 mm, the width is from 0.03 to 7 mm, and the height is from 0.03 to 1.0 mm. For example, powdery particles are usually in the range of 0.03 mm to 0.4 mm, jerky-type particles are in the range of 100 mm to 500, etc. Larger sized particles can be produced, and the biomass was grown in a rubber pool (66” diameter) that produced a single, perfectly round biomass of 66” diameter. Larger mats can be grown using larger containers.

[0088] The number of particles of reduced size produced per biomass depends on the size of the initial biomass and the purpose for which the particles of reduced size biomass are used.

[0089] Depending on the food product, the particles of reduced size have an average uncut filament length of at least 0.1 mm, such as 0.1 mm to 2.0 mm, 0.5 mm to 10 cm, 0.5 mm to 30 cm, 0.8 mm to 25 cm, 1.0 mm to 20 cm, 1.4 mm to 15 cm, 1.6 mm to 10 cm, 1.7 mm to 8 cm, 1.8 mm to 6 cm, 2.5 mm to 4 cm, 5 mm to 2 cm, 0.5 to 2.5 mm, 0.5 to 1.8 mm, 0.5 to 1.7 mm, 0.5 to 1.6 mm, 0.5 to 1.4 mm, 0.5 to 1.0 mm, 0.5 to 0.8 mm, 0.5 to 0.6 mm, 0.6 to 2.5 mm, 0.6 to 1.8 mm, 0.6 to 1.7 mm, 0.6 to 1.6 mm, 0.6 to 1.4 mm, 0.6 to 1.0 mm, 0.6 to 0.8 mm, 0.8 to 2.5 mm, 0.8 to 1.8 mm, 0.8 to 1.7 mm, 0.8 to 1.6 mm, 0.8 to 1.4 mm, 0.8 to 1.0 mm, 1.0 to 2.5 mm, 1.0 to 1.8 mm, 1.0 to 1.7 mm, 1.0 to 1.6 mm, 1.0 to 1.4 mm, 1.4 to 2.5 mm, 1.4 to 1.8 mm, 1.4 to 1.7 mm, 1.4 to 1.6 mm, 1.6 to 2.5 mm, 1.6 to 1.8 mm, 1.6 to 1.7 mm, 1.7 to 2.5 mm, 1.7 to 1.8 mm, or 1.8 to 2.5 mm, and contain a larger size distribution of 0.1 to 1.0 cm, 0.5 to 2.0 cm, 1.0 to 5.0 cm, 2.0 to 7.0 cm, 5.0 to 10.0 cm, 7.0 to 20 cm, 10.0 to 50.0 cm, and 15.0 to 100.0 cm.

[0090] Particles with reduced filamentous fungal biomass size also contain fragmented filaments, and in some cases, the fragmented filaments are present mainly, for example, as 100% fragmented filaments, 99% fragmented filaments, 98% fragmented filaments, 97% fragmented filaments, 96% fragmented filaments, and 95% fragmented filaments. Again, the size of the fragmented filaments is selected for the final food product to be produced. The average fragmented filament length is at least 0.01 mm, for example, in the range of 0.01 - 0.10 mm, 0.05 - 0.20 mm, 0.1 - 1.0 mm, 0.50 - 2.5 mm, 1.0 - 5.0 mm, 2.0 - 10.0 mm, 5.0 mm - 15.0 mm, 10.0 mm - 1.0 cm, 1.0 cm - 5.0 cm, 5.0 cm - 10.0 cm, 0.3 mm - 30 cm, 0.8 mm - 25 cm, 1.0 mm - 20 cm, 1.4 mm - 15 cm, 1.6 mm - 10 cm, 1.7 mm - 8 cm, 1.8 mm - 6 cm, 2.5 mm - 4 cm, 5 mm - 2 cm, 0.3 - 2.0 mm, 0.3 - 1.8 mm, 0.3 - 1.7 mm, 0.3 - 1.6 mm, 0.3 - 1.4 mm, 0.3 - 1.0 mm, 0.3 - 0.8 mm, 0.3 - 0.6 mm, 0.3 - 0.5 mm, 0.6 - 2.5 mm, 0.6 - 1.8 mm, 0.6 - 1.7 mm, 0.6 - 1.6 mm, 0.6 - 1.4 mm, 0.6 - 1.0 mm, 0.6 - 0.8 mm, 0.8 - 2.5 mm, 0.8 - 1.8 mm, 0.8 - 1.7 mm, 0.8 - 1.6 mm, 0.8 - 1.4 mm, 0.8 - 1.0 mm, 1.0 - 2.5 mm, 1.0 - 1.8 mm, 1.0 - 1.7 mm, 1.0 - 1.6 mm, 1.0 - 1.4 mm, 1.4 - 2.5 mm, 1.4 - 1.8 mm, 1.4 - 1.7 mm, 1.4 - 1.6 mm, 1.6 - 2.5 mm, 1.6 - 1.8 mm, 1.6 - 1.7 mm, 1.7 - 2.5 mm, 1.7 - 1.8 mm, or 1.8 - 2.5 mm.

[0091] In some cases, the average fragmented filament length of the reduced particles of filamentous fungal biomass is less than 1 µm, for example, less than 950 nm, less than 900 nm, less than 850 nm, less than 800 nm, less than 750 nm, less than 700 nm, less than 650 nm, less than 600 nm, less than 550 nm, less than 500 nm, or less than 400 nm.

[0092] The filamentous fungal biomass with reduced particle size can be added as a protein source to increase the protein content of food products or can be the sole protein component. In the case of foods consisting entirely of filamentous fungal biomass, the particles with reduced size can be optimized for a specific texture, mouthfeel, and chewiness. For example, a filamentous fungal biomass food shaped like a hamburger and flavored to resemble a hamburger can have 90% of its particles with a length of less than 1.5 mm, with the majority of the lengths being 1 mm or less, a width of less than 1 mm, and a depth of less than 0.75 mm. This type of food is characterized by having a higher perceived density in the mouth, being easier to chew, and providing a creamy mouthfeel and a more refined dining experience. The highly processed biomass particles have been compared to the types of burgers found in upscale dining establishments. For a more elaborate dining experience similar to the prepared types of burgers commonly found in burger restaurants or BBQs, 90% of the particles have a length of 4 mm to 10 mm, a width of 1.0 mm to 3 mm, and a depth of less than 0.75 mm. The ability to vary the texture, mouthfeel, and chewiness allows for customization to accommodate individuals with special dietary needs, such as those who have difficulty chewing, or those who require / desire a softer food while providing the same nutritional and taste experience, or those who desire a food with more texture, a better mouthfeel, and a better crunch. Due to the ability to easily control the particle size, foods augmented with or made solely from filamentous fungal biomass have a texture very similar to the standard protein foods they mimic, as can be seen from Table 2. Table 2 - Results of Stable Micro Systems TA XT+ Texture Analyzer

Table 2-1

Table 2-2

[0093] Food products that can be produced using only filamentous fungal biomass of reduced particle size, with or without the addition of flavorings, and / or food products that can be enhanced with filamentous fungal biomass of reduced particle size, examples of which are meat products (e.g., beef jerky, chicken jerky, turkey jerky, chicken nuggets, fish sticks or pâté, jerky, snacks (e.g., chips), soups, smoothies, beverages, milk-like foods, bread, pasta, noodles, dumplings, pastries (e.g., puff pastry), cookies, cakes, pies, desserts, frozen desserts, ice cream-like foods, yogurt, confectionery, and candies.

[0094] Food products enhanced with filamentous fungal biomass of reduced particle size can significantly increase their protein content, which is particularly important for individuals following a vegan diet. For example, increasing a cup of soup (227 g) with 68.1 g of MK7 liquid dispersion (i.e., 1 part MK7 to 3 parts water) adds 8.5 g of protein, and increasing a bowl of soup (340 g) with 136 g of MK7 liquid dispersion adds 17 g of protein. For example, using MK7 liquid dispersion as a main component in vegan soups, beverages, smoothies, etc. further increases the protein content of these food products. Changing the ratio of MK7 to water in turn changes the degree of protein enhancement.

[0095] Whether reduced-grit biomats are used to increase the protein content of foods or as the sole protein component, in some cases binders help achieve the desired texture. Approved food binders such as egg albumin, gluten, wheat flour, vegan binders, kudzu starch, gelatin, pectin, guar gum, carrageenan, xanthan gum, whey, chickpea water, ground flaxseed, egg substitutes, wheat flour, chia seeds, okra, etc. can be considered and can be used alone or in combination. In addition to food binders, reduced-grit filamentous fungal biomats can also be mixed with approved flavorings, spices, seasonings, fats, fat substitutes, preservatives, sweeteners, color additives, nutrients, emulsifiers, stabilizers, thickeners, pH regulators, acidulants, leavening agents, anti-caking agents, humectants, yeast nutrients, dough strengtheners, dough improvers, stabilizers, enzyme preparations, gases, and combinations thereof. Usually, binders, flavorings, spices, etc. are selected to meet the requirements of a particular group. For example, to accommodate individuals with dairy allergies / sensitivities, milk and / or milk solids are not used; to accommodate those with gluten allergies / sensitivities, wheat flour is not used, etc.

[0096] In some applications, reduced-grit filamentous fungal biomats are used in food products that mimic chicken nuggets, shrimp, pork, fish, burgers, sausages, jerky, bacon, etc. In this case, a single type of reduced grit, or various reduced-grit filamentous fungal biomats can be used. Similarly, the reduced grit can be derived from a filamentous fungal biomats of a single origin or a combination of filamentous fungal biomats of various origins, such as MK7 alone or MK7 + giant puffball biomats.

[0097] In some applications, reduced-grit filamentous fungal biomats are dried, ground to a sufficiently small size, and used as wheat flour in the production of baked foods with increased protein, such as bread, dinner rolls, muffins, cakes, cookies, pies, etc.

[0098] One aspect of introducing proteins into food products is to use a liquid dispersion made from filamentous fungal biomass as an alternative ingredient to milk or milk-like foods. The liquid dispersion can be used in several recipes including soups, ice creams, yogurts, smoothies, fudges, and candies such as caramels and truffles. In some cases, filamentous fungal biomass generated from various raw materials / carbon sources results in liquid dispersions with various flavors. For example, when the raw material / carbon source is glycerol, the liquid dispersion produced from Fusarium oxysporum strain MK7 has a sweeter taste, while the liquid dispersion obtained from Fusarium oxysporum strain MK7 grown on acid whey raw material / carbon source tends to be more acidic. The inherent sweetness or sourness of the filamentous fungus, such as Fusarium oxysporum strain MK7, transfers to the final food product. For example, the acid whey liquid dispersion tends to give the yogurt properties to itself, and the glycerol liquid dispersion tends to give the mousse, caramel, or fudge properties to itself.

[0099] Filamentous fungal biomass: By adjusting the ratio of water, a liquid dispersion with appropriate viscosity and density can be produced. The ratio can be 1:2 to 10:1, and the preferred ratios are 1:3, 1:4, and 7:3. For example, a relative density ratio of 1:3 follows that of ice cream-like foods, drinks, and yogurts.

[0100] In some cases, filamentous fungal biomass can be used as a source of oil, such as truffle oil produced from the surface-fermented edible fungal biomass of species of the genus Tuber.

[0101] The use of filamentous fungi as valuable microbial factories has been exploited conventionally, but generally required a considerable infrastructure and / or equipment, energy requirements, expensive reagents, and / or a significant amount of personnel. Filamentous fungi are known to have the greatest metabolic diversity among all microorganisms on earth, including the ability to produce a wide range of organic acids, antibiotics, enzymes, hormones, lipids, mycotoxins, vitamins, organic acids, pigments, and recombinant heterologous proteins [Wiebi (2002) Myco-protein from Fusarium venenatum: a well-established product for human consumption. Appl Microbiol Biotechnol 58, 421-427; El-Enshasy (2007) Chapter 9 - Filamentous Fungal Cultures - Process Characteristics, Products, and Applications. In. Bioprocessing for Value-Added Products from Renewable Resources. Editor: Shang-Tian Yang. Elsevier; Gibbs et al (2000) Growth of filamentous fungi in submerged culture: problems and possible solutions. Crit. Rev. Biotechnol. 20, 17-48], as well as the ability to degrade many types of recalcitrant materials such as lignocellulose and humic substances in soil.

[0102] Although widely used, there are still many challenges in submerged fermentation production, including important factors such as growth limitation due to limited oxygen utilization and excessive shear forces caused by agitation [Gibbs et al (2000) Growth of filamentous fungi in submerged culture: problems and possible solutions. Crit. Rev. Biotechnol. 20, 17-48]. Since the solubility of oxygen in water under Earth's surface conditions is approximately 8 mg / L, it is easily depleted during rapid growth in liquid culture. Therefore, continuous aeration using complex, expensive, and energy-intensive ventilation and agitation systems is required to maintain a high growth rate. Cultivation of filamentous fungi is even more difficult because the filamentous morphology imparts non-Newtonian rheological behavior that further inhibits oxygen transfer to the solution (Norregaard et al. (2014) Filamentous Fungi Fermentation. In Industrial Scale Suspension Culture of Living Cells, H.-P. Meyer, and D.R. Schmidhalter, eds. (Wiley-VCH Verlag GmbH & Co. KGaA), pp. 130-162). As the culture density increases, the amount of energy required to aerate and mix the culture increases non-linearly, and similarly, the energy requirement for aerating a high-density culture becomes very high. In the case of many filamentous fungal species, vigorous agitation and aeration of the culture are harmful to hyphal growth, resulting in a dramatic decrease in the growth rate. These and other challenges to submerged fermentation of filamentous microorganisms require innovative solutions to effectively utilize these organisms with limited resources available on spacecraft and space stations.

[0103] The disclosed hermetic reactor system (1) addresses these problems and has the following advantages: - Does not require active aeration or agitation of the liquid culture - In-situ aggregation of biomass into a single adherent mat with a fairly high tensile strength (>0.1 kg per 1 cm width of the biomatt) enables easy harvesting. - Texturized biomatt can be used for a wide variety of mission-critical products (i.e., for food, bioplastics, biofuels, dietary supplements, and as an expression platform for various pharmaceuticals). - Maintain high biomass production (80 - 120 g / m 2 / d or 0.55 g / L / h) while minimizing water use and minimizing and / or having no residual wastewater or nutrients from the process. - Can the growth rate be translated into the production of a sufficiently formed biomatt within 2 days at the earliest, or can it be further expanded for more than 10 days? - High biomass density (biomatt is usually 100 - 180 g / L) - A variety of filamentous fungi (including extreme environmental microorganisms) that have specific advantages for various processes can grow. - Scaling up or down is relatively easy and productivity does not decrease. - The process can use a very wide variety of C- and N-rich waste substrates resulting from natural disasters and / or space exploration missions.

[0104] The disclosed hermetic reactor system (1) provides an in-built biofilm-biomatt reactor that includes a container and raw materials, fungal inoculum, gas-permeable membrane (2), and optionally a liquid nutrient medium placed inside the container. Depending on the situation, the reactor can be a single-use reactor or a reusable reactor.

[0105] Typically, the container can be sealed and may include a lid for the container in addition to the seal. Examples of some containers are lidded trays, Petri dishes with lids, other types of lidded containers, or bags. For some applications or in some environments, the container has a plurality of growth chambers, for example, after a manifold design and / or a baffle system. To maximize efficiency under some environmental conditions, the container is produced from one or more raw materials, which may or may not be the same as the raw materials placed inside the container.

[0106] The raw material is inoculated with a fungal strain such as an ascomycete or basidiomycete fungal strain. Examples of ascomycete strains are Fusarium oxysporum MK7 strain (deposited at ATCC PTA - 10698, American Type Culture Collection 1081 University Boulevard, Manassas, Virginia, USA), Fusarium venenatum, Fusarium avenaceum, and / or combinations thereof. The inoculation of the raw material can be done when the raw material is placed inside the container or can be done some time after the raw material is placed. That is, the hermetic reactor (1) can be stimulated with a lyophilized filamentous fungal inoculum that reactivates upon contact with the raw material, or the raw material can be directly inoculated after being placed into the hermetic reactor channel (4), or can be placed into the hermetic reactor channel after being inoculated with the raw material.

[0107] Regarding the raw materials used in the reactor, the raw materials can be waste, such as naturally occurring urine and / or feces, food waste, plant materials, industrial waste, such as glycerol, and waste by-products, starch and / or by-products of starch hydrolysis, acid whey, sugar alcohols, and / or combinations thereof. Synthetic or manufactured waste substitutes, such as artificial urine, can also be used. The raw materials of plant materials are usually lignocellulose. Some examples of lignocellulose raw materials are agricultural crop residues (e.g., wheat straw, barley straw, rice straw, small grain straw, corn stover, corn fiber (e.g., corn fiber gum (CFG), distillers dried grain (DDG), corn gluten meal (CGM), switchgrass, sugar beet pulp, waste streams from palm oil production, hay alfalfa, sugarcane bagasse, non-agricultural biomass (e.g., algal biomass, cyanobacterial biomass, urban tree residues), forest products and industrial residues (e.g., softwood primary / secondary mill residues, hardwood softwood primary / secondary mill residues, recycled paper pulp sludge), lignocellulose-containing waste (e.g., newsprint, waste paper, brewing grains, used rubber tires (URT), municipal organic waste and by-products, household waste and by-products, clinical organic waste and by-products, and waste and by-products generated during the production of biofuels (e.g., processed algal biomass, glycerol), and combinations thereof.

[0108] The gas permeable membrane (2) enables the optimization of the system in several different ways shown in FIGS. 19-22. The hermetic reactor system shown in the figures has a total of nine channels (4), but as will be understood by those skilled in the art, depending on the available space for the arrangement of the hermetic reactor (1), any number of channels (4) can exist, from a single channel (4) to a large number of channels (4). Similarly, the shape of the channel (4) is not limited to a right prism or a cylinder, and can take any shape suitable for fitting into the available space of the hermetic reactor (1).

[0109] In some cases, the membrane (2) is arranged in direct contact with the surface of the raw materials, the appropriate liquid medium, and the inoculation material present in the container as shown in FIG. 16. The membrane can also be sealed in contact with the surface of the raw materials, for example, by attaching it to a plastic frame together with an integrated rubber gasket.

[0110] In other cases, the membrane is suspended above the raw material, allowing fungi to grow and consume oxygen, while the membrane descends towards the mat or onto a baffle system installed between the membrane and the raw material, thereby enabling the growth of aerial mycelium. Such a system is shown in Figure 19. Here, the hermetic reactor (1) consists of a number of channels (4) starting at the inlet valve (6) at the front (7) of the reactor and ending at the outlet valve (8) at the back (5) of the reactor, separated by baffles / walls (9). The gas-permeable membrane (2) forms the top of the reactor. The bottom (3) of the reactor can be formed of any suitable material including, but not limited to, both rigid and flexible plastics such as polyethylene terephthalate, high-density polyethylene, polyvinyl chloride, polyacetic acid, polycarbonate, acrylic, acetal, nylon, acrylonitrile butadiene styrene, glass, metals such as aluminum, titanium, stainless steel, etc. and / or combinations thereof. The baffles / walls (9) can be made of similar materials. Suitable front (6) and back (8) valves include, but are not limited to, one-way valves, two-way valves, ball valves, butterfly valves, gate valves, plug valves, globe valves, pinch valves, disk check valves, attachment valves, detachment valves, and / or combinations thereof. The inlet valve (6) serves to provide access to the channels (4) for the delivery of the raw material / media to the chamber, while the outlet valve (8) enables the removal of the discharged raw material and / or filamentous fungal biomass. The gas-permeable membrane (2) can consist of polymeric materials such as polypropylene, polyethylene, polytetrafluoroethylene, polycarbonate, polyamide, polypyrrolone, poly(amide amine) dendrimer composites, cellulose acetate, butadiene-acrylonitrile, Teflon AF2400, and nylon. The pore size of the gas-permeable membrane (2) is typically in the range of 0.05 - 1.5 μm, for example 0.2 μm, 0.45 μm, and 1.0 μm, while the membrane (2) can be in the form of a sterile cloth-like material or a paper-like material. In some applications, the surface of the membrane is smooth, while in other cases the surface is rough.In addition, the passageways for gas diffusion can vary from essentially straight to more convoluted passageways.

[0111] In other situations, the membrane facilitates the entry of oxygen and the release of other generated gases during fungal growth (Figure 18). In this situation, the hermetic reactor (1) has a gas collection chamber (14) just above the gas permeable membrane (2) (see Figure 20). The gas collection chamber (14) can be made of the same materials used for the walls / baffles (9) or bottom (3) of the reactor, i.e., both rigid and flexible plastics such as polyethylene terephthalate, high density polyethylene, polyvinyl chloride, polyacetic acid, polycarbonate, acrylic, acetal, nylon, acrylonitrile butadiene styrene, glass, metals such as aluminum, titanium, stainless steel, etc. and / or combinations thereof. Alternatively, the gas collection chamber can consist of channels (15) that faithfully reflect the channels (4) of the hermetic reactor (1) or that contain more than one of the hermetic reactor channels (20) (see Figure 21).

[0112] In yet other systems, another gas permeable membrane is used for the entry and release of gases. Figure 22 shows such a system. In this example, two different gas permeable membranes (2, 50) feed into separate gas collection chambers (30, 40) and are present above a single reactor channel (4). In this type of system, the entry, release, and / or collection and / or separation of different useful gases is made possible. As an example, one membrane regulates the passage of oxygen and the second membrane regulates the passage of carbon dioxide or hydrogen or other related gas systems.

[0113] The reactor (1) produces a biofilm-biomat that serves as a food source such as a protein source and / or an oil and fat source. However, the biofilm-biomat can also serve as a leather analogue, a bioplastic, a source of biofuel precursors, a biofuel, and / or a combination thereof. In still other embodiments, the biofilm-biomat serves to produce organic products such as organic acids, antibiotics, enzymes, hormones, lipids, mycotoxins, vitamins, pigments and recombinant heterologous proteins.

[0114] The disclosed biofilm-biomat reactor fermentation technology enables growth on standard as well as extreme raw materials and media, such as sewage (urine / feces), and produces highly established and textured products without the need for separation or concentration steps. A relatively high biomass production rate (0.55 g / L / h dry biomass) and high culture density (100 - 180 g / L) are achieved without the need for active aeration or agitation. Scale-up in the vertical, horizontal, and / or more than two dimensions of the system is straightforward and there is no decrease in productivity. The biofilm-biomat produced is typically 0.2 - 2.5 cm thick with a dry matter content of 10 - 30% and can be easily used for mission-critical needs such as meat substitutes, numerous other appetizing foods, and building materials.

[0115] The fungal biofilm-biomat grown in the disclosed reactor system is described as a pellicle suspended in a liquid culture at the gas-liquid interface, similar in many respects to microbial biofilms that grow on surfaces. For example, bacterial cells within the biofilm have been shown to withstand extreme disinfection treatments with sodium hypochlorite (bleach) and sodium hydroxide (Corcoran, 2013). The disclosed reactor system makes good use of biofilm organization and enables growth on harsh human and industrial wastes and by-products that can occur under extreme conditions, such as those generated in space exploration missions or by other severe conditions caused by natural disasters.

[0116] The disclosed reactor design incorporates a gas-permeable membrane that is positioned directly on or suspended just above the liquid surface. The encapsulated reactor design allows for gas exchange with the external atmosphere but is sealed to prevent contaminants from entering or gas / liquid from leaking out. The encapsulated reactor design can also separate consumable gases from the generated gases by means of the gas-permeable membrane. To achieve this, in some cases, valves and / or additional porous membranes with the same or different properties are used to form different layers between one or more raw materials of various properties and an appropriate liquid medium.

[0117] Rapid biofilm-bioplume growth using the disclosed reactor design has been demonstrated with various gas-permeable membrane materials. Figure 17 shows bioplume growth of approximately 7 mm thickness in a reactor where the vessel was a Petri dish covered with a polypropylene membrane placed directly on the raw material / liquid medium surface. The initial biofilm formed by adhering directly to the membrane and grew downward into the liquid medium over time (see Figure 16). By the end of the 5-day growth period, essentially all of the raw material / liquid medium was consumed and a high-density biomass completely filled the volume under the membrane.

[0118] The generated bioplume adhered only slightly to the membrane and was easily harvested by simply peeling the bioplume from the membrane (see Figures 17A - D). Additional experiments using polycarbonate membranes have yielded similar results (data not shown). Thus, the entire volume of the reactor can be efficiently utilized to produce a high-density biomass that is easily harvested.

[0119] The biofilm - biomat generally produced in the disclosed reactors is established (110 - 180 g / L) and exhibits a fibrous texture depending on the fungus and growth conditions. The production of fibrous biomass can be critical for certain mission - critical products such as foods that require a texture mimicking meat, as well as for fibrous materials such as leather and wood. The established properties of the biomass also enable easy harvest without the need for concentration steps (e.g., centrifugation, filtration).

[0120] Use of a Biofilm - Biomat Reactor in a Microgravity Environment The basic physical force that controls the formation and growth of biofilm - biomat in the disclosed reactors is adhesion to the membrane. Without being bound by theory, it is believed that growth within the disclosed reactors is not affected by the microgravity conditions experienced during spaceflight. Directional growth driven by gravity or growth controlled by physical mixing or flow is not the most important factor in the system because it tends to be within a gravity environment. Previous experiments in space have successfully demonstrated fungal growth [European Space Agency, Expeditions 25 - 28, Growth and Survival of Colored Fungi in Space (CFS - A)], providing additional reliable evidence that the disclosed reactor system functions in a space environment.

[0121] For ease of space exploration missions and deployment, lyophilized inoculum and essential components (if necessary) to support growth on specific raw materials can be pre-loaded into the reactor. As a result, astronauts and space travelers can prepare raw materials, inoculum, and any media components. Incubation times depend on the raw materials, microbial strain, and other growth parameters such as pH, temperature, and water content. Incubation conditions are simple in that fermentation is carried out under static conditions where the reactor is simply incubated in place. High-density established biomats are harvested simply by opening the reactor closure (e.g., ziplock®-type) and removing the mat.

Example

[0122] Example 1: Growth of Fusarium oxysporum MK7 strain and other fungi in a static tray reactor The biomats of the filamentous, acidophilic Fusarium oxysporum MK7 strain, Ganoderma lucidum (Reishi; Figure 1A), Pleurotus ostreatus (Pearl Oyster, Figure 1B; and Blue Oyster, Figure 1C), Flammulina velutipes (Cauliflower; Figure 1D), Hypsizygus tessellatus (Elm Oyster; Figure 1E), Boletus edulis (Giant Puffball; Figure 1F), and Fusarium venenatum were grown in shallow static tray reactors as described in International Application PCT / US2017 / 020050.

[0123] Example 2: Growth of Fusarium oxysporum MK7 strain biomats in a daily refreshed (semi-static conditions) nutrient medium A high-density Fusarium oxysporum MK7 strain biomatt with a thickness of approximately 3 cm was grown for 21 days in a nutrient medium refreshed daily. The biomatt was produced in 12.7×17.8 cm Pyrex® glass trays using sterile MK7-1 liquid medium (described in International Application PCT / US2017 / 020050) containing 7.5% glycerol at pH 3.0. To initiate the experiment, 200 mL of nutrient medium was inoculated with 5% (v / v) of a Fusarium oxysporum MK7 strain culture in the late exponential growth phase as previously described in International Application PCT / US2017 / 020050. The 200 mL of inoculated medium was added to each of three sterile trays lined with a sterile coarse nylon mesh screen. The cultures were incubated at room temperature (about 22 °C) for 4 days without disturbance to grow the first biomatt layer formed on the surface of the liquid. After 4 days of growth, the biomatt was gently lifted from the tray using the nylon mesh screen and tilted at a 45-degree angle to drain the liquid from the mat. The biomatt was drained of moisture at this position until less than 1 drop of liquid dripped every 5 seconds. On average, after about 3 minutes, it was sufficiently drained. The biomatt drained and dried by dripping on the screen was placed in a fresh pre-weighed 12.7×17.8 cm Pyrex® tray containing 200 mL of fresh MK7-glycerol medium (described in International Application PCT / US2017 / 020050). The tray containing the biomatt was reweighed. The process of moving the biomatt to another tray containing fresh medium was repeated at approximately daily intervals for more than 17 days. Samples were taken from one of the biomatts on days 12, 15, and 21, and the moisture content of these biomatts was determined. The average moisture content of the biomatt was 17.3% (standard deviation = 0.7), and this value was used to calculate dry biomass production over the duration of the experiment. Dry biomass production was linear from day 4 to day 18 (r 2 = 0.995), after which the biomass weight stabilized at approximately 2.5 Kg dry / m 2 (Figure 1, the y-axis is normalized per m 2 . Growth is usually exponential from day 0 to day 4). The average growth rate over this period of linear growth was 6.04 g / m2 / h. Figure 2 shows a bio-mat about 3 cm thick grown after 21 days of growth using this method.

[0124] Example 3: Growth of Bio-mat under Continuous Flow Conditions A continuous flow bioreactor system was assembled to demonstrate the growth of bio-mat on the surface of a flowing liquid medium. The system was assembled from a series of 2.44 m long transparent plastic roofing panels with pleats used as flow channels (Figure 3). The ends of each channel were sealed with silicon (100% silicone, DAP Products Inc., Baltimore, MD) so that the liquid could be retained within the channel. The flow was facilitated through the channel by delivering the liquid medium to one end of the channel by a peristaltic pump, and the liquid exited the other end of the channel through holes in the bottom of the channel. The entire plastic roofing panel system was inclined at an angle where it rises 1 cm for every 1 m forward, and approximately 500 mL of liquid was retained within each channel. The steady flow is a function of the amount of liquid and the angle of inclination.

[0125] The panel system was sterilized and wrapped in Saran (registered trademark) plastic wrap to isolate the system from the surrounding room environment. Sterile air was pumped under the plastic wrap at a rate of 400 mL / min to create a positive pressure on the system. Prior to starting the flow, a 500 mL volume of nutrient medium inoculated with the desired filamentous fungus was added to each channel and incubated for 4 days under resting / static conditions. After 4 days, a continuous pulsed flow of 400 mL / d was sent by a peristaltic pump to "feed" the bio-mat (ON, 2.016 mL / min, 49 min, 39 sec; OFF, 5 h 10 min 21 sec). Two independent experiments were conducted, and in each experiment, two separate flow channels were used as replicates (Figure 3).

[0126] The biomass established after 10 days of growth in the nutrient medium was harvested. (4 days under resting / static conditions and then 6 days under continuous flow; Figure 4). The average dry weight of the generated biomass was 2.38 g on average in the replicated flow channels. The average removal rates of C and N from the flowing liquid medium by the biomass growing during the continuous flow period (from 4 days to 10 days) were 11.9 and 1.2 mg / L / h, respectively. The C and N removal rates from the liquid medium were determined by measuring the liquid volume of the bioreactor system as well as the total C and N inflow and outflow using a Costech total C and N analyzer (ECS 4010, Costech Analytical Technologies, Valencia, CA). Thus, the continuous flow system supported the growth of the biomass on the surface. The experiment also served as a laboratory-scale demonstration of the continuous supply and production of the established biomass for the growth of the Fusarium oxysporum MK7 strain biomass. Note that other raw materials, flow rates, and achievable growth rates can be achieved with this type of system. For example, the expected yield at pH 2.8 with 10% glycerol in the MK7-1 medium (described in International Application PCT / US2017 / 020050) is more than 40 grams of dry biomass per day per meter 2 of dry biomass.

[0127] Example 4: Semi - continuous and continuous production of Fusarium oxysporum MK7 strain biomass The high-density Fusarium oxysporum MK7 strain biomats were grown and harvested semi-continuously over a 19-day period. The biomats were produced using acid whey supplemented with 1 / 2 strength MK7-1 medium salts (described in International Application PCT / US2017 / 020050) adjusted to pH 4.0 as the raw material / carbon source. To initiate the experiment, 200 mL of nutrient medium inoculated with the Fusarium oxysporum MK7 strain (5% v / v) in the late exponential growth phase was added to a sterilized 12.7×17.8 cm Pyrex® glass tray, which was then covered with Saran® wrap and incubated at room temperature. After 5 days of growth, 1 / 3 of the biomats were removed from one end of the tray by cutting out a 5.9×12.7 cm portion of the biomats (Figure 5A). The remaining 2 / 3 of the biomats were then physically moved towards the open area of the medium created by the removal of the 1 / 3 portion of the biomats. The biomats were physically grasped with gloved fingers and pulled until the biomats touched the end of the tray to transfer the biomats and open the medium so that no biomats were created at the other end of the tray (Figure 5B). The process of harvesting 1 / 3 of the most mature portion of the biomats and then moving the remaining 2 / 3 of the biomats to the open area was repeated periodically. 50 mL of medium was aseptically removed from the tray every 4 days and replaced with 50 mL of fresh sterile medium (acid whey containing 1 / 2 strength MK7-1) to replenish the nutrients removed from the liquid medium by the removal of the biomats. In dry biomass production using this method, 0.57 g / day or 25.2 g / d / m per tray was achieved in 5 - 19 days 2 (Figure 6). Thus, a semi-continuous production system was demonstrated in which the most mature end of the biomats was harvested at an average rate of 1.56 cm / day and fresh biomats growth was initiated in the open area of the medium at the other end of the tray.

[0128] This system is also suitable for the continuous harvesting and growth of the biomats, where continuous removal is facilitated by a roller wheel that adheres to the mature end of the biomats (Figure 7). The roller wheel rotates slowly, harvesting the mature biomats and simultaneously creating an open medium for the growth of new biomats at the other end of the tray. The roller wheel rotates and harvests the biomats at a rate of 1.56 cm / day to regenerate the semi - continuous system. The nutrients in the liquid medium are preferably replenished at the rate of nutrient removal by the biomats.

[0129] Example 5: Membrane - encapsulated bioreactor A high - density Fusarium oxysporum MK7 strain biomats was grown in a liquid growth medium encapsulated in a bioreactor system without a gas headspace. A sterile Petri dish bottom (55 mm in diameter) was filled to the brim with 57 mL of inoculated MK7 - 1 medium (described in International Application PCT / US2017 / 020050) containing 8% glycerol. Polypropylene and polycarbonate gas - permeable / semi - permeable membranes were placed directly on the surface of the liquid medium and sealed tightly with a rubber band. No gas headspace was provided at the start of the growth period.

[0130] After inoculating the medium and sealing the membrane, the bioreactor was left undisturbed until harvest. Figure 8 shows biomats of Fusarium oxysporum MK7 strain with a thickness of about 5 mm and about 1 mm grown for 5 days directly under polypropylene (Figures 17A - C) and polycarbonate (Figure 17D) membranes respectively. The biomats adhered slightly to the membranes and could be easily harvested by simply peeling the biomats from the membranes (Figure 17).

[0131] Example 6: Production of pigments and vitamin D2 by UVB irradiation of Fusarium oxysporum MK7 biomats Dye production was initiated using UVB light (290 - 320 nm) on a Fusarium oxysporum MK7 strain biomatt. The Fusarium oxysporum MK7 strain biomatt produced for 3 days in MK7-1 medium (described in International Application PCT / US2017 / 020050) with 7.5% glycerol was irradiated with UVB light for a period of 4 hours. The UVB light was emitted from a 50 W bulb (Slimline Desert 50 UVB T8 fluorescent lamp, 46 cm; Zilla, Franklin, WI) placed 10 cm above the biomatt. Orange dye deposition was visually detected after 0.5 h of irradiation and was prominent after 4 h of irradiation (Figure 9). Additionally, the biomatt not exposed to UVB light had a vitamin D2 content of less than 50 IU per 100 g of biomatt, but the vitamin D2 content increased to approximately 1.2 million IU per 100 g of biomatt after approximately 12 hours of UVB light exposure.

[0132] Example 7: Fusarium oxysporum MK7 strain biomatt grown on a mixture of glycerol, starch, and corn steep liquor The Fusarium oxysporum MK7 strain biomats were generated in just 4 days from a mixture of glycerol, starch, corn steep liquor, and MK7-1 salts (described in International Application PCT / US2017 / 020050). Glycerol was purchased from Duda Energy LLC (Decatur, AL; 99.7% Purity; USP Grade; Lot#466135376340); 100% Argo Corn Starch manufactured by Argo Food Companies, Inc (Memphis, TN) was purchased from an Albertson’s supermarket in Bozeman, MT, and corn steep liquor was purchased from Santa Cruz Biotechnology, Inc. (Dallas, TX; Lot#B0116). The growth medium was a mixture of 7.5% glycerol (weight / weight), 2.5% starch, 2.5% corn steep liquor, and MK7-1 salts. The mixture was adjusted to pH 3.3 by adding an appropriate amount of HCl and boiled in a suitable container for 15 minutes. After cooling to room temperature, the pH of the mixture was readjusted to 3.3 and then inoculated with 5% Fusarium oxysporum MK7 strain inoculum (vol / vol) prepared in International Application PCT / US2017 / 020050. Samples of 1.5 L of the inoculated medium were added to three sterilized 0.25 m 2 polypropylene trays and placed in a sterilized tray rack system completely covered with aluminum foil to create a dark condition and incubated at 23° ± 1°C. The filamentous fungal biomats that grew on the surface of the medium were harvested after 4 days simply by lifting the biomats out of the trays.

[0133] The average final pH of the remaining liquid in the three trays was 4.45 (standard deviation = 0.14). Three 56.7 cm 2 circular portions were cut out and removed from random positions on each biomats, and these portions were dried at 50°C for 48 h to obtain the dry weights. The average biomass dry weight (standard deviation) was 124.6 g / 0.25 m 2 (43.4) or 498.4 g / m 2(173.6). The average thickness of the wet biomass was 7.5 mm, and the average density on a dry weight basis was 0.66 g / cm 3 .

[0134] To expose the biomass filaments and enable inspection by field emission scanning electron microscopy (FE-SEM), the extracellular polymeric substances (EPS) between the filaments were removed by washing with ethanol. To achieve this, a 1 cm 2 portion (1 cm × 1 cm) of the biomass was excised with a razor blade just prior to harvesting, and the excised portion was subjected to a series of ethanol washes / dehydrations by successively submerging it in 40 mL of an ethanol mixture for the times noted below: 25% ethanol, 75% deionized H2O, 20 minutes; 50% ethanol, 50% deionized H2O, 20 minutes; 75% ethanol, 25% deionized H2O, 20 minutes; 95% ethanol, 5% deionized H2O, 20 minutes; 100% ethanol, 0% deionized H2O, 60 minutes. After repeating the 100% ethanol treatment two more times, the sample was stored in 100% ethanol.

[0135] To maintain the fine structure integrity of the biomass for FE-SEM, after ethanol washing / dehydration, a Tousimis Samdri-795 critical point dryer was used to perform critical point drying according to the manufacturer's instructions (Tousimis Samdri-795 Operations Manual; Tousimis, Rockville, MD). After critical point drying, the sample was placed directly on an aluminum stub or sliced with a razor blade into sections <0.3 mm thick and then placed on the stub. The sample was then coated with iridium (20 μm, EMITECH K575X, Electron Microscopy Sciences, Hatfield, PA) and inspected using a JEOL 6100 FE-SEM with an incident beam energy of 1 keV (JEOL USA, Inc., Peabody, MA).

[0136] The FE-SEM images revealed a complex network of intertwined hyphal filaments (Figure 10), which was very similar to the tissue revealed by light microscopy of the biomass grown on glycerol reported in International Application PCT / US2017 / 020050. Three different layers were observed: (a) an aerial hyphae layer on the upper surface, (b) a dense bottom layer, and (c) a transition layer between the upper and bottom layers. The transition layer was only lightly attached to the dense bottom layer, and thus easy separation of the bottom layer of the biomass from the rest was possible. The filament density of the transition layer ranged from slightly lower than that of the bottom layer in the zone where the two layers met to a density comparable to that of the aerial hyphae near the top of the biomass.

[0137] The excised samples were also prepared for light microscopy by slowly immersing them in the following solutions in the order and for the times shown below:

[0138] Xylene, 3 minutes; xylene, 3 minutes; 100% ethanol, 3 minutes; 100% ethanol, 3 minutes; 95% ethanol, 3 minutes; 95% ethanol, 3 minutes; 70% ethanol, 3 minutes; deionized water, 3 minutes; hematoxylin 1, 1.5 minutes; rinsing with tap water, 1 minute; clearing agent solution, 1 minute; rinsing with tap water, 1 minute; bluing solution, 1 minute; rinsing with tap water, 1 minute; immersing in 70% ethanol 30 times; immersing in 95% ethanol 30 times; immersing in 95% ethanol 30 times; immersing in 100% ethanol 30 times; immersing in 100% ethanol 30 times; immersing in 100% ethanol 30 times; immersing in xylene 30 times; immersing in xylene 30 times; immersing in xylene 30 times; covering with a cover glass.

[0139] Visualization was performed with a light microscope (B400B, Amscope, Irvine, CA) at a magnification of 100× according to the above procedure (Figure 11).

[0140] Approximately 2 cm of the biomass just prior to harvest 2The portion of the size was excised from the fresh biomats with a razor blade. Next, these portions were immersed in 35 mL of deionized water in a 50 mL conical-bottom centrifuge tube. The tubes were sonicated for 0, 40, 90, or 150 seconds (CP200T Ultrasonic Cleaner, Crest Ultrasonics, Ewing, NJ) to disperse the filaments in the liquid to enable microscopic observation. Samples of the liquid (about 100 uL) from these tubes were placed on glass slides, covered with cover glasses, and observed with an optical microscope (B400B, Amscope, Irvine, CA) at a magnification of 100×. When the average length (standard deviation) of the non-cut filaments was measured, it was determined to be 1.1 (0.6), 1.2 (0.4), 1.0 (0.4), and 1.2 (0.2) mm for sonication for 0, 40, 90, and 160 seconds, respectively. The maximum filament lengths observed in each treatment were 2.5, 1.4, 1.8, and 1.4 mm, respectively. These filament lengths were considerably longer compared to the growth of the Fusarium oxysporum MK7 strain by shaking flask culture in liquid with an average length of less than 0.02 mm.

[0141] Example 8: Production of chicken nuggets using the Fusarium oxysporum MK7 strain biomats grown on a mixture of glycerol, starch, and corn steep liquor Chicken nuggets were prepared using the Fusarium oxysporum MK7 strain biomass produced as described above. The wet biomass was steam-treated in a pot steamer at 97 °C for 0.5 h, cooled to room temperature, and used as a base to produce chicken nuggets. The steam-treated wet biomass (200 g) was finely chopped to a length of less than 0.5 mm and homogenized with 4% (weight / weight; 8 g) chicken base and 4% egg white protein (8 g). The resulting mixture contained more than 90% Fusarium oxysporum MK7 strain biomass. A portion (about 30 g) of this biomass mixture was shaped into nugget form and steam-treated in a pot steamer. The prepared nuggets were coated with egg white and then breaded by mixing with the breadcrumbs applied to the surface before frying in oil. The prepared nuggets exhibited a chicken-like texture (Figure 13A) and emitted a typical chicken smell. In a taste test by five people, the nuggets seemed to successfully mimic actual chicken-containing chicken nuggets in terms of taste and texture.

[0142] Example 9: Production of Fusarium oxysporum MK7 Strain Biomass Extract A highly concentrated viscous extract was produced from the Fusarium oxysporum MK7 strain biomass. The biomass harvested after 4 - 16 days of cultivation as described above was rinsed, steam-treated, dripped onto a porous plastic mesh for 5 minutes to dry, placed in a plastic bag, and sealed. The sealed bag was frozen at -20 °C or -80 °C for 24 h, and then incubated in the original sealed bag in an incubator at 60 °C for 48 h after adjusting the pH of the remaining culture liquid to pH 4 - 6. After heat treatment, the biomass was pressed through a filter with a pore size of <1.5 mm, and the resulting liquid was collected. The collected liquid was boiled in a non-reactive container for 10 minutes and then dried at 60 °C until the water content was about 6 - 8% to form a sticky paste extract. The nutritional value of the extract is similar to that of the steam-treated biomass and the powder made from the steam-treated biomass.

[0143] Example 10: Production of Yogurt from the Biomass of Fusarium oxysporum Strain MK7 Grown on Acid Whey Yogurt was produced using the Fusarium oxysporum strain MK7 biomass directly. The biomass was grown on acid whey raw material / carbon source generated as a by - product in the manufacture of Greek yogurt in trays, harvested after 6 days, and steam - treated within 20 minutes of harvest. 200 g of cooled wet biomass was blended with 600 g of potable - quality tap water to produce a milk - like suspension designated as the "MK7 liquid dispersion". The MK7 liquid dispersion was used alone or in combination with milk as an ingredient to produce yogurt.

[0144] Three mixtures containing various ratios of MK7 liquid dispersion / whole milk were prepared: 1) 25% MK7 liquid dispersion: 75% whole milk, 2) 50% MK7 liquid dispersion: 50% whole milk, and 3) 100% MK7 liquid dispersion. Using the mixtures, three batches of yogurt were made by heating each mixture to 83 °C and holding it at that temperature for 14 minutes with continuous stirring. After cooling the mixtures to 43 °C, a raw yogurt culture was added as an inoculum. The resulting mixtures were incubated in a yogurt maker (Model YM80; EuroCuisine, Los Angeles, CA) at 44 °C for 8 hours. All of the resulting mixtures had the appearance and texture of yogurt (Figure 14), as well as an odor and taste similar to typical yogurt.

[0145] Example 11: Growth of Mushroom Biomass on Glycerol A biomass biomat consisting of Baby Bella Brown cream mushrooms (mushrooms) and white mushrooms was generated for only 10 days using glycerol as the basic carbon source (raw material). These common edible mushrooms were purchased from an Albertson’s supermarket in Bozeman, MT and stored at 4 °C. The medium used to grow the mushrooms consisted of 1 L of 7.5% glycerol and MK7-1 salts (described in International Application PCT / US2017 / 020050), which was boiled for 10 minutes and then cooled to room temperature (about 23 °C). The pH of the mixture was adjusted to 2.7, and 200 mL of the pH-adjusted mixture was poured into two sterile 12.7 × 17.8 cm Pyrex® trays. The inoculation material consisted of 5 g of blended and surface-sterilized cream or white mushrooms, which was added to the medium in each tray. The mushroom inoculation material was prepared as follows: 1) 10 g of wet cream or white mushrooms was added to 200 mL of 5% bleach solution, and the suspension was stirred for 2 minutes to surface-sterilize the mushrooms, 2) then the mushrooms were transferred to 200 mL of sterile glycerol / MK7-1 salt medium (described in International Application PCT / US2017 / 020050) and rinsed by stirring for 2 minutes, 3) the surface-sterilized mushrooms were blended for 30 seconds in a coffee grinder sterilized by rinsing with 70% ethanol, 4) the ground mushroom biomass (aggregates <5 mm in length) was surface-sterilized again by repeating steps 1 and 2 using the ground biomass, 5) 5 grams of the ground mushroom biomass was added to the liquid medium in a Pyrex® tray (final pH = 4.0 - 4.1 after addition of the mushrooms), 6) the tray was covered and incubated in the dark at room temperature (22 ± 2 °C).

[0146] The biomats were observed to grow on the surface of the medium after 3 days of incubation, and the established biomats were harvested after 10 days of growth. The biomats of the Cream Mushroom covered the entire surface of the liquid medium in the tray, while the growth of the biomats of the White Mushroom covered approximately 1 / 2 of the liquid medium as five floating biomats islands. The average thickness of the biomats was 1.5 mm for Cream and 1.7 mm for White Mushroom. The biomass biomats were dried at 50 °C for 48 h, and the dry weight produced per tray was 1.14 g and 2.12 g for Cream and White Mushroom, respectively. The density of the dried biomass biomats on a dry weight basis was 0.033 and 0.111 g / cm 3 respectively.

[0147] Microscopic images revealed the mycelial characteristics of the biomats. The average mycelial thickness was 25.2 μm (standard deviation = 6.2) and 18.7 μm (4.0) for Cream and White Mushroom biomats, respectively.

[0148] The produced Cream biomats were used to make chicken nuggets. The biomats were steam-treated at 97 °C for 0.5 h, cooled to room temperature, and used as a base for producing chicken nuggets. The steam-treated wet biomass (2.5 g) was mixed with 3% (weight / weight; 75 mg) of Better Than Bouillon Chicken Base (Southeastern Mills, Inc. Rome, GA) and 3% of egg white protein (75 mg; Now Foods, Bloomingdale, IL), and minced into lengths less than 2 mm using a knife blade. The mixture was made into nuggets and steam-treated for 0.5 h. The prepared nuggets produced a typical chicken smell with a slight mushroom aroma. When tasted, the nuggets had a chicken or unclear flavor.

[0149] Example 12: Growth of Mushroom Biomats on Malt and Glycerol Medium A biomass biomat consisting of Ganoderma lucidum (Giant puffball), Pleurotus ostreatus (Pearl oyster), Pleurotus colombinus variety (Blue oyster), Hypsizygus ulmarius (Elm oyster), Flammulina velutipes (Cauliflower), and Ganoderma lucidum (Reishi) was produced in just 5 days using Malt Extract Medium 001, Glycerol Medium 002, Hansen’s Medium, MK7-SF Medium, and Malt Extract + NH4NO3 Medium 003 (Table 3). All final media contained 0.01% chloramphenicol. Table 3. Components added to deionized or potable-quality tap water to prepare nutrient media

[0150]

Table 3-1

Table 3-2

Table 3-3

Table 3-4

Table 3-5

Table 3-6

[0151] Using the recipe in Table 3 above, the medium was prepared by mixing the specified components in a specific volume of water according to the desired volume of the medium in a 2 L Pyrex® bottle or an 8 L stainless steel pot. The components were added to the water while continuously stirring the liquid with a stir bar or spoon. After each component of the medium was thoroughly mixed into the liquid, the next component was added, the pH of the MK7-SF medium was adjusted to 5.0, and the solution was autoclaved. All other pHs were left as the components were simply mixed. The medium and the container were autoclaved at 20 psi and 121 °C for at least 20 minutes. The osmotic pressure of the liquid was measured using an Advanced Instruments, Inc. osmometer Model 3250 (Two Technology Way, Norwood, MA).

[0152] After autoclaving, the medium was cooled to room temperature and inoculated with the mushroom species shown in Table 4 into individual containers.

[0153] Table 4. Mushroom spores (10 cc syringe) were purchased from MycoDirect (12172 Route 47, Ste 199 Huntley, Il 60142) and received on 8 / 2 / 2018. Elm oyster spores were purchased from Everything Mushrooms (1004 Sevier Ave Knoxville, TN 37920) and received on 8 / 3 / 2018.

Table 4

[0154] Inoculation of the growth medium was carried out using the following method applied under aseptic conditions. All aseptic work in these experiments was performed in a Class II safety cabinet. Using a spore syringe, it was directly inoculated into approximately 75 mL of growth medium in a previously autoclaved 12.7×17.8 cm Pyrex® glass tray. This was done by aseptically transferring the liquid medium to an autoclaved Pyrex® tray and inoculating it with a 2 cc suspension placed in the spore syringe. After covering the tray with sterile aluminum foil, it was gently swirled to mix the inoculated medium.

[0155] Malt Extract Agar (MEA; Table 5) plates were aseptically prepared by autoclaving MEA, cooling it to 50 °C, and pouring approximately 25 mL into sterile 100×15 mm Petri dishes.

[0156] Table 5. Ingredients used to prepare Malt Extract Agar

Table 5

[0157] 1 cc of liquid was sampled from the suspension in the spore syringe as a sample onto the plate to inoculate the MEA plate. The agar plate was then sealed with Parafilm® and placed in a clean, dark drawer at room temperature.

[0158] After the mycelium covered the entire surface of the MEA plate, it was used to inoculate 1.5 L of medium in a 2 L baffled shake flask. Approximately 2 cm 2 of the agar medium with mycelium on the surface was cut from the plate with a sterile scalpel blade, cut into approximately 2 mm 2 pieces with a scalpel, and then added to two flasks containing 1.5 L of Malt Extract 001 medium. The medium was incubated at room temperature (23 ± 1 °C) for 3 days with intermittent manual shaking (the flasks were shaken vigorously by hand at least 5 times a day for 1 minute).

[0159] Next, the cultures in the shaking flasks were used as inoculum for 6 L of Malt Extract medium 001 and 6 L of Malt Extract + NH4NO3 003 medium. The media were inoculated with 15% (vol:vol) of the inoculum cultures and mixed well. 2 liters of the inoculated media were poured into each of three 0.25 m 2 plastic trays placed in a trailer rack. The rack was wrapped with Saran (registered trademark) and incubated for 6 days. A relatively high-density biomass covered the entire surface within 4 days, and the biomass was harvested after 6 days.

[0160] The biomass in 12.7×17.8 cm Pyrex (registered trademark) glass trays and 0.25 m 2 plastic trays was lifted out of the trays and the biomass was harvested by gently squeezing it by hand. A portion (3 - 50 g) of the biomass was steam-treated for 20 minutes in boiling water (about 5 cm above the surface of the water) in a pot steamer set on a kitchen oven burner. After steam treatment, the biomass was cooled to room temperature and immediately placed in a Ziploc (registered trademark) bag and sent to Eurofins (Des Moines, IA) for protein analysis (N by combustion, Test Code QD252).

[0161] Table 6. Results of a series of giant puffball growth in various types of media in trays

Table 6

[0162] Example 13: Fusarium oxysporum MK7 strain chicken nuggets The Fusarium oxysporum MK7 strain with chicken flavor is a basic ingredient as a chicken substitute for several recipes starting with chicken for breaded or unbreaded chicken nuggets, Asian cuisine, or other chicken dishes. The Fusarium oxysporum MK7 strain biomass produced from various raw materials / carbon sources has somewhat different chicken flavors. Glycerol chicken is sweeter, and acid whey chicken tends to be slightly more acidic.

[0163] The amount of food processing and the blades used (i.e., sharp metal blades, dull metal blades, plastic blades) result in different chicken nugget textures. Furthermore, acceptable chicken nuggets can be produced from a wide variety of biomass sizes. That is, the biomass can be cut with a knife and lightly or highly food processed and still obtain an acceptable chicken analog.

[0164] The Fusarium oxysporum MK7 strain:chicken soup:binder in a ratio of 50 - 20:1:1 was used with or without an approximate 66.6% Fusarium oxysporum MK7 strain:fat ratio. Suitable fats include duck fat, coconut butter, and cocoa butter. After mixing, the mixture is steam treated for approximately 30 minutes to set the binder. However, some binders may require somewhat more time. Additional breading can then be applied, and typical food processing treatments for the resulting nuggets are carried out.

[0165] Example 14: Breakfast sausage and / or hot dog and / or burger To achieve the desired flavor, an appropriate spice mix is added to the reduced-size Fusarium oxysporum MK7 strain biomass as needed. This can be 10 wt.% spice mix relative to a certain amount of Fusarium oxysporum MK7 strain up to 20%, and can often be in a ratio of 10 Fusarium oxysporum MK7 strain:1 spice mix, with or without additional ingredients such as onion, binder, and fats like cocoa butter. The mixture is then fried in oil to remove an appropriate amount of moisture. Additional ingredients such as bulgur, vegetable broth, potatoes, etc. can then be added before shaping into the desired form and cooking.

[0166] Example 15: Ice cream and mousse Produce a Fusarium oxysporum MK7 strain biomass:water ratio of approximately 1:3 with a particle size where the average filament length is less than 900 microns. Gently heat this mixture until it no longer has a fungal smell, and then use it with cashew in a ratio of approximately 4:1, and optionally with an appropriate amount of xanthan gum and / or flavoring, to produce a mix that, after heating and cooling, may be used to produce a mousse. For frozen desserts, place the mix into an ice cream churn, and after churning, freeze to form a non-melting frozen dessert (Figure 14).

[0167] Example 16: Production of truffle oil from truffle biomass An oil extract can be prepared from the truffle (Tuber sp. biomass of the genus Tuber) grown as described above. In one example, the truffle biomass was grown on a tray for only 7 days using malt extract, glucose, and peptone as the basic carbon source (raw materials). Edible truffle mushrooms were purchased from IGB Trading LLC on the Amazon Marketplace and stored at 4°C. From the purchased truffles, approximately 3 mm of truffle (cut with a sterile razor blade) 3A pure culture of a fungus of the genus Penicillium was prepared by placing the portion on Malt Extract Agar + 0.01% chloramphenicol (used to inhibit the growth of bacteria). Malt Extract Agar was prepared by mixing 20 g of malt extract, 20 g of glucose, 1 g of peptone and 20 g of agar in 1 L of deionized water, autoclaving for 30 minutes, cooling to 50 °C and then adding 0.01% chloramphenicol. The sterile mixture was then poured into 9 cm diameter Petri plates, cooled and allowed to solidify.

[0168] Growth of the fungus on the tray was observed after 3 days. After 4 days of growth, the hyphae were collected with a sterile microbiological loop and streaked onto a fresh series of Malt Extract Agar + chloramphenicol plates. After growing the fungus on the plates for 5 days, the hyphae were collected with a microbiological loop and used to confirm the culture purity by DNA sequencing. Confirmation was carried out by extracting and purifying the DNA (FastDNA Spin Kit, MP Biomedicals), sequencing the ITS region of the metagenome and then performing phylogenetic classification of the sequences using Blast (NCBI database).

[0169] To prepare Malt Extract Broth, 20 g of malt extract, 20 g of glucose, and 1 g of peptone were mixed in 1 L of deionized water and sterilized. Also, using a scrape of mycelium scraped with a microbial loop, it was inoculated into 50 mL of sterile Malt Extract Broth in a sterile baffled shake flask covered with a sterile gauze material. The use of sterile gauze allows for the exchange of gases entering and leaving the shake flask. The shake flask was then rotated at 185 rpm for 5 days. Then, the rotated culture was used to inoculate 350 mL of sterile Malt Extract Broth in a sterile 12.7×17.8 cm Pyrex® glass tray. The inoculum density of this culture medium was 7.5% inoculum with respect to 92.5% broth. After growing in the tray for 7 days, the biomatt produced on the surface was harvested by lifting the filamentous biomatt from the liquid medium. The harvested biomatt was dried at 40 °C for 48 h. Lipids / oils were extracted from these harvested biomatts by solvent extraction using a mechanical press or hexane, although other extraction methods can be used. Another extraction method sent by Yuval can also be used.

[0170] Example 17: MK7 powder Using the Fusarium oxysporum MK7 strain biomass generated as described above, a dry powder with a particle size and particle size distribution similar to that of a standard baking powder was prepared. At this time, the wet biomass was steam-treated with a pot steamer at 97 °C for 0.5 hours, cooled to room temperature, and dehydrated with a Cuisinart dehydrator (model DHR-20) for 2 to 8 hours with an average dehydration time of 4 hours. The dehydration time is a function of the amount of biomass loaded into the dehydrator, the distribution of the biomass in the dehydrator affecting the air flow in the dehydrator, the water content of the biomass (average water content is approximately 75%), and the room temperature. The water content after dehydration varies from 4% to 14%, and the average water content after dehydration is less than 12%. The dehydrated biomass was reduced in size using a coffee grinder (KRUPS, Electric coffee and spice grinder, stainless steel blade F2034251) until it was finely ground. The average particle size of the ground biomass powder was in the range of 75 microns to 120 microns. A small portion of the larger particles, approximately 5 wt%, had a particle size larger than 180 microns. A small portion of the smaller particles, approximately 5 wt%, had a particle size smaller than 75 microns. The smaller particles were outside the size that could keep the small particles floating for a long time. The particle size was determined by passing a 100-gram sample of the biomass with reduced size through sieves with openings of 180 μm, 120 μm, and 75 μm for 5 minutes. Since a higher water content can cause aggregation of the dried and ground biomass, a water content of less than 6% after dehydration and after size reduction is preferred.

[0171] Next, biomatt powder was used as an additive to other standard flours (King Arthur flour, Bob’s Red Mill Flour & Bob’s Red Mill Wheat Flour) to prepare various baked foods. When loaded with biomatt powder at 5 wt%, 10 wt%, 20 wt% and 30 wt%, there was no detrimental effect on the taste, fermentation, texture, appearance or smell of the final baked foods. The products included bread (7-grain, white and wheat), pastry (puff pastry), cookies, pasta and dumplings. The resulting products passed the taste test, and the fact that they contained MK7 flour was not discernible to the people who tasted the products.

[0172] Example 18: MK7 extender Using the Fusarium oxysporum MK7 strain biomass generated as described above, particulate biomass was created for use as a bulking agent (i.e., increasing the total amount of a food product by adding MK7 to other existing food products) and added to meat and fish. The wet biomass was steam-treated in a pot steamer at 97 °C for 0.5 hours and cooled to room temperature. The size of the biomass was reduced to a desired particle size distribution (i.e., chopped with a knife or processed with a food processor). The reduced-size biomass was then added to various food products to increase the amount of meat in the case of a meat bulking agent or fish in the case of a fish bulking agent. As an example of meat bulking, 10%, 20%, 30%, 40% and 50% addition of the reduced-size biomass was added to hamburger meat. The reduction in biomass size was evaluated with several different size distributions. Smaller particle sizes tended to result in a denser and creamier texture. Larger particles tended to produce products with more texture, more mouthfeel and required more chewing before swallowing. The bulked meat was then processed as if no biomass had been added. In the case of hamburger bulking, spices or binders may be added, and the bulked meat was formed into patties or meatballs and cooked until the meat was cooked to the desired temperature for the consumer. Cooking methods included on a stove, in an oven, frying in oil and grilling. Taste tests indicated that acceptable food products were produced at all loading levels and all size distributions of added biomass. Chicken and pork bulking were also tested at similar loading levels with similar cooking and tasting results.

[0173] The fish weight gain was also demonstrated at loads of 10%, 20%, 30% and 40%. Fillets and fish meatballs were produced by adding processed MK7 in various different size distributions ranging from small particles (less than 1.0 mm) to large particles (larger than 2 mm), and there was no harmful effect on taste, color, smell or all food experiences. In the case of adding small particle sizes, the resulting food products had a creamier texture. In the case of adding large particle sizes, the resulting food products had a firmer texture characterized by larger particles that required more chewing before swallowing. The taste tests showed that acceptable food products were produced at all tested load and size distribution levels.

[0174] Example 19: MK7 Jerky Using the Fusarium oxysporum MK7 strain biomatt produced as described above, jerky-like mushrooms with appearance and taste similar to meat jerky (i.e., beef jerky, buffalo jerky, pork jerky, chicken jerky, turkey jerky, etc.) were created. The wet biomatt was steam-treated in a 97 °C pot steamer for 0.5 hour and cooled to room temperature. The size of the biomatt was reduced to a size consistent with that found in normal jerky products. The reduced-size biomatt pieces were seasoned as appropriate and dehydrated in a Cuisinart dehydrator (model DHR-20) for 20 - 200 minutes, with an average dehydration time of 40 - 120 minutes. The dehydration time is a function of the amount of biomass loaded into the dehydrator, the distribution of the biomatt within the dehydrator affecting the air flow within the dehydrator, the water content of the biomatt (average water content was approximately 75%), room temperature, and the desired water content of the final product. The water content after dehydration varied from 8% - 12% depending on the desired product characteristics. Optionally, holes were made in the biomass prior to dehydration to produce a product that was more easily consumable by being more easily torn into smaller pieces. The perforation of the biomass was done by using a fork, knife, or meat tenderizer that perforates the biomass and breaks the filamentous network to make it more easily torn. A variety of spice mixtures (i.e., Cajun, cheese, soy, vinegar, herbs, sour cream and onion, smoke liquid, vegetarian meat flavor, etc.) were evaluated. The spice mixtures were evaluated both before and after dehydration. Samples with spices added before dehydration provided more flavor and adhered better to the biomass than those treated after dehydration. All of the resulting jerky passed the taste test.

[0175] Example 20: Mushroom Chips The Fusarium oxysporum MK7 strain biomatt generated as described above was used for chips whose appearance and taste were similar to potato chips or corn chips. The wet biomatt was steam-treated for 0.5 hour with a pot steamer at 97°C and cooled to room temperature. The size of the biomatt was reduced to a size consistent with that found in normal chip products, highly processed into a paste, and formed into a chip-like shape. Then the mushroom chips were placed in hot oil (the temperature was approximately equal to 380°F) in a frying pan until browned. The cooking time varied as a function of the shape of the biomass but was extremely fast and was usually cooked within less than 15 seconds. It was proven that the chips fried in the generated oil had an extremely good mouthfeel and could provide a wide variety of taste experiences depending on the spices added to or applied to the biomass before frying.

[0176] Example 21: Sealed and closed bioreactor: Biomatt A Pyrex (registered trademark) glass tray 12.7×17.8 cm and a 100×15 mm Petri dish are used as the base tray. The glass tray is loaded with 200 mL of raw materials mixed with a liquid nutrient medium (if necessary) and an inoculation material. The tray is covered and sealed with a gas-permeable membrane attached to a plastic frame with an integrated rubber gasket. The sealing system provides an effective sterile seal between the membrane and the glass tray and allows for easy assembly and opening / closing of the reactor for the purpose of sample collection and harvesting.

[0177] A series of different gas-permeable membrane materials with different thicknesses, pore sizes, and textures (surface roughness) are used as materials at the gas-liquid interface. Initially, eight polymeric materials are used, including polypropylene, polyethylene, polytetrafluoroethylene, polycarbonate, polyamide, polypyrrolone, poly(amidoamine) dendrimer composites, and cellulose acetate (e.g., Specialty Silicone Products, Inc. Corporate Technology Park, NY; Thermo-Fisher, Waltham, MA; Merck Millapore, Burlington, MA). Three pore sizes (0.2, 0.45, 1.0 μm) are used in materials that facilitate gas transfer in addition to the direct diffusion of gases through the polymer itself, excluding microorganisms. Additionally, sterile cloth-like materials with different rough surface textures and tortuous pathways for gas diffusion are used. Abundant such materials are commercially available from other companies including 3M, Solvay, Ube Industry, and Saint-Gobain.

[0178] To analyze and determine parameters for different environmental and mission conditions, the tray reactor is fitted with sensors to monitor temperature, dissolved oxygen, and pH as a function of depth across the tray. The ports for the wires entering the reactor across the sensors and membranes are sealed with silicone, epoxy, and / or adhesives depending on the membrane material. The septum integrated into the membrane is used as a sampling port to collect liquid samples for GC-MS, ICP-MS, IC, and total C / N analysis. Standard and microelectrodes are used to measure in real-time the pH and electron acceptor flux (O2) across the gas-permeable membrane and within the biomat at regular time intervals (e.g., 6, 12, 24, 36, 48 hours). Flux information is important to match the real-time metabolic requirements to the gas permeability of the membrane and the changing concentrations and distributions of the electron donors (organic matter) and nutrients (inorganic matter) required for optimal growth and raw material conversion.

[0179] Example 22: Un-instrumented Reactor Reactors without equipment are used for growth studies using the fungal strain Fusarium oxysporum MK7 as a model filamentous organism. The MK7 strain is a facultative anaerobic fungus that has been shown to grow on a wide variety of raw materials, including urine, food waste, cyanobacteria and algal biomass, as well as lignocellulosic materials. The MK7 strain biomatt has also been shown to have resistance to high urea levels (at least 26 g / L), as well as high dissolved organic carbon, and osmotic pressure (300 g / L glycerol).

[0180] The raw materials tested include 1) substitute human urine as a basic nitrogen source, 2) substitute food waste (dog food) as a basic carbon source, and 3) plant material (lignocellulose) as an additional carbon source. All raw materials are extensively analyzed for organic and inorganic constituents, pH, and biological oxygen demand. Substitute human urine is prepared using a media composition recommended by NASA scientists or scientists involved in mission waste research.

[0181] The effectiveness of various gas-permeable membranes is measured by conducting comparative biofilm-biomatt growth studies in which various membranes are sealed to the surfaces of trays and Petri dishes containing various raw materials and MK7 inoculum material. The membrane is brought into direct contact with the liquid phase and serves as the only means of gas exchange between the gas / vapor external environment and the biofilm-biomatt / liquid medium. The reactor is destroyed over time to collect samples and measure growth (dry biomass weight, biomatt thickness). The growth rate is compared to a control tray without a membrane. A factorial experimental design consisting of combinations of raw materials and membranes is tested to provide the best match of raw materials and membranes. Additional variables are also evaluated, including initial raw material pH and inorganic nutrient addition. Furthermore, the experiment tracks the number of viable bacterial cells from the raw material as a function of time to quantify the disinfection kinetics associated with biomatt growth.

[0182] Example 23 The membrane / raw material combinations with the best performance are used in additional experiments. The gas fluxes are first quantified through the selected gas permeable membranes and modeled by abiotic experiments. The flux of O2 from the vapor phase outside the reactor into the liquid phase is measured using the initial slope method, with a dissolved oxygen probe and a medium that is initially oxygen-free. The flux of CO2 from the liquid phase saturated with CO2 into the vapor phase is also measured using the initial slope method, with a pH probe (a measure of carbonic acid) by total inorganic carbon analysis. The dissolved inorganic carbon phase is initially 0%, 0.5%, and 5% CO2. The data are incorporated into a moving front fungal growth model to obtain more accurate parameters.

[0183] Next, the observed membrane / raw material combinations with the best performance are used in detailed biological optimization experiments assisted by the fungal growth model. Both glass trays and Petri dishes are used. The smaller Petri dishes facilitate intensive destructive sampling for biomass and liquid analysis over time. It is confirmed that conditions are created where almost all of the added carbon and nutrients are converted to biomass with minimal waste. Here, the carbon and electron fluxes and reactor conditions are evaluated by measuring the biomass produced per electron donor and the biomass produced per electron acceptor yield. The elemental composition of the biomass is measured using commercial services (e.g., Microanalysis Inc., Wilmington DE) to satisfy the mass balance. The parameters of interest are the volume of the liquid phase and the concentrations of the available raw materials and nutrients (carbon substrate, nitrogen source, inorganic nutrients, oxygen). The data obtained are used in a moving front mathematical model of fungal mat growth that facilitates quantitative comparison and ultimately optimization of the growth conditions.

Claims

1. An edible formulation of filamentous fungi containing edible filamentous fungal particles, wherein the edible filamentous fungal particles are particles of an aggregative edible filamentous fungal biomass, and the filamentous fungi contain at least 46% protein.

2. The formulation according to claim 1, wherein the filamentous fungi are Fusarium sp. MK7 strain (ATCC deposit number PTA-10698).

3. The formulation according to claim 1, wherein the filamentous fungi are Fusarium venenatum.

4. The formulation according to claim 1, wherein the filamentous fungi are selected from the group consisting of mushrooms (cream and white), Boletus edulis (porcini), Cantharellus cibarius (chanterelle), Gyroporus cyanescens (giant puffball), Cyclocybe aegerita (velvet pioppini), Ganoderma lucidum (reishi), Grifola frondosa (maitake), Morchella spp. (morel), Clitocybe nebularis (clouded agaric), Pleurotus ostreatus (oyster mushroom), Laetiporus spp. (chicken of the woods), Pleurotus eryngii (king oyster mushroom), Hypsizygus tessellatus (pearl oyster and blue oyster), Pholiota nameko (forest pholiota), Sparassis crispa (cauliflower), and Tuber spp. (truffle).

5. The formulation according to claim 1, which is a liquid.

6. The formulation according to claim 1, which is a solid.

7. The formulation according to claim 1, which is a paste, powder, aerated mass or hard mass.

8. A food product containing the edible filamentous fungal formulation according to claim 1.

9. The food product according to claim 8, wherein the filamentous fungi are Fusarium sp. MK7 strain (ATCC deposit number PTA-10698).

10. The food product according to claim 8, wherein the filamentous fungi are Fusarium venenatum.

11. The food product according to claim 8, wherein the filamentous fungus is selected from the group consisting of mushroom (cream and white), Boletus edulis (porcini), Cantharellus cibarius (chanterelle), Gyroporus cyanescens (giant puffball), Cyclocybe aegerita (velvet pioppini), Ganoderma lucidum (reishi), Grifola frondosa (maitake), species of the genus Morchella (morel), Clitocybe nuda (blewit), Pleurotus ostreatus (oyster mushroom), species of the genus Laetiporus (chicken of the woods), Flammulina velutipes (enoki mushroom), Hericium erinaceus (lion's mane mushroom), Pleurotus cornucopiae (pearl oyster and blue oyster), Pholiota nameko (forest pholiota), Sparassis crispa (cauliflower), and species of the genus Tuber (truffle).

12. The food product according to claim 8, which is a meat substitute food, a meat extender, bread, pasta, pastry, dumpling, jerky, snack, drink, yogurt, or dessert.

13. The food product according to claim 12, wherein the meat substitute food mimics the texture of a burger, sausage, hot dog, chicken nugget, or fish fillet.

14. The food product according to claim 12, wherein the dessert is a mousse, pudding, frozen dessert, or ice cream-like food.

15. The food product according to claim 12, wherein the dessert is a confectionery or candy.

16. A method for manufacturing a food product, the method comprising contacting the formulation according to claim 1 with one or more other ingredients.

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