JP2021527399A - Method for producing abundant cell culture medium using chemosynthetic autotrophic microorganisms - Google Patents

Abstract

The production of a nutrient-rich medium from the first minimal medium suitable for culturing heterotrophic cells is described. These methods use carbon in common industrial emissions to feed growing biomass, gas fermentation of chemosynthetic autotrophic microorganisms and / or chemosynthetic autotrophic microorganisms under chemosynthetic autotrophic conditions. use. Microorganisms also convert some of the carbon into organic nutrients that are released into the minimal medium, thereby enriching the minimal medium. In a further method, heterotrophic cells are cultured in nutrient-rich medium.
[Selection diagram] Fig. 3

Description

Cross-reference to related applications This application is a US patent filed on June 17, 2018, claiming the interests of US Provisional Patent Application No. 62 / 686,508 filed on June 18, 2018. A partial continuation of US Patent Application No. 15 / 641,114, alleging the interests of Application No. 16 / 443,658 and filed on July 3, 2017, all three by reference. Incorporated herein.

Fields of Invention The present invention generally relates to the fields of microbial fermentation and industrial biotechnology, and more particularly to the production of nutrient-rich growth media for the cultivation of heterotrophic organisms.

Cell culture is a method of growing, growing and maintaining cells in a liquid or on a solid or semi-solid substrate such as agar. In performing cell culture, the liquid or substance in which the cells are cultured is called the medium. All dependent vegetative cells, i.e. all cells other than independent vegetative cells, require some form of medium containing chemical energy and carbon, which are small molecules such as formates, acetates, and methanol, or sugars and starches. It can be provided by more complex and larger molecules such as, and in some cases very complex and larger chemicals such as proteins.

Cell culture media contain many different compositions containing a variety of components, including inorganic salts, sugars, amino acids, peptides, proteins, polysaccharides, hormones, growth factors, and complex components such as bovine serum, tryptone and yeast extracts. Can have. Medium compositions containing only water and mineral salts are called "minimal media" and are essentially inadequate for heterotrophic cells because they lack some form of chemical energy and carbon. The minimum medium does not contain organic compounds. Mediums containing organic compounds such as sugars, yeast extracts, enzymatically digested proteins and other energy sources and complex compounds are called "rich media" or "composite media", and composite media or rich media are microorganisms. Is a medium that essentially contains all the energy, carbon and other factors needed to grow yeast. Proteins are of particular importance in culturing cell lines of multicellular organisms such as vertebrates, mollusks and arthropods.

The following additional definitions apply herein:

"Heterotrophic" is defined to mean "requiring complex organic compounds of nitrogen and carbon (such as those obtained from yeast, plant or animal material) for metabolic synthesis".

The "autotrophic", that "the carbon source as carbon dioxide or a carbonate (C ₁ compounds) requires only require simple inorganic nitrogen compounds in the metabolic synthesis of organic molecules (such as glucose)" means.

"Chemosynthetic autotrophic" is defined as "autotrophic and oxidizes an inorganic compound as an energy source". In the case of hydrogen-oxidizing bacteria, the inorganic compound as an energy source is H ₂ Is included, and _{a combination of CO 2} , H ₂ , and O ₂ can be consumed. For example, there is an anaerobic acetogen that consumes _{CO 2} for _{carbon and H 2 for energy.} Other inorganic energy sources chemoautotrophic organisms, reduced small molecules, e.g., H 2 _S, ammonium, or first iron. In some cases, the input of carbon and energy to chemoautotrophic organisms are combined into a single C ₁ molecule. For example, carboxydotrophs (carbon monoxide assimilation) and carboxydovores consume CO (carbon monoxide) in both carbon and energy, and methanotrovs are O ₂ (oxygen molecules). together or other oxygen donor compounds consume CH 4 a _(methane). Chemosynthesis Autotrophic metabolism is known in bacteria and archaea and may also be present in other organisms as an undiscovered trait or as an ability conferred by genetic modification. Examples of chemoautotrophic organisms, Cupriavidus, Rhodobacter, Methylobacterium, Methylococcus, Methylosinus, Nitrosomonas, Nitrosococcus, Nitrobacter, Nitrococcus, Paracoccus, Hydrogenothermus, Hydrogenovibrio, Clostridium, Rhodococcus, Rhodospirillum, Alcaligines, Rhodopseudomonas, and many, such as Thiobacillus It is found in the genus Bacteria, as well as in many genera of paleococci, including methane-producing bacteria. Examples of chemical synthesis autotrophs, Cupriavidus necator, Cupriavidus basilensis, Rhodococcus opacus, Methylococcus capsulatus, Methylosinus trichosporium, Methylobacterium extorquens, Hydrogenothermus marinus, Rhodospirillium rubrum, Rhodopseudomonas palustrus, Paracoccus zeaxanthinifaciens, Rhodobacter sphaeroides, Rhodobacter capsulatus, and Clostridium Autoethanogenum Is included.

"Fermentation" is defined as "a metabolic process that causes a chemical change in an organic substrate by the action of an enzyme", and in biochemistry, it is narrowly defined as extracting energy from carbohydrates in the absence of oxygen. In the context of food manufacturing, it is more broadly referred to as any process in which the activity of microorganisms results in the desired changes in food or beverage. In an even broader sense, for the purposes of the present invention, "fermentation" refers to special containers (glass, metal) under controlled process conditions to optimize their growth and maximize efficiency. Or made of plastic, a process for culturing cells in a fermenter or bioreactor). Controlled process conditions include asepticity, temperature, agitation rate, pH, input gas composition and flow rate, nutritional components, cell density, dissolved gas concentration, biomass removal rate (for continuous or semi-continuous harvesting), etc. .. In the latter case, the fermentation is aerobic or anaerobic.

"Gas fermentation" refers to fermentation in a bioreactor in which the metabolic processes of chemically synthesized autotrophic cells extract energy and carbon from the gas inputs supplied to them. Gas fermentation refers to an anaerobic or aerobic process of culturing microorganisms on gas. By combining these gas inputs with simple inorganic salts in the medium, chemosynthetic autotrophic cells convert these basic inputs into more complex biomass and other cell products. Gas fermentation is either aerobic or anaerobic, depending on the microorganisms used and the source gas available for fermentation. Gas fermentation is a particularly advantageous form of chemically synthesized autotrophic fermentation. This is because the main input is provided by widely available and low cost gas.

"Culture" is "the act or method of culturing organisms (bacteria, viruses, etc.) in the prepared nutrient medium", and "nutrition" is "a substance that promotes growth, supplies energy, and sustains life." "Or component", "medium" means "nutrition system for artificially culturing cells or organisms, especially bacteria", and the medium is liquid, semi-solid or solid (for example, agar, beads or other scaffolds). ). Solid or semi-solid medium can provide a growth support for cells.

Note that in a typical heterotrophic fermentation, cells grow in a medium containing complex organic molecules such as sugars, amino acids, peptides and organic acids. Heterotrophic cells generally extract most of the "high energy" type of carbon from the medium, increasing the cell's biomass and turning the catabolic carbon into carbon dioxide, carbon acetate, or other simple, low-value waste products. As released, the medium remaining after recovery of biomass from the bioreactor after the heterotrophic fermentation process is commonly referred to as "used medium" because it is generally very low in nutritional value for further heterotrophic culture. Even if heterotrophs are selected or designed to excrete high value products into the medium, they are nevertheless on fairly high cost media (including complex molecules such as sugar or protein). Must be cultivated in, thus limiting the profitability of the production process.

In chemosynthetic autotrophic fermentation, cells grow in the same way and produce biomass. However, the high degree of anabolic metabolism of chemosynthetic autotrophs produces excess nutritionally valuable products, some of which leak out or are excreted in the medium.

"Higher organism" or "higher organism" means eukaryotes such as yeast, fungi, microalgae, plants and animals.

A significant cost in the commercial culture and maintenance of cells, especially in the culture of cells isolated from multicellular organisms such as plants, fish, soft animals and arthropods, is the cost of growth medium. Such media often include water and various inorganic salts, as well as many different peptide growth factors, amino acids, sugars, yeast extracts, animal or plant-derived protein digests, animal-derived serum, proteins (eg, eg). Contains trypton and peptone, or albumin such as bovine serum albumin), as well as other metabolites important for cell growth. An important part of the high manufacturing costs of these media is the use of animal materials such as those processed from blood and other fluids and tissues.

For example, basal medium Eagle (BME) is a synthetic basal medium that is widely used to support the growth of many different mammalian cells. BME contains 8 B-vitamins and 10 essential amino acids + cystine, tyrosine, and glutamine.

FIG. 1 is a schematic representation of an exemplary embodiment of a system according to various embodiments of the present invention. FIG. 2 is a schematic representation of an exemplary bioreactor according to various embodiments of the present invention. FIG. 3 is a flow chart representation of the method according to various embodiments of the present invention. FIG. 4 is a graph of experimental results showing the culture of Lactobacillus brevis in a medium produced by the exemplary method of the invention.

The present invention describes the production of a nutrient-rich medium suitable for culturing heterotrophic cells from exhaust gas. The methods described herein are free of carbon in industrial waste, photosynthetic autotrophic microorganisms or chemosynthetic autotrophic microorganisms, on that carbon under chemosynthetic autotrophic conditions, and initially free of organic nutrients. It is used as the bottom of the food chain, which is initiated by gas fermentation in the medium. Microorganisms can grow to convert this carbon to larger biomass, and under appropriate conditions, some of the carbon can also be converted to organic nutrients as waste by-products, which translates heterotrophic cells further into the food chain. The medium can be concentrated to be suitable for culturing above.

An exemplary method of the present invention comprises providing a minimal medium to the bioreactor, inoculating an inoculum containing chemically synthesized independent vegetative cells and / or photosynthetic independent vegetative cells into the minimal medium in the bioreactor, and in the medium. In order to grow the biomass in the bioreactor by providing a gas input into the bioreactor until the cell density of the biomass in the bioreactor meets the threshold, the inoculum is cultured and chemically synthesized and independently cultured. , Thereby enriching the minimum medium during culture to a concentrated medium. In various embodiments, the method further comprises preparing an inoculum prior to inoculating the minimal medium. Various methods may further comprise preparing a minimal medium. A further embodiment comprises sterilizing the minimal medium and the bioreactor prior to inoculating the minimal medium with the inoculum. In a further embodiment, the minimal medium comprises a gel. In various embodiments, the minimal medium is added to the bioreactor along with the cell growth support.

In various embodiments, chemosynthetic autotrophic cells or photosynthetic autotrophic cells produce growth factors, hormones, antibiotics, amino acids, peptides, proteins, vitamins, colorants, carotinoids, fatty acids, or oils. In various embodiments, the inoculum also comprises heterotrophic cells. The inoculum can also contain photosynthetic autotrophic cells, and in these embodiments, the culture is carried out in the absence of light in the bioreactor.

In various embodiments, culturing the inoculum comprises adding a beneficial molecule to the concentrated medium, and in some of these embodiments, the beneficial molecule comprises glucose. Culturing the inoculum comprises adjusting the pH of the enriched medium in some embodiments. In some cases, the inoculum contains chemically synthesized autotrophic cells and the gas influx contains CH ₄ and O ₂ . In some of these embodiments, chemosynthetic autotrophic cells include cells of Methylococcus capsulatus. In various embodiments, the gas input comprises CO, the gas input comprises CO ₂ and H ₂ S, and the gas input comprises CO ₂ , H ₂ and O ₂ . In still other embodiments, the inoculum comprises Cupriavidus necator cells, Rhodobacter capsaturus cells, or both cells. Inoculum can include, in various embodiments, carboxydotrophs (carbon monoxide assimilation) cells or Rhodococcus opacus cells.

Various embodiments of the invention further include destroying cells in the concentrated medium such that their contents are released into the concentrated medium. Destroying a cell can optionally include lysing the cell. In various embodiments, the concentrated medium comprises one or more of growth factors, hormones, antibiotics, amino acids, peptides, proteins, vitamins, colorants, carotinoids, fatty acids, or oils.

Various embodiments further include separating insoluble biomass from the concentration medium by methods such as centrifugation, filtration, or gravity-based separation. In some of these embodiments, the concentrated medium after separation contains at least 1 gram of D-glucose per liter. In some embodiments, the method is to add mineral salts to the concentrated medium after separation, adjust the pH of the concentrated medium, filter, provide enzymatic treatment to the concentrated medium, chromatograph to the concentrated medium. It further comprises one or more of performing separations or performing selective precipitation from concentrated media. In the method of separating insoluble biomass from the concentrated medium, some methods further include removing ammonium ions from the concentrated medium.

In various embodiments, the method further comprises culturing the cells of the heterotrophic organism in a concentrated medium separated from the biomass, thereby depleting the concentrated medium. In some of these methods, heterotrophs include yeast, fungi, algae, archaea, bacteria, or mammals. Heterotrophic cells, in a further embodiment, are derived from the cell lineage of multicellular aquatic organisms.

In addition, the present invention is directed to a variety of concentrated media produced by the methods described herein.

The present invention describes the production of nutrient-rich media suitable for culturing heterotrophic cells from waste gas. In the production of such a medium, initially, chemically synthesized autotrophic cells and / or photosynthetic autotrophic cells were cultured in a minimum medium through gas fermentation, and then sufficient culture was performed. Later, it involves abundant harvesting of such media. The concentrated medium can then be used to culture heterotrophic organisms such as yeast, fungi, microalgae, plants and animals. Cells cultured in a bioreactor may contain a single species or single strain of chemosynthetic autotrophic microorganisms or photosynthetic autotrophic microorganisms, or they may contain multiple strains or species. In addition, these cells can be co-cultured with one or more species or strains of various selected dependent trophic microorganisms, the purpose of which is a medium not produced solely by chemically synthesized autotrophic cells or photosynthetic autotrophic cells. To supply additional desired nutritional components (such as probiotics). Co-cultures or communities with heterotrophic cells can be designed to add value to the end product rather than being consumed by the heterotrophic cells. In some embodiments, the intracellular contents of the resulting biomass can be added to the concentrated medium.

Here, the "first medium" is the first medium used to supply the basic nutrients for the first fermentation. The "second medium" used herein is the supernatant concentrated medium obtained from the enrichment of the first medium, which remains after separation from all or most of the biomass. Therefore, during chemosynthetic autotrophic fermentation, cells proliferate and also produce highly assimilated products. The medium remaining after enrichment from chemosynthetic autotrophic fermentation is referred to as the "second medium" to distinguish it from the nutritionally inferior "used medium" obtained from dependent autotrophic fermentation. After removal of most or all of the biomass, the second medium contains many complex substances suitable to support the growth of heterotrophic cells. Therefore, this second medium is collected during or after fermentation and processed to remove any residual cells or cell debris (if desired) and nutritionally for culturing dependent vegetative cells. It can be reused as an advantageous growth medium or additive. As used herein, "concentrated medium" refers to the first medium after the onset of gas fermentation and broadly includes both the medium during culture and the second medium after the separation process.

Chemosynthetic and autotrophic microorganisms cultured on industrial waste gas are those microorganisms from simple and generally inexpensive inputs (eg, hydrogen, carbon dioxide, oxygen, water and inorganic salts). It can be a particularly abundant and beneficial source of nutrients, as all of its cell constituents (including sugars, fatty acids, carotenoids, cofactors, vitamins, peptides and proteins) must be newly produced. The production of this chemosynthetic autotrophic mode also has the advantage that vitamins and proteins can be synthesized at a lower cost than, for example, by fermentation of heterotrophic microorganisms on sugar.

The second medium produced according to the present invention can contain similar or identical components as compared to the rich medium of the prior art and can provide equivalent nutritional value and therefore various cell cultures. It can supplement or completely replace animal-derived and other expensive ingredients for use. In some cases, this not only reduces production costs, but also provides a source of culture medium that does not require killing or harming the animal. In other cases, the components available in the medium can provide advantages not found in known rich media, and therefore the product of this method is itself novel to the prior art. The medium may be completely liquid, or may be formulated into a gel (eg, using agar) for use as a cell growth support, or a solid or semi-solid substance (eg, using agar). , Beads).

It is important to note that the typical prior art growth medium for heterotrophs has an initial composition designed to be depleted as the growing cells are cultured. According to the present invention, fermentation of chemosynthetic autotrophic cells is initiated in a minimal medium. Supply combined with chemical synthetic autotrophic growth of cells supplying mineral salts and feedstock gas, and any additional heterotrophic cell growth and product supply from chemically synthesized autotrophic growth that may be included as part of the consortium. The feed gas input actually accumulates the nutrient quality of the enriched medium as the culture progresses.

FIG. 1 shows a schematic view of an exemplary system 100 of the present invention. System 100 includes a bioreactor 110 containing photosynthetic autotrophic cells and / or chemosynthetic autotrophic cells 120. System 100 also includes a carbon source 130 such as an industrial source that produces a waste stream 140 containing one or more carbon oxides, carbon monoxide and carbon dioxide. Examples of carbon source 130 are cement production facilities, power plants that burn fossil fuels, iron and metal products production (eg casting and forging), non-ferrous product production, food production, ethanol or other bio production production plants. Includes gasification of biomass, gasification of coal, and chemical production such as petroleum refining, carbon black production, ammonia production, methanol production and coke production.

The system 100 further comprises any molecular hydrogen source 150 such as a hydrogen storage tank, a hydrogen pipeline, a steam methane reformer, a gasifier, or an electrolytic system. The hydrogen source 150 produces a hydrogen stream 160 containing molecular hydrogen as an energy source for chemosynthetic autotrophic, ie, chemosynthetic autotrophic cells or photosynthetic autotrophic cells grown in the absence of light. In various embodiments of the invention, cells in a chemically synthesized autotrophic bioreactor derive both carbon and energy from a single waste stream 140, such as methane or carbon monoxide, in these embodiments. The hydrogen molecule source 150 is not required. In a further embodiment, the waste stream 140 comprises a carbon source and a hydrogen source such that it can be produced by a gasifier. FIG. 1 also schematically shows that the two output streams of System 100 are the accumulated biomass 170 and the concentrated or "second" medium 180 after removal and separation from the bioreactor 110. In some embodiments, the biomass 170 is gasified (not shown) and the product is used as a second carbon source 130.

FIG. 2 shows a schematic representation of the bioreactor 200 as an example of a suitable bioreactor for the method of the invention. Bioreactor 200 comprises either a synthetic vessel for the production of cells for inoculation into the first medium and a synthetic vessel for use in conjunction with a separate growth vessel for the production of biomass in the concentrated medium. The bioreactor 200 can include containers suitable for both synthetic and growth stages. In FIG. 2, the bioreactor 200 includes a container 205 that holds a constant amount of liquid medium 210 in culture containing chemosynthetic autotrophic cells or photosynthetic autotrophic cells and optionally other dependent autotrophic cells during operation. The bioreactor 200 also has an inlet 215 capable of introducing gas 220 into container 205 for introduction into medium 210, a medium inlet 225 capable of introducing fresh medium 230 into container 205, and medium 210. Includes, for example, a medium outlet 235 capable of separating the concentrated medium from the insoluble biomass. The bioreactor 200 can also include a headspace 240 and an outgassing valve 245 for discharging gas from the headspace 240. In some embodiments, an outgassing valve 245 is attached to the recirculation system to return the vented gas to the input port 215 and a manifold for making additions to optimize the gas composition (not shown). Can be included.

In various embodiments, the bioreactor 200 can be a continuous stirring tank reactor, a loop bioreactor, or any other design suitable for gas fermentation. The bioreactor 200 can further include controlled agitation for mixing, various probes for measuring pH, dissolved gas, and culture density, as well as controls for gas, temperature control, and the like. Agents for controlling foaming can also be added to the bioreactor 200.

FIG. 3 shows an exemplary method 300 of the present invention. Some steps are listed as options, but steps not listed as options are not always required. Method 300 includes any step 305 of preparing the inoculum and any step 310 of preparing the minimal medium. Method 300 then includes step 315 of adding minimal medium to the bioreactor and optional step 320 of sterilizing the minimal medium and bioreactor. Method 300 then includes step 325 of inoculating the inoculum with the sterile minimal medium by supplying gas to the bioreactor and step 330 of fermenting until the cell density meets a threshold. Method 300 then comprises an optional step 335 of releasing the cell contents into a concentrated medium, followed by an optional step 340 of separating the biomass from the second medium. In step 345 of the option, the second medium is inoculated with the type of heterotrophic cells designed to be supported by the second medium.

In step 305, sufficient inoculum material is prepared to inoculate a bioreactor such as bioreactor 200. This step is optional as long as the particular embodiment of Method 300 can be initiated from a prefabricated inoculum. The inoculum comprises cells of at least one species of photosynthetic autotrophic microorganism or chemosynthetic autotrophic microorganism and can be prepared using either rich medium or minimal medium with the gas feedstock. The particular species of microorganism selected for the inoculum, and any other dependent microbial microorganisms contained therein, may be a second, such as a bacterium, archaea, microalgae, fungus, mold, or yeast. It is later selected to produce a suitable second medium tuned for the benefit of culturing a particular higher organism in the medium. To prepare inoculum is to co-culture chemically synthesized autotrophic cells, photosynthetic autotrophic cells, and / or dependent autotrophic cells together in the same medium or in one or more strains in separate media. May include. In the latter case, preparing the inoculum means preparing the amounts of chemosynthetic autotrophic cells, photosynthetic autotrophic cells, and / or heterotrophic cells at different times, and preparing those amounts for all use. It can include storing until possible.

In some embodiments, the chemically autotrophic microorganism has been genetically modified to produce a beneficial heterologous product, such as Cupriavidus necastor, Methylococcus capsaturus, Rhodobacter capsaturus, or Rhodobacter capsus, or chemically autotrophic. Includes a single chemical autotrophic cell line, such as an organism. A chemically synthesized autotrophic strain may be natural, contain mutations, be genetically modified, or contain one or more genes edited via CRISPR. Valuable small molecules, growth factors, hormones, antibiotics, amino acids, peptides, sugars, polysaccharides, proteins, vitamins, colorants, carotinoids, fatty acids, organic acids, nucleic acids, oils, glycolic acids, hydrocarbons, polyhydroxyalkanoic acids, It may be to produce facins, gene transfer agents (GTAs) or other biomolecules that can be utilized as growth substrates and growth regulators by non-autotrophic microorganisms and other autotrophic cells. Examples of photosynthetic autotrophic microorganisms that can be used in this way include Rhodobacter rubrum, Rhodopseudomonas pulustrus, Paracoccus zeaxanthinifaciens, Rhodobacter sphaeradias, Rhodobacter sphaeroides, Rhodobacter.

Deposit of biomaterials The following microorganisms are American Type Culture Collection, 10801 University Boulevard, Manassas,
Deposited with VA 20111-2209, USA (ATCC):
Table 1:
Designation of microorganisms: Rhodobacter capsulatus OB-213
ATCC number: PTA-12049
Deposit date: August 25, 2011

This deposit was made under the provisions of the Budapest Treaty on International Approval of the Deposit of Microorganisms in Patent Procedures and the rules below it (Budapest Treaty). This guarantees the maintenance of viable cultures for 30 years from the date of deposit. Organisms have been made available by the ATCC under the terms of the Budapest Treaty, Okbio, Inc. Follow the agreement between the ATCC and the ATCC. This guarantees the permanent and unlimited availability of the progeny of the culture at the time of issuance of the relevant US patent, or at the time of publication of either the US patent or the foreign patent application, 35 USC §122 and the Director in accordance therewith. To ensure the availability of descendants to those determined by the Director of the United States Patent and Trademark Office, in accordance with the rules of 886 OG 638, including 37 CFR §1.12, Okbio, Inc. Follow the agreement between the ATCC and the ATCC.

The assignee of this application shall promptly, if the deposited cultures are killed, lost or destroyed when cultured under appropriate conditions, as soon as they are notified that they are viable specimens of the same culture. Agreed to be exchanged. The availability of deposited strains should not be construed as a license to work the invention in violation of the rights granted under government authority in accordance with patent law.

In step 310, a minimal medium containing mineral salts in water is prepared. Also, this step is optional as long as the particular embodiment of Method 300 can begin with a prefabricated minimal medium. Examples of minimal media for chemosynthetic autotrophs include Repasuke media for hydrotrophs and NMS media for metanotrovs. These exemplary minimal media recipes are generally available, for example, through the American Type Culture Collection (ATCC).

In step 315, the minimal medium is added to the bioreactor and in step 320, the minimal medium and the bioreactor are sterilized. The minimal medium can be sterilized, for example, by heat, radiation, or by passing the minimal medium through a sterile filter (eg, a 0.2 μm filter device). In step 315, the minimal medium can include a gel. Also, in step 315, the minimal medium can be added to the bioreactor along with a growth support such as beads.

In step 325, the sterile medium in the bioreactor is inoculated with the inoculum as prepared in step 305. In some embodiments, inoculating the bioreactor with the inoculum comprises continuously introducing differently prepared amounts of different cells.

In step 330, the inoculum is provided by supplying the bioreactor with a gas feedstock containing one or more of _{CO 2} , CO, CH ₄ , H ₂ , H ₂ S, O ₂ , N ₂ , or NH _3. Is fermented and cultured in biomass. When two or more gases are included in the feedstock, some gases are supplied in the appropriate combination and proportion for the cell species to be cultured. Specific examples include _{a mixture of CO 2} , H ₂ , and O _2, a mixture of CH ₄ and O ₂ , and a mixture of CO ₂ and H ₂ S, with biomass cells typically 0.5 per liter. Fermentation is maintained until a threshold density of more than a gram of cell dry weight (CDW / L), preferably more than about 2 grams of CDW / L, is achieved. During step 330, beneficial molecules are secreted into the minimal medium by the growing biomass cells to create a concentrated medium. In a further embodiment, during step 330, additional beneficial molecules can be added to the concentrated medium to further increase its nutritional quality. For example, glucose can be added to make a suitable culture medium for mammalian cells to increase the glucose concentration to acceptable levels for rapid growth. In other cases, the addition of iron or other minerals may be required. Similarly, the pH can be adjusted by adding an acid or base and can include additional inorganic salts, yeast extract, tryptone, phenol red or other ingredients.

In any step 335, the medium further comprises intracellular amino acids, proteins, nucleic acids, polyhydroxyalkanoates, organic acids, and other factors that make the medium more useful for culturing cells of higher organisms. In addition, cells in concentrated medium can be sterilized or processed to sterilize, lyse, or inactivate or destroy cells in a manner that releases their contents.

In step 340, the second medium is collected. In some embodiments, this is achieved by a separation process, such as, for example, centrifugation, filtration, or gravity-based separation to remove biomass. In some embodiments, this treatment may include sterile filtration through a 0.2 μm filter membrane, so that the resulting liquid does not contain any microbial cells. In some embodiments, the second medium recovered in step 340 is modified by the addition of inorganic salts, pH adjustment, filtration, enzymatic treatment, chromatographic separation, selective precipitation, and / or other operations. The second medium can be made more useful by culturing cells of higher organisms. For example, if heterotrophic fermentation is inhibited by high concentrations of this component, it may be advantageous to selectively remove ammonium ions from the second medium, such as by washing steps. The same applies to lactate. In some embodiments, the second medium comprises chemosynthetic autotrophs or other cells from the original inoculum.

The second medium produced by the chemically synthesized independent nutrition methods described herein is more than 1 gram of D-glucose per liter, as well as significant amounts of vitamin B2, vitamin B3, vitamin B12, biotin, pantothenic acid. Contains peptides starting from a first medium that are completely free of acid salts, glutamates, methionines, and glucose, vitamins, amino acids or proteins. Dependent bacterium, yeast (Phafia, Saccharomyces), fungus (Aspergillus), bacterium (Lactobacillus, Bacillus, Bifidobacterium, Brevundimonas, Eschericia) are produced by culturing a chemically synthesized autotrophic bacterium. It can be cultured in a medium. The second medium of the invention can also be used as a basis for, or as a supplement to, a more complex medium preparation for higher organisms.

In any step 345, the second medium is inoculated with cells of higher life form. Cells are cultured in a second medium until the cells are harvested, or the entire culture is harvested, or until some of its components (eg, recombinant proteins) are isolated from the resulting medium. NS. These microorganisms can include bacteria, archaea, microalgae, fungi, molds, yeasts or the like. Examples of such microorganisms include:
Ascomycota, Aspergillus niger, Aspergillus oryzae, Bacillus coagulans, Bacillus lentus, Bacillus licheniformis, Bacillus magaterium, Bacillus pumilus, Bacillus subtilis, Bacteroides amylophilus, Bacteroides capillosus, Bacteroides ruminocola, Bacteroides suis, Basidiomycota, Bifidobacterium adolescentis, Bifidobacterium amimalis, Bifidobacterium bifidum, Bifidobacterium infantis, Bifidobacterium lactis, Bifidobacterium longum, Bifidobacterium thermophilum, Bifidobacterium breve, Saccharomyces cerevisiae, Blaques
is, Kluyveromyces lactis, Hensenula polymorpha, Lactobacillus acidophilus, Lactobacillus brevis, Lactobacillus bulgaricus, Lactobacillus casei, Lactobacillus cellobiosus, Lactobacillus curvatus, Lactobacillus delbruekii, Lactobacillus fermentum, Lactobacillus helveticus, Lactobacillus johnsonii, Lactobacillus lactis, Lactobacillus paracasei, Lactobacillus parafarraginis, Lactobacillus plantarum, Lactobacillus reuterii, Lactobacillus rhamnosus, Lactobacillus salivarius, Lactobacillus sporogenes, Lactobacillus lactis, Leuconostosticocicessocicescence
cerevisiae, Pediococcus pentosaceus, Propionibacterium shermanii, Propionibacterium freudenreichii, Saccharomyces boulardii, Streptococcus cremoris, Streptococcus diacetylactis, Streptococcus faecium, Streptococcus intermedius, Streptococcus lactis, Streptococcus thermophiles.

Example 1: Preparation of a second medium using gas fermentation of chemosynthetic autotrophic bacteria

In this first example, gas fermentation was performed using a New Brunswick BioFlo 4500 30L continuous stirring tank bioreactor modified for gas fermentation. Cupriavidus necator as chemoautotrophic species, Rhodobacter capsulatus, Rhodospirillum rubrum, Rhodobacter sphaeroides, Rhodopseudomonas palustris, heterotrophic Bacillus megaterium as (probiotic) species, Lactobacillus acidophilus, using Lactobacillus casei subsp casei, chemoautotrophic and Inoculum for dependent nutrients was prepared.

To prepare inoculum, bacterial cultures are cultured from frozen stock inoculated into 15 ml sterile Luria Bertani medium and shaken overnight at 200 rpm and 30 ° C. in a temperature controlled incubator or at 620 nm. The cells were grown until the absorbance reached 0.6 absorbance units (au). These were further cultured in chemosynthetic autotrophic growth medium containing less than or equal to 1 liter:
Phosphate from 10x solution (PO ₄ ) 100 mL
Ammonium chloride (NH ₄ Cl) 200 ml from 10x solution
2 ml of _{sodium bicarbonate (NaHCO 3} ) from a 20 g / 200 mL solution
100mM nickel from _{(NH 4) 2 Ni (SO} 4) 2 · 6H 2 O solution 4 0.166 mL
Distilled water (diH ₂ O) 674 ml

After sterilizing the medium, the following sterile inorganic salts were added:
Trace element 2mL from Schlegel Solution'E'
CaCl ₂ · _{2H 2} O 0.1 ml of a solution 5 of 200 g / L
MgSO from 100 times the solution 6 ₄ · _7H 2 O 10 ml
FeSO ₄ · _7H 2 O 12 ml of a solution of 0.1 g / 100 mL

Many of the above components were added from much higher concentration stock solutions. The stock of 10 times phosphate is 40 g of dibasic sodium phosphate (Na ₂ HPO _{4 ) anhydride mixed in 1 L of diH 2} O and monobasic potassium phosphate (KH ₂ PO ₄ ) anhydride 66. Prepared from 7 g. 10 volumes of ammonium chloride stocks were prepared from of _NH 4 Cl 18g mixed in diH ₂ O in 1L. Sodium bicarbonate stock solution was prepared from NaHCO ₃ in 20g mixed in diH ₂ O in 200 mL. For nickel, 100 _{μL of 100 mM NiCl 2} may be added. Calcium chloride can be obtained from 200 g of CaCl ₂ · H ₂ O; 1 L of diH ₂ O; 10,000 times concentrated stock solution. Finally, it is possible by adding appropriate amount from a solution containing MgSO ₄ · 7H ₂ O in 113.05G, preparing a (stock solution concentrated to 100-fold) magnesium sulfate.

The bioreactor was filled with ~ 20 L of medium, then sterilized for 30 minutes using the bioreactor's onboard sterilization cycle, cooled to room temperature, and then the remaining inorganic salts were added. Various components of the consortial inoculum were then added through sterile ports.

Hydrogen gas was supplied to the bioreactor by a 9 kW proton S40 electrolytic cell supplied with ultrapure water. O ₂ and CO ₂ were supplied from a compressed gas cylinder equipped with a gas regulator and reduced to about 20 psi. Gas mixing was controlled by a set of three mass flow controllers according to a ratio of 80:10:10 _{(H 2} : CO ₂ : O _2). The gas flow rate to the bioreactor increased from 1-8 SLPM as the fermentation progressed. The gas head pressure in the bioreactor was 10 psi. A temperature of 30 ° C., a pH of 6.8, and an impeller stirring rate of 300 rpm were maintained.

The bioreactor was run in semi-continuous sampling mode for 32 days. Every 24 hours, 10 L of reactor contents were removed through a sterile port and the same volume of sterile fresh medium was returned to the bioreactor. Bacterial biomass was separated from the concentrated medium by centrifugation and then lyophilized for later use. The aliquots of the remaining supernatant medium were sterilized by filtering through a sterilized disposable 0.2 μm filter and frozen at −20 ° C. to give a “second medium”.

Frozen samples of the second medium from days 11 and 23 of fermentation were subjected to spent medium analysis and the following results were obtained:

The BCA protein assay (Pierce) showed that the sample on day 11 also contained 0.83 g / L of protein. Analysis of protein molecular weight by SDS-PAGE by Coomassie Blue staining (Invitrogen) showed that the majority of proteins consisted of small molecular weight peptides less than about 10 kD (not shown).

Bacterial biomass from each sample was separated from the second medium by centrifugation and lyophilized for later use (approximately 8 g CDW for each liter harvested). A second medium that was already cell-free was collected and stored at 4 ° C.

Example 2: Heterotrophic culture of Lactobacillus brevis in a second medium from day 11 of gas fermentation

Cells in a frozen stock of heterotrophic Lactobacillus brevis were inoculated into 30 ml sterile second medium from day 11 of fermentation as described in Example 1 at a ratio of 1:50. The inoculum was shaken in a 250 ml baffled culture flask in a temperature controlled incubator on a rotary shaker at 28 ° C. at 100 rpm. No additives were added to one flask (“no glucose”), and the other two flasks contained the same second medium as the first flask, but with an additional 0.5% (w / v) glucose. And 1.0% (w / v) glucose, respectively (above the 0.14% level already present in the second medium). Samples were removed at various time points and the optical densities of 200 μL aliquots at 620 nm (A620) were measured with a microplate reader. The growth curve is shown in FIG.

The results in FIG. 4 show that heterotrophic bacteria have their densities 3 on a second medium without glucose supplementation.
Indicates that it has been increased more than double. Additional glucose stimulated their growth above this level, especially in the late stages of 0.5% addition, but 1% glucose may become less effective as the culture period increases. This demonstrates that the second medium can be used to culture heterotrophic cells as either complete medium or significant medium components. This method also allows heterotrophic cells to be indirectly and effectively cultured on gas, thereby utilizing feedstocks and culture conditions in which they are not metabolically suitable. The present invention also makes it possible to extract additional value from the product of gas fermentation beyond the original extracted biomass.

Although the invention is described herein with reference to a particular embodiment thereof, one of ordinary skill in the art will recognize that the invention is not limited thereto. The various features and aspects of the invention described above can be used individually or jointly. Moreover, the invention can be used in any number of environments and uses beyond those described herein, without departing from the broader spirit and scope of the specification. Therefore, the specification and drawings are considered to be exemplary rather than limiting. It will be appreciated that the terms "include", "include", and "have" as used herein are specifically intended to be read as open-ended technical terms.

Claims (32)

Providing the minimum medium to the bioreactor and
Inoculating the minimal medium in the bioreactor with an inoculum containing chemosynthetic autotrophic cells and / or photosynthetic autotrophic cells.
The inoculum is cultivated chemically and autotrophically to grow the biomass in the bioreactor by providing gas uptake into the bioreactor until the cell density of the biomass in the medium meets a threshold. As a result, the minimum medium is concentrated during culturing to become a concentrated medium.
How to include. The method of claim 1, wherein the chemosynthetic autotrophic cell or the photosynthetic autotrophic cell produces a growth factor, hormone, antibiotic, amino acid, peptide, protein, vitamin, colorant, carotinoid, fatty acid, or oil. .. The method of claim 1 or 2, wherein the inoculum also comprises heterotrophic cells. The method of claim 3, wherein the inoculum comprises the photosynthetic autotrophic cells and the culture is carried out in the absence of light in the bioreactor. The method of claim 1-3 or 4, wherein culturing the inoculum comprises adding a beneficial molecule to the concentrated medium. The method of claim 5, wherein the beneficial molecule comprises glucose. The method of claim 1-5 or 6, wherein culturing the inoculum comprises adjusting the pH of the concentrated medium. The method of claim 1-6 or 7, wherein the inoculum comprises chemosynthetic autotrophic cells and the gas input comprises CH ₄ and O _2. The method of claim 8, wherein the chemosynthetic autotrophic cells include cells of Methylococcus capsulatus. The method according to claim 1-6 or 7, wherein the gas input comprises CO. The method of claim 1-6 or 7, wherein the gas injection comprises CO ₂ and H _{2 S.} The method of claim 1-6 or 7, wherein the gas injection comprises CO ₂ , H ₂ and O _2. The method of claim 1-11 or 12, wherein the inoculum comprises cells of Cupriavidus necator, cells of Rhodobacter capsuleus, or both cells. The method according to claim 1-11 or 12, wherein the inoculum comprises carbon monoxide assimilating (carboxydotrov) cells. The method of claim 1-11 or 12, wherein the inoculum comprises cells of Rhodococcus opacus. The method of claim 1-14 or 15, further comprising preparing the inoculum material prior to inoculation with the minimal medium. The method of claim 1-15 or 16, further comprising preparing the minimal medium. The method of claim 1-16 or 17, further comprising sterilizing the minimal medium and the bioreactor prior to inoculating the minimal medium with the inoculum. The method of claim 1-17 or 18, further comprising destroying the cells in the concentrated medium such that the contents of the cells are released into the concentrated medium. 19. The method of claim 19, wherein destroying the cells comprises lysing the cells. The method of claims 1-19 or 20, wherein the concentrated medium comprises one or more of growth factors, hormones, antibiotics, amino acids, peptides, proteins, vitamins, colorants, carotenoids, fatty acids, or oils. The method of claim 1-20 or 21, further comprising separating the insoluble biomass from the concentrated medium. 22. The method of claim 22, wherein the concentrated medium contains at least 1 gram of D-glucose per liter after separation. After separation, the inorganic salt is added to the concentrated medium, the pH of the concentrated medium is adjusted, the concentrated medium is filtered, the concentrated medium is subjected to enzyme treatment, and the concentrated medium is chromatographed. 22 or 23. The method of claim 22 or 23, further comprising one or more of performing separation or selectively precipitating from said concentrated medium. The method of claim 22, 23 or 24, wherein separating the biomass from the concentrated medium comprises centrifugation, filtration, or gravity-based separation. 22-24 or 25. The method of claim 22-24 or 25, further comprising removing ammonium ions from the concentrated medium. 22-25 or 26. The method of claim 22-25 or 26, further comprising culturing the cells of the heterotrophic cells in the concentrated medium separated from the biomass, thereby depleting the concentrated medium. 27. The method of claim 27, wherein the heterotrophic cells include yeast, fungi, algae, archaea, bacteria, or mammals. 27. The method of claim 27, wherein the heterotrophic cells are derived from a multicellular aquatic cell line. The method of claim 1-28 or 29, wherein the minimal medium comprises a gel. The method of claim 1-29 or 30, wherein the minimal medium is added to the bioreactor along with the growth support of the cells. A concentrated medium produced by the method according to any one of claims 1-31.

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