JP7520367B2 - Methods for producing rich cell culture media using chemoautotrophic microorganisms - Google Patents

Description

CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of U.S. Provisional Application No. 62/686,508, filed June 18, 2018, which claims the benefit of U.S. Provisional Application No. 16/443,658, filed June 17, 2019, and is a continuation-in-part of U.S. Application No. 15/641,114, filed July 3, 2017, all three of which are incorporated herein by reference.

FIELD OF THEINVENTION The present invention relates generally 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 the process of growing, propagating, and maintaining cells in a liquid or on a solid or semi-solid substrate such as agar. In the practice of cell culture, the liquid or substance in which the cells are cultured is called the medium. All heterotrophic cells, i.e. all cells other than autotrophic cells, require a medium containing some form of chemical energy and carbon, which can be provided by small molecules such as formate, acetate, and methanol, or more complex and larger molecules such as sugars and starches, and sometimes very complex and large chemicals such as proteins.

Cell culture media can have many different compositions with various components, including inorganic salts, sugars, amino acids, peptides, proteins, polysaccharides, hormones, growth factors, and complex components such as bovine serum, tryptone, and yeast extract. Media compositions containing only water and mineral salts are called "minimal media" and are essentially insufficient for heterotrophic cells because they lack any form of chemical energy and carbon. Minimal media do not contain organic compounds. Media containing organic compounds such as sugars, yeast extract, enzymatically digested proteins, and other energy sources and complex compounds are called "rich media" or "complex media" and complex or rich media are media that essentially contain all the energy, carbon, and other factors required for microorganisms to grow. Proteins are particularly important when culturing cell lines of multicellular organisms such as vertebrates, mollusks, and arthropods.

The following further 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."

"Autotrophic" means "requiring only carbon dioxide or carbonate ( _C1 compounds) as a carbon source and simple inorganic nitrogen compounds for the metabolic synthesis of organic molecules (such as glucose)."

"Chemoautotrophy" is defined as "autotrophic and oxidizing inorganic compounds as an energy source", and in the case of hydrogen oxidizing bacteria, the inorganic compounds as an energy source include _H2 , and they can consume a combination of _CO2 , _H2 , and _O2 . For example, there are anaerobic acetogens that consume _CO2 for carbon and _H2 for energy. Other inorganic energy sources for chemoautotrophs include reduced small molecules such as _H2S , ammonium, or ferrous iron. In some cases, the carbon and energy inputs to chemoautotrophs are combined into a single _C1 molecule. For example, carboxydotrophs and carboxydovores consume CO (carbon monoxide) for both carbon and energy, and methanotrophs consume _CH4 (methane) along with _O2 (molecular oxygen) and other oxygen-donating compounds. Chemoautotrophic metabolism is known in bacteria and archaea, and may exist in other organisms either as undiscovered traits or as an ability conferred by genetic modification. Examples of chemoautotrophs are found in numerous genera of bacteria such as Cupriavidus, Rhodobacter, Methylobacterium, Methylococcus, Methylosinus, Nitrosomonas, Nitrosococcus, Nitrobacter, Nitrococcus, Paracoccus, Hydrogenothermus, Hydrogenovibrio, Clostridium, Rhodococcus, Rhodospirillum, Alcaligines, Rhodopseudomonas, and Thiobacillus, as well as many genera of archaea, including the methanogens. Specific examples of chemoautotrophic organisms include Cupriavidus necator, Cupriavidus basilensis, Rhodococcus opacus, Methylococcus capsulatus, Methylosinus trichospori um, Methylobacterium extorquens, Hydrogenothermus marinus, Rhodospirillium rubrum, Rhodopseudomonas palustrus, Paracoccus zea xanthinefaciens, Rhodobacter sphaeroides, Rhodobacter capsulatus, and Clostridium autoethanogenum.

"Fermentation" is defined as "a metabolic process that produces chemical changes in organic substrates by the action of enzymes" and in biochemistry is narrowly defined as the extraction of energy from carbohydrates in the absence of oxygen. In the context of food production, it is more broadly referred to as any process in which microbial activity produces a desired change in a food or beverage. Even broader, for the purposes of this invention, "fermentation" is a process for cultivating cells in specialized vessels (made of glass, metal or plastic and known as fermenters or bioreactors) under controlled process conditions to optimize their growth and maximize efficiency. Controlled process conditions include sterility, temperature, agitation speed, pH, input gas composition and flow rate, nutrient content, cell density, dissolved gas concentrations, biomass removal rate (for continuous or semi-continuous harvesting), etc. In the latter case, fermentation can be aerobic or anaerobic.

"Gas fermentation" refers to fermentation in a bioreactor in which the metabolic processes of chemoautotrophic cells extract energy and carbon from gas inputs fed to them. Gas fermentation refers to the anaerobic or aerobic process of culturing microorganisms on gas. By combining these gas inputs with simple inorganic salts in the medium, the chemoautotrophic cells convert these basic inputs into more complex biomass and other cellular products. Gas fermentation can be either aerobic or anaerobic, depending on the microorganism used and the feed gas available for fermentation. Gas fermentation is a particularly advantageous form of chemoautotrophic fermentation because the major input is provided by widely available, low-cost gas.

"Culturing" means "the act or method of culturing organisms (bacteria, viruses, etc.) in a prepared nutrient medium," "nutrient" means "substances or components that promote growth, provide energy, and sustain life," and "medium" means "a nutrient system for the artificial cultivation of cells or organisms, especially bacteria." A medium can be liquid, semi-solid, or solid (e.g., agar, beads, or other scaffolding). A solid or semi-solid medium can provide a support for the growth of cells.

It should be noted that in a typical heterotrophic fermentation, cells are grown in a medium containing complex organic molecules such as sugars, amino acids, peptides, organic acids, etc. Since heterotrophic cells generally extract most of the "energy-rich" forms of carbon from the medium, increasing the cellular biomass and releasing the dissimilated carbon as carbon dioxide, carbon acetate, or other simple, low-value waste products, the medium remaining after the biomass is harvested from the bioreactor after the heterotrophic fermentation process is usually called "spent medium" since it generally has very low nutritional value for further culturing of the heterotrophs. Even when heterotrophs are selected or engineered to excrete high-value products into the medium, they nevertheless must be cultured on a fairly high-cost medium (containing complex molecules such as sugars or proteins), thus limiting the profitability of the production process.

In chemoautotrophic fermentation, cells grow in a similar manner and produce biomass; however, the highly anabolic metabolism of chemoautotrophs results in the production of excess nutritionally valuable products, some of which leak out or are excreted into the medium.

"Higher life forms" or "higher organisms" refers to eukaryotic organisms such as yeasts, fungi, microalgae, plants, and animals.

A significant expense in the commercial cultivation and maintenance of cells, especially those isolated from multicellular organisms such as plants, fish, mollusks, and arthropods, is the cost of the growth media. Such media often contain, in addition to water and various inorganic salts, many different peptide growth factors, amino acids, sugars, yeast extracts, protein digests of animal or plant origin, serum of animal origin, proteins (e.g., tryptone and peptone, or albumins such as bovine serum albumin), and other metabolic products important for cell growth. A significant portion of the high production costs of these media is the use of materials processed from animal materials, such as 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 eight B-vitamins and ten essential amino acids plus cystine, tyrosine, and glutamine.

FIG. 1 is a schematic diagram of an exemplary embodiment of a system in accordance with various embodiments of the present invention. FIG. 2 is a schematic diagram of an exemplary bioreactor, according to various embodiments of the present invention. FIG. 3 is a flowchart representation of methods according to various embodiments of the present invention. FIG. 4 is a graph of experimental results showing the cultivation of Lactobacillus brevis in a medium produced by an exemplary method of the present invention.

The present invention describes the production of a nutrient-rich medium suitable for culturing heterotrophic cells from exhaust gases. The method described herein uses carbon in industrial waste as the base of a food chain that starts by fermenting photoautotrophic or chemoautotrophic microorganisms under chemoautotrophic conditions on the carbon and in a medium that is initially free of organic nutrients. The microorganisms grow to convert this carbon into larger biomass, and under appropriate conditions can also convert some of the carbon into organic nutrients as waste by-products, which can enrich the medium to make it suitable for culturing heterotrophic cells further up the food chain.

An exemplary method of the invention includes providing a minimal medium to a bioreactor, inoculating the minimal medium in the bioreactor with an inoculum comprising chemoautotrophic and/or photoautotrophic cells, and cultivating the inoculum chemoautotrophically to grow a biomass in the bioreactor by providing a gas input into the bioreactor until the cell density of the biomass in the medium meets a threshold, whereby during cultivation the minimal medium is enriched to become a concentrated medium. In various embodiments, the method further includes preparing an inoculum prior to inoculating the minimal medium. Various methods may further include preparing a minimal medium. Further embodiments include sterilizing the minimal medium and the bioreactor prior to inoculating the minimal medium with the inoculum. In yet further embodiments, the minimal medium includes a gel. In various embodiments, the minimal medium is added to the bioreactor along with a growth support for the cells.

In various embodiments, the chemoautotrophic or photoautotrophic cells produce growth factors, hormones, antibiotics, amino acids, peptides, proteins, vitamins, colorants, carotenoids, fatty acids, or oils. In various embodiments, the inoculum also includes heterotrophic cells. The inoculum can also include photoautotrophic cells, and in these embodiments, the culture is performed in the absence of light in the bioreactor.

In various embodiments, culturing the inoculum includes adding a beneficial molecule to the enriched medium, and in some of these embodiments, the beneficial molecule includes glucose. Culturing the inoculum includes, in some aspects, adjusting the pH of the enriched medium. In some cases, the inoculum includes chemoautotrophic cells and the gas input includes _CH4 and _O2 . In some of these embodiments, the chemoautotrophic cells include Methylococcus capsulatus cells. In various embodiments, the gas input includes CO, the gas input includes _CO2 and _H2S , and the gas input includes _CO2 , _H2 , and _O2 . In still other embodiments, the inoculum includes Cupriavidus necator cells, Rhodobacter capsulatus cells, or both. The inoculum can, in various embodiments, include cells of carboxydotrophs or cells of Rhodococcus opacus.

Various embodiments of the invention further include disrupting the cells in the enriched medium to release their contents into the enriched medium. Disrupting the cells can optionally include lysing the cells. In various embodiments, the enriched medium includes one or more of a growth factor, a hormone, an antibiotic, an amino acid, a peptide, a protein, a vitamin, a colorant, a carotenoid, a fatty acid, or an oil.

In various embodiments, the method further comprises separating the insoluble biomass from the enrichment medium by methods such as centrifugation, filtration, or gravity-based separation. In some of these aspects, the enrichment medium after separation comprises at least 1 gram of D-glucose per liter. In some embodiments, the method further comprises one or more of adding mineral salts to the enrichment medium after separation, adjusting the pH of the enrichment medium, filtering, providing an enzyme treatment to the enrichment medium, performing a chromatographic separation on the enrichment medium, or performing selective precipitation from the enrichment medium. In the methods of separating the insoluble biomass from the enrichment medium, some methods further comprise removing ammonium ions from the enrichment medium.

In various embodiments, the method further comprises culturing cells of a heterotroph in an enrichment medium separated from the biomass, thereby depleting the enrichment medium. In some of these methods, the heterotroph comprises a yeast, a fungus, an algae, an archaea, a bacterium, or a mammal. The heterotrophic cells are, in further embodiments, derived from a cell line of a multicellular aquatic organism.

Additionally, the present invention is directed to various enriched media produced by the methods described herein.

The present invention describes the production of a nutrient-rich medium suitable for cultivating heterotrophic cells from waste gas. The production of such a medium involves initially cultivating chemoautotrophic and/or photoautotrophic cells chemoautotrophically via gas fermentation in a minimal medium, followed by harvesting such medium in abundance after sufficient cultivation. The concentrated medium can then be used for cultivating heterotrophic organisms such as yeasts, fungi, microalgae, plants and animals. The cells cultured in the bioreactor may comprise a single species or strain of chemoautotrophic or photoautotrophic microorganisms, or they may comprise multiple strains or species. Furthermore, these cells may be co-cultured with one or more species or strains of various selected heterotrophic microorganisms, the purpose of which is to supply additional desired nutritional components (such as probiotics) to the medium that are not produced by the chemoautotrophic or photoautotrophic cells alone. The co-culture or consortium with heterotrophic cells can be designed to provide added value to the end product than is consumed by the heterotrophic cells. In some embodiments, the intracellular contents of the resulting biomass can be added to the enrichment medium.

Here, the "first medium" is the initial medium used to provide basic nutrients for the initial fermentation. The "second medium" as used herein is the supernatant enriched medium obtained from the enrichment of the first medium, remaining after separation from all or most of the biomass. Thus, during chemoautotrophic fermentation, the cells grow and also produce highly assimilable products. The medium remaining after enrichment from chemoautotrophic fermentation is called the "second medium" to distinguish it from the nutritionally poor "spent medium" obtained from heterotrophic fermentation. After most or all of the biomass has been removed, the second medium contains many complex substances suitable for supporting the growth of heterotrophic cells. This second medium can therefore be collected during or after fermentation, treated (if desired) to remove any remaining cells or cell debris, and reused as a nutritionally favorable growth medium or additive for the cultivation of heterotrophic cells. As used herein, the "enriched medium" refers to the first medium after gas fermentation has begun, and broadly encompasses both the medium during the cultivation and the second medium after the separation process.

Chemoautotrophic and photoautotrophic microorganisms cultivated on industrial waste gases can be a particularly rich and valuable source of nutrients, since they must produce all of their cellular components (including sugars, fatty acids, carotenoids, cofactors, vitamins, peptides and proteins) de novo from simple and generally inexpensive inputs (e.g., hydrogen, carbon dioxide, oxygen, water and inorganic salts). This chemoautotrophic mode of production also has the advantage that vitamins and proteins can be synthesized at lower cost than, for example, by heterotrophic fermentation on sugars.

The second medium produced according to the present invention may contain similar or identical ingredients compared to the rich medium of the prior art and may provide comparable nutritional value, thus supplementing or completely replacing animal-derived and other expensive ingredients for various cell culture applications. In some cases, this not only reduces production costs, but also provides a source of culture medium that does not require killing or harming animals. In other cases, the ingredients available in the medium may provide advantages not found in known rich media, and thus the product of this method is itself novel over the prior art. The medium may be entirely liquid, or may be formulated into a gel (e.g., using agar) or may contain solid or semi-solid materials (e.g., beads) for use as a growth support for cells.

It is important to note that typical prior art growth media for heterotrophic organisms have an initial composition designed to be depleted as the growing cells are cultured. In accordance with the present invention, fermentation of chemoautotrophic cells is initiated in a minimal medium. The chemoautotrophic growth of cells supplying mineral salts and feed gas, as well as the input of feed gas combined with the growth of any additional heterotrophic cells that may be included as part of the consortium and the supply of products from the chemoautotrophic growth, actually builds up the nutritional qualities of the enriched medium as the culture progresses.

Figure 1 shows a schematic diagram of an exemplary system 100 of the present invention. The system 100 includes a bioreactor 110 containing photoautotrophic and/or chemoautotrophic cells 120. The 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 sources 130 include cement manufacturing facilities, power plants burning fossil fuels, ferrous metal product manufacturing (e.g., casting and forging), non-ferrous product manufacturing, food manufacturing, fermentation plants producing ethanol or other bioproducts, biomass gasification, coal gasification, and chemical manufacturing such as petroleum refining, carbon black production, ammonia production, methanol production, and coke production.

The system 100 further comprises an optional molecular hydrogen source 150, such as a hydrogen storage tank, a hydrogen pipeline, a steam methane reformer, a gasifier, or an electrolysis system. The hydrogen source 150 produces a hydrogen stream 160 containing molecular hydrogen as an energy source for chemoautotrophic or photoautotrophic cells grown chemoautotrophically, i.e., in the absence of light. In various embodiments of the invention, the cells in the bioreactor grown chemoautotrophically derive both carbon and energy from one waste stream 140, such as methane or carbon monoxide, and in these embodiments, the molecular hydrogen source 150 is not required. In further embodiments, the waste stream 140 includes a carbon source and a hydrogen source, such as may be produced by a gasifier. FIG. 1 also shows diagrammatically that the two output streams of the 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 the second carbon source 130.

2 shows a schematic diagram of a bioreactor 200 as an example of a suitable bioreactor for the method of the present invention. The bioreactor 200 can include either a synthetic vessel for the production of cells for inoculating a first medium and a synthetic vessel for use in conjunction with a separate growth vessel for the production of biomass in a concentrated medium, or the bioreactor 200 can include vessels suitable for both the synthesis and growth stages. In FIG. 2, the bioreactor 200 includes a vessel 205 that holds a volume of liquid medium 210 containing chemoautotrophic or photoautotrophic cells and optionally other heterotrophic cells in culture during operation. The bioreactor 200 also includes an inlet 215 through which a gas 220 can be introduced into the vessel 205 for introduction into the medium 210, a medium inlet 225 through which fresh medium 230 can be introduced into the vessel 205, and a medium outlet 235 through which the medium 210 can be removed, for example to separate the concentrated medium from the insoluble biomass. Bioreactor 200 can also include a headspace 240 and a gas release valve 245 for venting gas from headspace 240. In some embodiments, gas release valve 245 is attached to a recirculation system to return vented gas to input port 215 and can include a manifold (not shown) for additions to optimize gas composition.

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

Figure 3 illustrates an exemplary method 300 of the present invention. Some steps are listed as optional, while steps not listed as optional are not necessarily required. Method 300 includes optional step 305 of preparing an inoculum and optional step 310 of preparing a minimal medium. Method 300 then includes step 315 of adding the minimal medium to a bioreactor and optional step 320 of sterilizing the minimal medium and the bioreactor. Method 300 then includes step 325 of inoculating the sterilized minimal medium with the inoculum by supplying gas to the bioreactor, and step 330 of fermenting until the cell density meets a threshold. Method 300 then includes optional step 335 of releasing the contents of the cells into an enrichment medium, and then optional step 340 of separating the biomass from the second medium. In optional step 345, the second medium is inoculated with heterotrophic cells of the type that the second medium is designed to support.

In step 305, an inoculum sufficient to inoculate a bioreactor, such as bioreactor 200, is prepared. This step is optional, as long as certain embodiments of method 300 can start with a pre-made inoculum. The inoculum contains cells of at least one species of photoautotrophic or chemoautotrophic microorganisms and can be prepared using either a rich or minimal medium with a gas feedstock. The particular species of microorganism selected for the inoculum, and any other heterotrophic microorganisms contained therein, are selected to produce an appropriate second medium tailored for the benefit of subsequently culturing a particular higher organism in the second medium, such as bacteria, archaea, microalgae, fungi, molds, or yeasts. Preparing the inoculum can include co-cultivating chemoautotrophic, photoautotrophic, and/or heterotrophic cells together in the same medium or with one or more strains in separate media. In the latter case, preparing the inoculum can include preparing quantities of chemoautotrophic cells, photoautotrophic cells, and/or heterotrophic cells at different times and storing those quantities until ready for all use.

In some embodiments, the chemoautotrophic microorganism comprises a single chemoautotrophic cell line such as Cupriavidus necastor, Methylococcus capsulatus, Rhodobacter capsulatus, or Rhodococcus opacus, or a chemoautotroph that has been genetically modified to produce a beneficial heterologous product. Chemoautotrophic strains may be naturally occurring, contain mutations, be genetically modified, or contain one or more genes that have been edited via CRISPR, and may be for producing valuable small molecules, growth factors, hormones, antibiotics, amino acids, peptides, sugars, polysaccharides, proteins, vitamins, colorants, carotenoids, fatty acids, organic acids, nucleic acids, oils, glycolic acid, hydrocarbons, polyhydroxyalkanoates, phasins, gene transfer agents (GTAs), or other biomolecules that can be utilized as growth substrates and growth regulators by non-autotrophic microorganisms and other heterotrophic cells. Examples of photoautotrophic microorganisms that can be used in this way include cyanobacteria such as Rhodospirillium rubrum, Rhodopseudomonas palustrus, Paracoccus zeaxanthinifaciens, Rhodobacter sphaeroides, Rhodobacter capsulatus, spirulina, and anabaena.

Deposit of Biological Materials The following microorganisms are deposited at the American Type Culture Collection, 10801 University Boulevard, Manassas, USA.
Deposited in the USA (ATCC) under the accession number VA 20110-2209:
Table 1:
Microorganism designation: 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 the International Recognition of the Deposit of Microorganisms for the Purposes of Patent Procedure and the Regulations Thereunder (Budapest Treaty), which guarantees maintenance of a viable culture for 30 years from the date of deposit. The organisms will be made available by the ATCC under the terms of the Budapest Treaty, and are subject to an agreement between Oakbio, Inc. and the ATCC, which guarantees perpetual and unlimited availability of the progeny of the culture upon issuance of the relevant U.S. patent, or upon publication of either a U.S. patent or foreign patent application, and guarantees availability of the progeny to such extent as may be determined by the U.S. Commissioner for Patents and Trademarks pursuant to 35 U.S.C. § 122 and the Commissioner's regulations thereunder (including 37 CFR § 1.12, with particular reference to 886 OG 638).

The assignee of this application has agreed that if the deposited cultures die or are lost or destroyed when cultured under appropriate conditions, they will be promptly replaced upon notice with viable specimens of the same cultures. The availability of the deposited strains shall not be construed as a license to practice the invention in violation of rights granted under government authority pursuant to the Patent Act.

In step 310, a minimal medium is prepared that includes mineral salts in water. Again, this step is optional insofar as certain embodiments of method 300 can begin with a pre-made minimal medium. Examples of minimal media for chemoautotrophs include Repaske medium for hydrogenotrophs and NMS medium for methanotrophs. Recipes for these exemplary minimal media are publicly available, for example, through the American Type Culture Collection (ATCC).

In step 315, minimal medium is added to the bioreactor, and in step 320, the minimal medium and the bioreactor are sterilized. The minimal medium may be sterilized, for example, by heat, radiation, or by passing the minimal medium through a sterile filter (e.g., a 0.2 μm filter device). In step 315, the minimal medium may include a gel. Also, in step 315, the minimal medium may 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 an inoculum, such as that prepared in step 305. In some embodiments, inoculating the bioreactor with an inoculum includes sequentially introducing separately prepared amounts of different cells.

In step 330, the inoculum is fermented and cultivated into biomass by feeding a gas feedstock containing one or more of _CO2 , CO, _CH4 , _H2 , _H2S , _O2 , _N2 , or _NH3 to the bioreactor. When more than one gas is included in the feedstock, the several gases are fed in a combination and ratio appropriate for the species of cells being cultivated. Specific examples include a mixture of _CO2 , _H2 , and _O2 , a mixture of _CH4 and _O2 , and a mixture of _CO2 and _H2S , and fermentation is maintained until the cells of the biomass reach a threshold density, usually greater than 0.5 grams cell dry weight per liter (CDW/L), preferably greater than about 2 grams CDW/L. During step 330, beneficial molecules are secreted into the minimal medium by the cells of the growing biomass, creating an enriched medium. In further embodiments, additional beneficial molecules can be added to the enriched medium during step 330 to further increase its nutritional quality. For example, to make the culture medium suitable for mammalian cells, glucose can be added to increase the concentration of glucose to an acceptable level for rapid growth. In other cases, the addition of iron or other minerals may be required. Similarly, the pH can be adjusted by adding acids or bases, and additional inorganic salts, yeast extract, tryptone, phenol red, or other ingredients can be included.

In optional step 335, the medium may be sterilized or treated to kill, lyse, or inactivate or destroy the cells in the enriched medium in such a manner as to release their contents so that the enriched medium further contains 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 step 340, the second medium is harvested. In some embodiments, this is accomplished by a separation process, such as, for example, centrifugation, filtration, or gravity-based separation to remove biomass. In some embodiments, this process may include sterile filtration through a 0.2 μm filter membrane, so that the resulting liquid is free of any microbial cells. In some embodiments, the second medium recovered in step 340 may be modified by addition of inorganic salts, adjustment of pH, filtration, enzymatic treatment, chromatographic separation, selective precipitation, and/or other manipulations to make the second medium more useful for culturing cells of higher organisms. For example, it may be advantageous to selectively remove ammonium ions from the second medium, such as with a washing process, if heterotrophic fermentation is inhibited by high concentrations of this component. The same is true for lactate. In some embodiments, the second medium contains chemoautotrophic organisms or other cells from the original inoculum.

The second medium produced by the chemoautotrophic method described herein contains more than 1 gram of D-glucose per liter, as well as significant amounts of vitamin B2, vitamin B3, vitamin B12, biotin, pantothenate, glutamate, methionine, and peptides starting from a first medium that does not contain any glucose, vitamins, amino acids, or proteins. Heterotrophic bacteria, yeasts (Phaffia, Saccharomyces), fungi (Aspergillus), bacteria (Lactobacillus, Bacillus, Bifidobacterium, Brevundimonas, Escherichia) can be cultivated in the unimproved second medium produced by culturing chemoautotrophic bacteria. The second medium of the present invention can also be used as a basis for, or as a supplement to, more complex medium preparations for higher organisms.

In an optional step 345, the second medium is inoculated with cells of a higher life form. The cells are cultured in the second medium until the cells are harvested, or until the entire culture is harvested, or some component thereof (e.g., recombinant protein) is isolated from the resulting medium. These microorganisms may include bacteria, archaea, microalgae, fungi, molds, yeasts, or others. Examples of such microorganisms include:
Ascomycota, Aspergillus niger, Aspergillus oryzae, Bacillus coagulans, Bacillus lentus, Bacillus licheniformis, Bacillus magat erium, 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, Bl akeslea trispora, Pichia pastor
is, Kluyveromyces lactis, Hensenula polymorpha, Lactobacillus acidophilus, Lactobacillus brevis, Lactobacillus bulgaricus, Lacto bacillus casei, Lactobacillus cellobiosus, Lactobacillus curvatus, Lactobacillus delbruekii, Lactobacillus fermentum, Lactobacillus us helveticus, Lactobacillus johnsonii, Lactobacillus lactis, Lactobacillus paracasei, Lactobacillus parafarraginis, Lactobacillus us plantarum, Lactobacillus reuterii, Lactobacillus rhamnosus, Lactobacillus salivarius, Lactobacillus sporogenes, Lactococcus la ctis, Leuconostoc mesenteroides, Pediococcus acidilactici, Pediococcus
cerevisiae, Pediococcus pentosaceus, Propionibacterium shermanii, Propionibacterium freudenreichii, Saccharomyces boulardii, St. Reptococcus cremoris, Streptococcus diacetylactis, Streptococcus faecium, Streptococcus intermedius, Streptococcus lactis, Streptococcus ococcus thermophiles.

Example 1: Production of a second medium using gas fermentation with chemoautotrophic bacteria

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

To prepare the inoculum, bacterial cultures were grown from frozen stocks inoculated into 15 ml of sterile Luria Bertani medium and grown in a temperature-controlled incubator with shaking at 200 rpm and 30° C. overnight or until the cultures reached an absorbance of 0.6 absorbance units (au) at 620 nm. These were further cultured in a chemically synthesized autotrophic growth medium containing the following per liter:
Phosphate ( _PO4 ) from 10x solution 100mL
Ammonium chloride ( _NH4Cl ) from 10x solution 200ml
Sodium bicarbonate (NaHCO ₃ ) from a 20 g/200 mL solution 2 ml
Nickel from 100 mM ( _NH4 ) _2Ni ( _SO4 ) _2.6H2O solution 4 _0.166 ml
Distilled water ( _diH2O ) 674 ml

After the medium was sterilized, the following sterile inorganic salts were added:
Trace elements from Schlegel Solution 'E' 2mL
0.1 ml of CaCl _{2.2H 2} O from solution 5 at 200 g/L
10 ml of MgSO _{4.7H 2} O from 100x solution 6
12 ml _{of FeSO4.7H2O} from a 0.1 g/100 mL solution

Many of the above ingredients were added from much more concentrated stock solutions. Phosphate 10X stock was prepared from _{40 g of dibasic sodium phosphate (Na2HPO4) anhydrous and 66.7 g of monobasic potassium phosphate (KH2PO4 ) anhydrous mixed} in 1 L of _diH2O . Ammonium chloride 10X stock was prepared from 18 g of _NH4Cl mixed in 1 L of _diH2O . Sodium bicarbonate stock solution was prepared from ₂₀ g of NaHCO3 mixed in 200 mL of _diH2O . Nickel may be added as 100 _μL of 100 mM _NiCl2 . Calcium chloride can be obtained from 200 g of _CaCl2.H2O ; 1 L of _diH2O ; 10,000x concentrated stock solution. Finally, magnesium sulfate (100x concentrated stock solution) can be prepared by adding an appropriate amount from a solution containing _113.05 g _MgSO4.7H2O .

The bioreactor was filled with ~20 L of medium, then sterilized for 30 min using the bioreactor's on-board sterilization cycle, cooled to room temperature, and the remaining inorganic salts were then added. The various components of the consortial inoculum were then added through the sterilization port.

Hydrogen gas was supplied to the bioreactor by a 9 kW Proton S40 electrolyzer fed with ultrapure water. _O2 and _CO2 were supplied from compressed gas cylinders equipped with gas regulators and depressurized to approximately 20 psi. The gas mixture was controlled by a set of three mass flow controllers according to a ratio of 80:10:10 ( _H2 : _CO2 : _O2 ). The gas flow rate to the bioreactor was 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 agitation speed of 300 rpm were maintained.

The bioreactor was operated in semi-continuous harvest 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. The bacterial biomass was separated from the concentrated medium by centrifugation and then lyophilized for later use. An aliquot of the remaining supernatant medium was sterilized by filtration through a sterile disposable 0.2 μm filtration device and frozen at −20°C to obtain the “second medium”.

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

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

The 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). The second medium, now cell-free, was collected and stored at 4°C.

Example 2: Heterotrophic cultivation of Lactobacillus brevis in the second medium from the 11th day of gas fermentation

Frozen stock cells of heterotrophic Lactobacillus brevis were inoculated at a 1:50 ratio into 30 ml of sterile second medium from day 11 of the fermentation described in Example 1. The inoculum was shaken at 100 rpm at 28° C. on a rotary shaker in a temperature-controlled incubator in a 250 ml baffled culture flask. One flask contained no additives ("No Glucose"), while the other two flasks contained the same second medium as the first flask, but with additional 0.5% (w/v) and 1.0% (w/v) glucose, respectively (above the level of 0.14% already present in the second medium). Samples were removed at various time points, and the optical density at 620 nm (A620) of 200 μL aliquots was measured with a microplate reader. The growth curves are shown in FIG. 4.

The results in FIG. 4 show that heterotrophic bacteria increased their density by 3.5% on the second medium without glucose supplementation.
The results show that the fermentation yield increased by more than 2-fold. Additional glucose stimulated their growth above this level, especially at the later stages of 0.5% addition, but 1% glucose may be less effective as the culture period increases. This demonstrates that a second medium can be used to culture heterotrophic cells, either as a complete medium or as a significant medium component. This method also allows heterotrophic cells to be effectively cultured indirectly on gas, thereby utilizing feedstocks and culture conditions for which they are metabolically unsuited. The invention also allows for the extraction of additional value from the products of gas fermentation beyond the original extracted biomass.

In the foregoing specification, the invention has been described with reference to specific embodiments thereof, but those skilled in the art will recognize that the invention is not limited thereto. The various features and aspects of the invention described above may be used individually or jointly. Moreover, the invention may be utilized in any number of environments and applications beyond those described herein without departing from the broader spirit and scope of the specification. The specification and drawings are therefore to be regarded as illustrative rather than restrictive. It will be recognized that the terms "comprises", "including" and "having" as used herein are specifically intended to be read as open-ended terms of art.

Claims (22)

providing a medium free of organic compounds to a bioreactor;
inoculating the medium in the bioreactor with an inoculum comprising chemoautotrophic cells;
chemoautotrophically cultivating an inoculum to grow a biomass in the bioreactor by providing a gas input to the bioreactor until a cell density of the biomass in the medium meets a threshold value, thereby concentrating the medium during cultivation to become a concentrated medium;
Including,
the inoculum also comprises heterotrophic cells;
the gas input comprises _CH4 and _O2 ;
The method for obtaining said enriched medium, wherein said chemoautotrophic cells comprise Methylococcus capsulatus cells, Cupriavidus necator cells, Rhodobacter capsulatus cells, and Rhodococcus opacus cells. The method of claim 1, wherein the chemoautotrophic cells produce a growth factor, a hormone, an antibiotic, an amino acid, a peptide, a protein, a vitamin, a colorant, a carotenoid, a fatty acid, or an oil. The method of claim 1 or 2, wherein culturing the inoculum comprises adding a beneficial molecule to the enrichment medium. The method of claim 3, wherein the beneficial molecule comprises glucose. The method of claims 1 to 3 or 4, wherein growing biomass in the bioreactor includes adjusting the pH of the medium. 5. The method according to claim 1, wherein the inoculum comprises carbon monoxide-utilizing (carboxydotrophic) cells.
Or the method according to item 5. The method according to claim 1 to 5 or 6, further comprising preparing the inoculum prior to inoculating the medium. The method according to claim 1 to 6 or 7, further comprising preparing the medium. The method of claim 1 to 7 or 8, further comprising sterilizing the medium and the bioreactor before inoculating the medium with the inoculum. The method of claims 1 to 8 or 9, further comprising disrupting cells in the enrichment medium to release the contents of the cells into the enrichment medium. The method of claim 10, wherein destroying the cells comprises lysing the cells. The method of claims 1 to 10 or 11, wherein the enriched medium contains one or more of a growth factor, a hormone, an antibiotic, an amino acid, a peptide, a protein, a vitamin, a colorant, a carotenoid, a fatty acid, or an oil. The method of claims 1 to 11 or 12, further comprising separating insoluble biomass from the enrichment medium. The method of claim 13, wherein the enriched medium contains at least 1 gram of D-glucose per liter after separation. The method of claim 13 or 14, further comprising one or more of the following steps after separation: adding inorganic salts to the enrichment medium, adjusting the pH of the enrichment medium, filtering the enrichment medium, subjecting the enrichment medium to an enzymatic treatment, performing a chromatographic separation on the enrichment medium, or selectively precipitating from the enrichment medium. The method of claim 13, 14 or 15, wherein separating the biomass from the enrichment medium comprises centrifugation, filtration, or gravity-based separation. The method according to claims 13 to 15 or 16, further comprising removing ammonium ions from the concentrated medium. The method of claims 13 to 16 or 17, further comprising culturing cells of heterotrophic cells in the enrichment medium separated from the biomass, thereby depleting the enrichment medium. The method of claim 18, wherein the heterotrophic cells comprise yeast, fungi, algae, archaea, bacteria, or mammals. The method of claim 18, wherein the heterotrophic cells are derived from a cell lineage of a multicellular aquatic organism. The method of claim 1 , wherein the medium comprises a gel. The method of claims 1 to 20 or 21, wherein the culture medium is added to the bioreactor together with a growth support for the cells.

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