阅读说明：本技术 用于在好氧生物合成期间控制氧浓度的方法 (Method for controlling oxygen concentration during aerobic biosynthesis ) 是由 加里·J·史密斯 保罗·S·珀尔曼 格雷戈里·S·柯比 于 2019-04-01 设计创作，主要内容包括：本公开提供了用于在好氧生物合成(例如发酵)期间控制氧浓度的方法。所述方法可包括将含氧气体进料到包含发酵原料的容器中,以及使所述发酵原料与所述含氧气体反应以形成发酵液,所述发酵液包含分散在所述发酵液内的气相。所述气相可包含来自所述含氧气体的任何未反应的氧气。所述方法还包括将分散气相中的未反应的氧气的浓度降低至小于易燃性的极限氧浓度(“LOC”),然后将所述气相从所述发酵液中分离。所述气相中的未反应的氧气的浓度通过采用氧去除方案或氧稀释方案来降低。(The present disclosure provides methods for controlling oxygen concentration during aerobic biosynthesis (e.g., fermentation). The method can include feeding an oxygen-containing gas into a vessel containing a fermentation feedstock, and reacting the fermentation feedstock with the oxygen-containing gas to form a fermentation broth comprising a gas phase dispersed within the fermentation broth. The gas phase may comprise any unreacted oxygen from the oxygen-containing gas. The process further comprises reducing the concentration of unreacted oxygen in the dispersed gas phase to less than the limiting oxygen concentration of flammability ("LOC"), and then separating the gas phase from the fermentation broth. The concentration of unreacted oxygen in the gas phase is reduced by employing an oxygen removal scheme or an oxygen dilution scheme.)

1. A method for controlling oxygen concentration during aerobic biosynthesis, the method comprising:

feeding an oxygen-containing gas to a bioreactor containing microorganisms, wherein a combustible gas fermentation feedstock component is within the bioreactor;

reacting at least a portion of the oxygen from the oxygen-containing gas with the microorganisms;

forming a fermentation broth comprising a gas phase dispersed within the fermentation broth, the gas phase comprising unreacted oxygen from the oxygen-containing gas;

reducing the concentration of the unreacted oxygen in the gas phase to less than the limiting oxygen concentration of flammability (LOC) of the combustible gas feed component, wherein the reducing comprises diluting the unreacted oxygen with a diluent; and

separating the gas phase from the fermentation broth.

2. The method of claim 1, wherein the diluent comprises a gas stream comprising one or more of nitrogen, carbon dioxide, and hydrogen.

3. The method of claim 1 or 2, wherein the diluent comprises less than 5% oxygen by volume.

4. The process of any of claims 1-3, wherein the oxygen-containing gas comprises greater than 21 vol% oxygen.

5. The method of any of claims 1-4, wherein the oxygen-containing gas comprises air.

6. The process of any one of claims 1-5, wherein the gas phase separated from the fermentation broth comprises oxygen at a concentration of less than 85% of the LOC.

7. The process of any of claims 1-6, wherein the gas phase separated from the fermentation broth comprises less than 6% oxygen by volume.

8. The method of any one of claims 1-7, wherein the step of reducing the concentration of unreacted oxygen occurs prior to the step of separating the gas phase from the fermentation broth.

9. The method of any one of claims 1-8, wherein the microorganism comprises cuprinophilus hookeri (C.necator) or cuprinobulinus metallothioneine (C.metallidurans).

10. The method of any one of claims 1-9, wherein the bioreactor is selected from the group consisting of: single fermenter, multiple fermenters in series, stirred tank fermenters, non-stirred tank fermenters, membrane fermenters, fixed bed fermenters, fluidized bed fermenters, single autoclaves, multiple autoclaves in series, plug flow fermenters, pneumatically stirred fermenters, airlift fermenters with external circuit with forced circulation, bubble column fermenters, fixed (packed) bed tower fermenters, horizontal single fermenters with multiple compartments, and multistage tower fermenters.

11. The method of any one of claims 1-10, wherein the separating comprises separating the gas phase from the fermentation broth into a headspace of the bioreactor.

12. The method of any one of claims 1-11, further comprising feeding a combustible gas composition into the bioreactor.

13. The method of claim 12 wherein said combustible gas composition comprises hydrogen.

14. The process of claim 12 or 13, wherein the oxygen-containing gas and the combustible gas composition are continuously fed to the bioreactor as separate feeds.

15. A method for controlling oxygen concentration during aerobic biosynthesis, the method comprising:

feeding an oxygen-containing gas to a bioreactor containing microorganisms, wherein a combustible gas fermentation feedstock component is within the bioreactor;

reacting at least a portion of the oxygen from the oxygen-containing gas with the microorganisms;

forming a fermentation broth comprising a gas phase dispersed within the fermentation broth, the gas phase comprising unreacted oxygen from the oxygen-containing gas;

reducing the concentration of the unreacted oxygen in the gas phase to less than the limiting oxygen concentration of flammability (LOC) of the combustible gas feed component, wherein the reducing comprises adsorbing the unreacted oxygen with a solid oxygen catalyst or reacting the unreacted oxygen with a solid oxygen catalyst; and

separating the gas phase from the fermentation broth.

16. The method of claim 15, wherein the oxygen-containing gas comprises greater than 21% oxygen by volume.

17. The method of claim 15 or 16, wherein the oxygen-containing gas comprises air.

18. The process of any one of claims 15-17, wherein the gas phase separated from the fermentation broth comprises oxygen at a concentration of less than 85% of the LOC.

19. The process of any one of claims 15-18, wherein the gas phase separated from the fermentation broth comprises less than 6% oxygen by volume.

20. The method of any one of claims 15-19, wherein the step of reducing the concentration of unreacted oxygen occurs prior to the step of separating the gas phase from the fermentation broth.

21. The method of any one of claims 15-20, wherein the microorganism comprises cuprinophilus hookeri (c.necator) or cuprinobulinus metallothioneine (c.metallidurans).

22. The method of any one of claims 15-21, wherein the bioreactor is selected from the group consisting of: single fermenter, multiple fermenters in series, stirred tank fermenters, non-stirred tank fermenters, membrane fermenters, fixed bed fermenters, fluidized bed fermenters, single autoclaves, multiple autoclaves in series, plug flow fermenters, pneumatically stirred fermenters, airlift fermenters with external circuit with forced circulation, bubble column fermenters, fixed (packed) bed tower fermenters, horizontal single fermenters with multiple compartments, and multistage tower fermenters.

23. The method of any one of claims 15-22, wherein separating comprises separating the gas phase from the fermentation broth into a headspace of the bioreactor.

24. The method of any one of claims 15-23, further comprising feeding a combustible gas composition into the bioreactor.

25. The method of claim 24 wherein said combustible gas composition comprises hydrogen.

26. The process of claim 24 or 25, wherein said oxygen-containing gas and said combustible gas composition are continuously fed to said bioreactor as separate feeds.

27. A method for controlling oxygen concentration during aerobic biosynthesis, the method comprising:

feeding an oxygen-containing gas to a bioreactor containing microorganisms, wherein a combustible gas fermentation feedstock component is within the bioreactor;

reacting at least a portion of the oxygen from the oxygen-containing gas with the microorganisms;

forming a fermentation broth comprising a gas phase dispersed within the fermentation broth, the gas phase comprising unreacted oxygen from the oxygen-containing gas;

reducing the concentration of said unreacted oxygen in said gas phase to less than the flammable LOC of said combustible gas feedstock components, wherein said reducing comprises absorbing said unreacted oxygen in an oxygen-absorbing liquid; and

separating the gas phase from the fermentation broth.

28. The method of claim 27, wherein the oxygen-containing gas comprises greater than 21% oxygen by volume.

29. The method of claim 27 or 28, wherein the oxygen-containing gas comprises air.

30. The process of any one of claims 27-29, wherein the gas phase separated from the fermentation broth comprises oxygen at a concentration of less than 85% of the LOC.

31. The process of any one of claims 27-30, wherein the gas phase separated from the fermentation broth comprises less than 6% oxygen by volume.

32. The method of any one of claims 27-31, wherein the step of reducing the concentration of unreacted oxygen occurs prior to the step of separating the gas phase from the fermentation broth.

33. The method of any one of claims 27-32, wherein the microorganism comprises cuprinopsis hookeri (c.necator) or cuprinopsis metalaxyl (c.metallidurans).

34. The method of any one of claims 27-33, wherein the bioreactor is selected from the group consisting of: single fermenter, multiple fermenters in series, stirred tank fermenters, non-stirred tank fermenters, membrane fermenters, fixed bed fermenters, fluidized bed fermenters, single autoclaves, multiple autoclaves in series, plug flow fermenters, pneumatically stirred fermenters, airlift fermenters with external circuit with forced circulation, bubble column fermenters, fixed (packed) bed tower fermenters, horizontal single fermenters with multiple compartments, and multistage tower fermenters.

35. The method of any one of claims 27-34, wherein the separating comprises separating the gas phase from the fermentation broth into a headspace of the bioreactor.

36. The method of any one of claims 27-35, further comprising feeding a combustible gas composition into the bioreactor.

37. The method of claim 36 wherein said combustible gas composition comprises hydrogen.

38. The process of claim 36 or 37, wherein said oxygen-containing gas and said combustible gas composition are continuously fed to said bioreactor as separate feeds.

Technical Field

The present disclosure relates generally to methods for controlling oxygen concentration during aerobic biosynthesis. In particular, the present disclosure relates to a process for reducing the oxygen concentration in a gas phase of a fermentation broth comprising a combustible gas such as hydrogen to below the limiting oxygen concentration ("LOC"), and then separating the gas phase from the fermentation broth.

Background

In gas feed fermentation, carbon-rich gases such as carbon dioxide, carbon monoxide and methane are converted by microorganisms into a wide variety of products such as fuels, proteins and compounds, for example alcohols and organic acids. These products are used by industries in the chemical, petrochemical, pharmaceutical, animal feed, environmental and agricultural fields. Gas fermentation processes can utilize a variety of feedstocks, including domestic, industrial, or agricultural wastes, thereby reducing dependence on fossil sources of carbon and reducing greenhouse gas emissions. Fermentation processes are typically operated at lower reaction temperatures and pressures when compared to high temperature and high pressure chemical catalytic reactions.

The microorganisms used in the fermentation process are grown in fermentors under various engineering and physical conditions, such as stirring, mixing, aeration, pressure, shear, temperature, and pH. Some microorganisms grow under anaerobic conditions, while others grow under aerobic conditions. For aerobic reactions, air is usually used as the oxygen source, but oxygen-enriched air or pure oxygen may also be used. It is generally preferred to operate at the highest possible oxygen concentration to maximize oxygen mass transfer, thereby optimizing productivity. This is because the rate of oxygen mass transfer from the gas phase to the liquid phase is the rate limiting step for most aerobic microbial biosynthetic reactions.

During aerobic biosynthesis, any unreacted oxygen (e.g., air) from the oxygen source is separated from the fermentation broth into the headspace of the bioreactor (e.g., fermentor). Unreacted oxygen mixes with other unreacted gases (e.g., off-gases) in the headspace of the bioreactor. In certain instances where the feed gas comprises a potentially flammable component, the sum of the feed gases (e.g., the oxygen-containing gas, the hydrogen-containing gas, and the carbon dioxide-containing gas) may have an oxygen concentration greater than the LOC of the composition comprising the flammable component, e.g., a 6 vol% oxygen concentration for an air/hydrogen system. The gaseous mixture in the headspace of the fermentor and any unreacted oxygen in the off-gas can result in a flammable mixture, especially when flammable gases (e.g., hydrogen), flammable volatile organic products, or intermediates are used or produced during aerobic biosynthesis. Even when operated on a small scale (e.g. laboratory), there is still a risk of explosion from flammable gas mixtures, but the extent of this risk is mitigated due to the small scale of the bioreactor and the reduced gas volume. However, when scaling up the size of bioreactors for pilot or commercial use, flammability and risk of explosion are major concerns for safe operation of the process. Furthermore, when designing large scale systems operating above LOC, the necessary equipment design features can be extremely capital intensive, especially at higher operating pressures, e.g., explosion proof electronics, explosion proof valves, thicker steel, etc. may be required.

Therefore, there is a need to improve control of oxygen concentration to below the limiting oxygen concentration of flammability (LOC) of the combustible gas component prior to separation of the gas phase from the fermentation broth into the headspace of the bioreactor, while achieving acceptable productivity, capital cost (capital efficiency), and operating cost.

Disclosure of Invention

In some embodiments, the present disclosure relates to a method for controlling oxygen concentration during aerobic biosynthesis, the method comprising: feeding an oxygen-containing gas to a bioreactor comprising a fermentation feedstock; reacting a fermentation feedstock with an oxygen-containing gas to form a fermentation broth comprising a gas phase dispersed within the fermentation broth, the gas phase comprising unreacted oxygen from the oxygen-containing gas; reducing the concentration of unreacted oxygen in the gas phase to less than the limiting oxygen concentration of flammability ("LOC"); and separating the gas phase from the fermentation broth. In some aspects, the step of reducing the concentration of unreacted oxygen occurs before the step of separating the gas phase from the fermentation broth. In some aspects, the step of reducing the concentration of unreacted oxygen in the gas phase comprises adsorbing the unreacted oxygen with an oxygen reduction catalyst or reacting the unreacted oxygen with an oxygen reduction catalyst. In some aspects, the step of reducing the concentration of unreacted oxygen in the gas phase comprises absorbing the unreacted oxygen in an oxygen-absorbing liquid separated from the fermentation broth with a liquid impermeable gas membrane. In some aspects, the step of reducing the concentration of unreacted oxygen in the gas phase comprises diluting the unreacted oxygen with a diluent. In some aspects, the diluent comprises a gas stream comprising one or more of nitrogen, carbon dioxide, and hydrogen. In some aspects, the diluent comprises less than 5 vol%, for example less than 3 vol% oxygen. In some aspects, the oxygen-containing gas is air. In some aspects, the oxygen-containing gas comprises an oxygen concentration greater than 21 vol%. In some aspects, the gas phase separated from the fermentation broth comprises less than 6% by volume, such as less than 5.5%, less than 5%, less than 4.5%, less than 4%, less than 3.5%, less than 3%, less than 2%, or less than 1% by volume oxygen. In some aspects, the feedstock comprises microorganisms including cupreous hookeri (c.necator) or cupreous metalaxyl (c.metallidurans). In some aspects, the bioreactor is selected from the group consisting of a single fermentor, multiple fermentors in series, a stirred tank fermentor, a non-stirred tank fermentor, a membrane fermentor, a fixed bed fermentor, a fluidized bed fermentor, a single autoclave, multiple autoclaves in series, a plug flow fermentor, a pneumatically stirred fermentor, an airlift fermentor with an external loop with forced circulation, a bubble column fermentor, a fixed (packed) bed tower fermentor, a horizontal single fermentor with multiple compartments, and a multi-stage tower fermentor. In some aspects, the gas phase is separated from the fermentation broth into the headspace of the bioreactor. In some aspects, the method further comprises feeding a combustible gas to the bioreactor. In some aspects, the combustible gas comprises hydrogen.

In some embodiments, the present disclosure relates to a method for controlling oxygen concentration during aerobic biosynthesis, the method comprising: feeding a fermentation feedstock to a bioreactor comprising microorganisms; feeding a combustible gas to a bioreactor; feeding an oxygen-containing gas into a bioreactor, the oxygen-containing gas having an oxygen concentration greater than 21% by volume; reacting a fermentation feedstock with an oxygen-containing gas and a flammable gas to form a fermentation broth comprising a gas phase dispersed within the fermentation broth, the gas phase comprising unreacted oxygen from the oxygen-containing gas and/or the flammable gas; reducing the concentration of unreacted oxygen in the gas phase to less than a limiting oxygen concentration ("LOC"); and separating the gas phase from the fermentation broth into the upper headspace of the bioreactor. In some aspects, the combustible gas comprises hydrogen. In some aspects, the oxygen-containing gas and the flammable gas are continuously fed to the bioreactor as separate feeds. In some aspects, the step of reducing the concentration of unreacted oxygen in the gas phase comprises adsorbing or reacting unreacted oxygen with an oxygen reduction catalyst or absorbing unreacted oxygen with an oxygen absorbing liquid separated from the fermentation broth with a liquid impermeable gas membrane. In some aspects, the step of reducing the concentration of unreacted oxygen in the gas phase comprises diluting the unreacted oxygen with a diluent comprising less than 5% by volume, for example less than 3% by volume, of oxygen.

Drawings

The disclosure will be better understood from the attached non-limiting drawings, in which:

fig. 1 shows a graph of reducing oxygen concentration in an exhaust gas by an oxygen destruction scheme (option 1) or an oxygen dilution scheme (option 2), according to an embodiment of the present disclosure.

Fig. 2 illustrates an oxygen destruction scheme using a solid oxygen reduction catalyst, according to embodiments of the present disclosure.

Fig. 3 illustrates an oxygen destruction scheme using a liquid impermeable gas membrane, according to embodiments of the present disclosure.

Fig. 4 illustrates an oxygen gas dilution scheme according to an embodiment of the present disclosure, wherein the dispersed gas phase within the fermentor is diluted with nitrogen to reduce the oxygen concentration below LOC in the dispersed gas phase.

Detailed Description

The present disclosure relates to methods for controlling oxygen concentration during aerobic biosynthesis (e.g., fermentation). In some aspects, the oxygen concentration in the gas phase of the fermentation broth is reduced below the LOC of the combustible gas composition, e.g., 6 vol% for a hydrogen/oxygen mixture, and then separated from the fermentation broth into the headspace of a bioreactor (e.g., fermentor). A method for controlling oxygen concentration during aerobic biosynthesis can include feeding an oxygen-containing gas into a bioreactor including a microorganism and a combustible fermentation feedstock, and reacting oxygen of the oxygen-containing gas with the microorganism to form a fermentation broth, the fermentation broth comprising a gas phase, wherein unreacted oxygen is dispersed within the fermentation broth. The gas phase dispersed within the fermentation broth can also include a flammable gas, such as at least a portion of a flammable fermentation feedstock. The process further comprises reducing the concentration of unreacted oxygen in the dispersed gas phase to less than LOC and then separating the gas phase from the fermentation broth into the headspace of the bioreactor.

Control parameter of oxygen concentration

As described herein, the oxygen concentration in the bioreactor is controlled within a specified range. The dissolved oxygen concentration in the fermentation broth is controlled to be at least the minimum value required for the microorganism to function. A minimum is required because the microorganisms are aerobic and require a certain amount of oxygen. The gaseous oxygen concentration in the headspace of the bioreactor is controlled using the oxygen control scheme described above to safely operate below LOC. In some aspects, LOC is about 6.0 vol% of oxygen in the gas mixture outside the fermentation broth. As a safety measure, the gaseous oxygen concentration in the headspace may be measured and controlled to be less than 90% of LOC, such as less than 85%, less than 80%, less than 75%, or less than 70%. In some aspects, the gaseous oxygen concentration in the headspace is controlled to be in the range of 60% to 95%, such as 65% to 90%, 70% to 85%, 70% to 80%, or 75% to 85% of LOC.

At least one feed stream comprising an oxygen-containing gas can be introduced into the bioreactor by suitable means in order to generate microbubbles and enhance the gas-liquid interface between the gas phase and the bulk liquid. In addition, gas-liquid mass transfer depends on the reactor configuration. There are seven general steps of delivering the gas mass to the reaction site.

1. Diffusion through the bulk gas within the bubble to the gas-liquid interface.

2. Moves across the gas-liquid interface.

3. The solute gas diffuses through a relatively unmixed liquid region (membrane) adjacent to the gas bubble and into the well-mixed bulk liquid.

4. Solute gases are transported through the bulk liquid to the stagnant membrane surrounding the cells.

5. The transport is through a second unmixed liquid membrane associated with the cells.

6. Transport across cell membranes.

7. Transported by the cell to the reaction site.

Gaseous oxygen concentration in the headspace

The upper limit of the gaseous oxygen concentration in the headspace of the bioreactor is limited by safety considerations. In general, the literature cites H of the starting gas mixture₂/CO₂/O₂A ratio of 7: 1 or 8: 1 (hydrogen/carbon dioxide/oxygen) to obtain optimal growth/production conditions for the gas fermentation of cupprilus hookeri (c.necator) (Ishizaki et al, 2001), but this ratio may vary due to regulatory and/or reaction requirements. This means that the hydrogen/oxygen ratio is within the flammable range of hydrogen and oxygen concentrations. The critical oxygen concentration when mixed with hydrogen and carbon dioxide as diluents was 5.9% by volume (Jones and Kenny, 1935). Thus, 5.9 vol% LOC is defined herein as the minimum oxygen concentration at which a combustible gas mixture can be formed from a fermentation process mixture according to the present disclosure. These fermentation processes include a gas phase comprising, for example, a mixture of oxygen, nitrogen, hydrogen, carbon dioxide, and water vapor, which rises to the headspace of a bioreactor (e.g., fermentor). Temperature and pressure conditions in the bioreactor may also affect the relative concentrations of the components in the headspace.

Unreacted components (e.g., oxygen, nitrogen, hydrogen, carbon dioxide, and/or water vapor) are in a dispersed gas phase (e.g., bubbles) within the fermentation broth before the gas phase in the fermentation broth rises to the headspace of the bioreactor. The oxygen concentration in the dispersed gas phase is reduced below LOC and then separated from the fermentation broth into the headspace of the reactor and mixed with other flammable gases. In particular, the oxygen concentration in the gas phase is reduced below the LOC of the headspace gas mixture, e.g. 6.0 vol% oxygen. To maintain a safety margin, the bioreactor may be operated within 65% to 85%, or even less than 65% of LOC. In some aspects, the gaseous oxygen concentration in the headspace is controlled to be 3.5 to 4.5 vol.% oxygen, e.g., 3.75 to 4.25 vol.%, 3.85 to 4.15 vol.%, 3.95 to 4.05 vol.%, or about 4 vol.% oxygen. The bioreactor off-gas also has the same LOC.

In some aspects, the step of transferring the mass of gas to the reaction site comprises diffusion of bulk gas within the gas bubble to a gas-liquid interface, movement across the gas-liquid interface, diffusion of solute gas through a relatively unmixed liquid region (membrane) adjacent the gas bubble and into the well-mixed bulk liquid, transport of solute gas through the bulk liquid to a stagnant membrane surrounding the cell, transport through a second unmixed liquid membrane associated with the cell, transport across the cell membrane, and transport through the cell to the reaction site.

Gas-liquid mass transfer also depends on the fermenter configuration, and the gas mixture should be introduced into the fermenter by suitable means to generate small bubbles or microbubbles (with high specific surface area) to increase the gas-liquid interfacial surface area available for gas mass transfer. It is desirable to operate at the highest possible oxygen concentration in order to maximize oxygen mass transfer and thus the productivity of the gas fermentation reaction in the fermentor.

In some aspects, the concentration of unreacted oxygen in the gas phase is reduced by adsorbing the unreacted oxygen with an oxygen reduction catalyst or reacting the unreacted oxygen with an oxygen reduction catalyst. The oxygen reduction catalyst may be fed to or present at the top of the bioreactor in a sufficient amount to reduce the oxygen concentration in the gas phase to below LOC. In some aspects, the oxygen reduction catalyst is a solid oxygen reduction catalyst. The solid oxygen reduction catalyst may be disposed in a portion of the bioreactor that is within or above the fermentation broth (e.g., directly above or adjacent to the fermentation broth) to trap any unreacted oxygen before a separated gas phase forms in the headspace and bioreactor. In this way, bulk gas is only formed in the headspace after the gas phase of the fermentation broth is contacted with the reaction zone of the solid oxygen reduction catalyst.

In some aspects, the concentration of unreacted oxygen in the gas phase is reduced by adsorbing the unreacted oxygen with a liquid impermeable gas membrane. The liquid impermeable gas membrane comprises an oxygen absorbing liquid region that prevents the fermentation broth and the oxygen absorbing liquid from blending, but allows gas (e.g., from collapsing bubbles in the fermentation broth) to escape from the fermentation broth through the membrane into the liquid region. In other words, a liquid impermeable gas membrane allows gas to pass through but is not liquid permeable. A liquid impermeable gas membrane may be disposed in a portion of the bioreactor directly above or adjacent the fermentation broth to trap any unreacted oxygen before a separated gas phase (e.g., bulk gas phase) forms in the headspace of the bioreactor. As used herein, a "headspace" is a portion of a bioreactor that does not include fermentation broth, e.g., the volume above fermentation broth in a vertical bioreactor. In some aspects, the oxygen dilution scheme and/or the oxygen removal (e.g., destruction) scheme are particularly suitable for vertical reactors with oxygen concentration gradients, such as loop, lift, or tubular reactors with vertical separation zones.

In some aspects, the concentration of unreacted oxygen in the gas phase is reduced by diluting the gas phase comprising unreacted oxygen with a diluent. The diluent may comprise a low oxygen gas stream fed to the bioreactor in sufficient quantity to reduce the concentration of unreacted oxygen in the gas phase below the LOC of the combustible gas component. In some aspects, the diluent may be one or more of nitrogen, carbon dioxide, and hydrogen. In some aspects, the diluent comprises oxygen at a concentration lower than the LOC of the oxygen/combustible gas mixture.

Conventionally, to safely operate aerobic microbial biosynthetic processes, such as gas fermentation, with explosive head spaces or gas volumes, bioreactors are designed with strong walls to withstand the pressure and temperature from a deflagration or explosion. In some cases, the walls of the bioreactor may be reinforced, for example constructed with a larger width or made of a particular material to withstand a deflagration or explosion. However, utilizing fermentors with stronger walls may increase capital equipment costs and operating costs. Additional strategies include keeping below the flammability limit, separating the flammable gas from oxygen, generating hydrogen in situ, or directly using electrons as an energy source. However, all of these alternative strategies have economic or productivity disadvantages and may be incompatible with the microorganisms used for fermentation. For example, operating a bioreactor below LOC will reduce oxygen mass transfer from the gas phase to the liquid phase and reduce the overall productivity of the final product.

The inventors have now found that reducing the oxygen concentration in the fermentation broth prior to separation of the gas phase from the fermentation broth can greatly reduce the flammability of the headspace gases and off-gases while maintaining a high oxygen mass transfer in the fermentation broth. The safe operation of the process is achieved by reducing the unreacted oxygen concentration to below the flammability limit using an oxygen removal scheme or an oxygen dilution scheme prior to separation of the gas phase from the fermentation broth. It was found that diluting the gas phase with an inert gas (e.g. nitrogen) to an oxygen concentration below LOC prevents deflagration or explosion in the headspace of the bioreactor. Advantageously, by diluting the gas phase late in the fermentation process, e.g. after the maximum amount of oxygen has been consumed by the microorganisms, the process maintains the maximum amount of oxygen in the fermentation broth, which is then separated into the headspace of the bioreactor. It has also been found that removal of oxygen from the gas phase by adsorption or absorption (e.g., using an oxygen reduction catalyst or an oxygen absorbing liquid in combination with a liquid impermeable gas membrane) can also prevent deflagration or explosion in the headspace of the bioreactor. Advantageously, this also allows the bioreactor design to include a variety of materials and is not limited to current enhanced bioreactor designs that accommodate controlled explosions.

The process advantageously controls the oxygen concentration in the fermentation broth to ensure safe operation of the bioreactor, while maintaining the oxygen concentration to achieve high conversion of the carbon source by the microorganisms. In a typical gas fermentation process, a fermentation feedstock (e.g., gaseous CO) comprising microorganisms is introduced₂) With a combustible gas (e.g., hydrogen and an oxygen-containing gas) to form a fermentation broth. The flammable gas and the oxygen-containing gas are in a dispersed gas phase (e.g., bubbles) in the fermentation broth and any unreacted gas eventually rises as off-gas to the headspace of the bioreactor (e.g., fermentor). "off-gas" refers to a gaseous mixture of gases separated from the fermentation broth during the fermentation process. If the oxygen concentration is relatively high, for example, above the limiting oxygen concentration ("LOC") of the combustible components in the exhaust gas, it is prone to combustion. Advantageously, the method reduces the amount of oxygen in the dispersed gas phase prior to rising to the upper portion (e.g., headspace) of the bioreactor to prevent combustion. The process of the present invention enables the use of higher concentrations of excess oxygen (e.g., LOC above flammable components) in the feed stream entering the bioreactor to promote higher reaction rates, followed by dilution or removal of oxygen from the dispersed gas phase of the fermentation broth, which is then separated to form bulk gas in the headspace of the bioreactor.

The method of the invention can greatly improve the efficiency of the method and realize safe operation of the fermentation process. The process utilizes an oxygen-containing gas having a high oxygen concentration above LOC (e.g., greater than 6% oxygen by volume in a hydrogen/air mixture) to promote reaction with the microorganisms, and then provides a means for removing or diluting unreacted oxygen prior to separation of the gas phase from the fermentation broth into the headspace. In some aspects, the oxygen-containing gas may comprise greater than 6 vol%, such as greater than 10 vol%, greater than 20 vol%, greater than 40 vol%, greater than 60 vol%, greater than 80 vol%, greater than 90 vol%, greater than 95 vol%, and greater than 99 vol% oxygen. In some aspects, the oxygen-containing gas comprises pure oxygen.

In some cases, the fermentation process is an air-fed fermentation reaction with aerobic microorganisms in a large, unstirred fermentor. Large unstirred fermentors may include a fermentation broth having a dispersed gas phase in the flammability range. The oxygen concentration of the dispersed gas phase within the fermentation broth is reduced before the gas phase is separated from the fermentation broth into the headspace of the fermentor. The oxygen concentration in the gas phase is reduced to a concentration of LOC lower than the flammability of the flammable component. For example, for a hydrogen-rich gas stream containing a hydrogen concentration above the lower flammability limit of hydrogen, the oxygen concentration in the dispersed gas phase of the fermentation broth is reduced to less than 6% by volume oxygen in that gas phase. In some aspects, the oxygen concentration in the gas phase of the fermentation broth is reduced to less than 6 vol.%, e.g., less than 5.9 vol.%, less than 5.5 vol.%, less than 5.0 vol.%, less than 4.0 vol.%, less than 3.0 vol.%, less than 2.0 vol.%, less than 1.0 vol.%, less than 0.5 vol.%, less than 0.1 vol.%, less than.01 vol.% oxygen. In some aspects, the oxygen concentration in the gas phase is controlled to be less than 90%, such as less than 85%, less than 80%, less than 75%, or less than 70% of the LOC of the flammability of the gaseous mixture in the headspace.

Microorganisms

The bioreactor described herein is provided with microorganisms for use in aerobic biosynthesis, such as fermentation. For aerobic reactions, air is usually used as the oxygen source, but oxygen-enriched air or pure oxygen may also be used. It is generally preferred to operate in the fermentor at the highest possible oxygen concentration in the dispersed gas phase to maximize oxygen mass transfer and thereby optimize productivity. This is because the rate of oxygen mass transfer from the gas phase to the liquid phase is a known rate limiting step for most aerobic microbial biosynthetic reactions. As a result of the oxygen concentration being higher than the LOC of the gaseous composition comprising the flammable component (e.g., greater than 6 vol% oxygen), any unreacted oxygen in the fermentor headspace and the exhaust stream can lead to the formation of unsafe flammable mixtures when flammable gases (e.g., hydrogen), flammable volatile organic products, or intermediates are present.

The microorganism may be cuppric hookeri (c.necator) or an organism with similar properties thereto. Hookworm cuprum bacteria (previously known as hydrogenotrophus eutrophus (Hydrogenomonas eutrophus), alcaligenes eutrophus (alcaligenes eutropha), Ralstonia eutropha (Ralstonia eutropha) and waterworth (wautersia eutropha) are gram-negative flagellated soil bacteria of the beta proteobacteria In one embodiment, a host of the cuppridinium hookeri (c.necator) H16 strain is used, wherein at least a portion of the phaC1AB1 locus is knocked out (Δ phaCAB), as described in U.S. patent application serial No. 15/717,216, the teachings of which are incorporated herein by reference. The organism may be selected from a non-pathogenic member of the genera Ralstonia (Ralstonia), wautersia (Wausteria), cupriasis (Cupriavidus), Alcaligenes (Alcaligenes), Burkholderia (Burkholderia) or panduraea (Pandoraea).

Feed stream

As mentioned above, oxygen is required for fermentation and is introduced into the bioreactor via the feed stream. In order to introduce the gaseous feed stream into the bioreactor in a safe manner, at least two different continuous streams are used. At least one of the continuous feed streams comprises a combustible gas (e.g., hydrogen) and at least one of the continuous feed streams comprises gaseous oxygen, e.g., an oxygen-containing gas. At least one continuous flow comprising a combustible gas will comprise hydrogen (combustible gas), optionally oxygen at a concentration below the limiting oxygen concentration of flammability ("LOC") of the gas flow, and optionally CO₂All or a portion of the gas feed. The at least one continuous flow comprising gaseous oxygen may be an air feed flow. Such streams will not be packetizedContaining hydrogen above the lower flammability limit of hydrogen, but optionally containing CO₂All or a portion of the gas feed. Each gas feed stream is introduced into the bioreactor by means such as a microbubble generator, venturi nozzle or porous gas bubbler. By separating hydrogen and oxygen into separate feed streams, a flammable gas mixture cannot be formed in the feed system, and a gas mixture containing both hydrogen and oxygen is only present in the fermentation broth and in the small volume bubbles in the headspace and waste gas streams.

In some aspects, an oxygen-containing gas (e.g., air) can be fed directly into the fermentation broth. In some aspects, the oxygen-containing gas may be a rich oxygen source, such as oxygen-enriched air or pure oxygen. In some aspects, the oxygen-containing gas may comprise greater than 6 vol%, such as greater than 10 vol%, greater than 20 vol%, greater than 40 vol%, greater than 60 vol%, greater than 80 vol%, or greater than 90 vol% oxygen. In some aspects, the oxygen-containing gas may be pure oxygen.

In fermentation processes, air is typically used as the source of oxygen, but in some cases pure or oxygen-enriched air may be used. Any unreacted oxygen (along with nitrogen present in the air) leaves the reactor or reactors as a gaseous effluent. Unreacted oxygen is commonly referred to as the oxygen concentration in the exhaust or "oxygen breakthrough". Any gasification products in the gaseous effluent may be condensed and recovered, and the off-gas exits the system to an abatement system. The remaining product in the fermentation broth may be recovered from the liquid effluent from the bioreactor.

Bioreactor

As described herein, the temperature and pressure parameters of the bioreactor may vary, for example, from sub-atmospheric to super-atmospheric pressures, and at temperatures of 20 ℃ to 50 ℃. The type of bioreactor to be used may be selected based on the desired operating temperature and pressure, as well as additional factors. Examples of additional factors include whether mechanical agitation or stirring is desired, whether the microorganisms will be immobilized, and how many oxygenation points are desired.

Examples of bioreactors, such as the type of gas fermentor, include single fermenters, multiple fermenters in series, stirred tank fermenters, non-stirred tank fermenters, membrane fermenters, fixed bed fermenters, fluidized bed fermenters, single autoclaves, multiple autoclaves in series, plug flow fermenters, pneumatically stirred fermenters with an internal draft tube loop or an external loop, such as gas (air) lift fermenters, airlift fermenters with an external loop with forced circulation, bubble column fermenters, fixed (packed) bed tower fermenters, horizontal single fermenters with multiple compartments, and multistage tower fermenters. Furthermore, the fermentor may be operated in batch mode, fed-batch mode, and continuous mode.

Removal of oxygen from fermentation broths

As described herein, the fermentation broth comprises a feed stream that is combined with aerobic microorganisms in a bioreactor. In some aspects, the feed streams (e.g., the carbon source feed stream, the combustible gas-containing stream, and the oxygen-containing gas feed stream) react with microorganisms in the bioreactor to at least partially form a fermentation broth (which may also include other products, byproducts, and other media fed to the bioreactor). Unreacted oxygen or oxygen not consumed by the microorganisms is present as both dissolved and gaseous oxygen in the dispersed gas phase within the fermentation broth. The same is true for other gases that are soluble. The dispersed gas phase containing unreacted components (e.g., oxygen, nitrogen, hydrogen, carbon dioxide, and/or water vapor) rises to the headspace of the bioreactor.

The oxygen concentration in the gas phase is reduced to less than the limiting oxygen concentration ("LOC") less than the flammability of the combustible component of the dispersed gas composition. As a safety measure, the gas phase rising to the headspace can be measured and controlled to be less than 90% of LOC, such as less than 85%, less than 80%, less than 75% or less than 70% of LOC. In some aspects, the gaseous oxygen concentration in the headspace is controlled to be in the range of 60% to 95%, such as 65% to 90%, 70% to 85%, 70% to 80%, or 75% to 85% of LOC. In some aspects, LOC should be less than 6.0 vol%, such as less than 5.9 vol%, less than 5.5 vol%, less than 5.0 vol%, less than 4.0 vol%, less than 3.0 vol%, less than 2.0 vol%, less than 1.0 vol%, less than 0.5 vol%, less than 0.1 vol%, less than.01 vol% oxygen, or no oxygen. In certain aspects, hydrogen is the only flammable gas in the fermentation reaction system. In terms of ranges, the oxygen concentration in the gas phase is reduced to a range of.01 vol% to 6.0 vol%, such as 0.1 vol% to 5.9 vol%, 0.5 vol% to 5.5 vol%, 1.0 vol% to 5.0 vol%, 2.0 vol% to 4.0 vol%, or 3.0 vol% to 4.0 vol%.

Fig. 1 shows a graph of reduced oxygen concentration in a gas phase, according to an embodiment of the present disclosure. During fermentation, the oxygen concentration in the fermentation broth may be greater than LOC, e.g., 6 vol.% oxygen for a hydrogen/oxygen mixture. Since the rate of oxygen mass transfer from the gas phase to the liquid phase is the rate-limiting step in the fermentation process, it is generally preferred to provide the highest possible gas phase oxygen concentration for the fermentation to maximize oxygen mass transfer, thereby optimizing the yield of the final product. However, when the gas phase of the fermentation broth comprises a high concentration of oxygen, this may produce an off-gas mixture in the headspace of the bioreactor, which is a flammable mixture. In certain aspects, the sum of the feed gas in the fermentation broth is greater than LOC. In some aspects, the process of the invention reduces the oxygen concentration in the dispersed gas phase to below LOC, or to below the safety margin of LOC, before it is separated from the fermentation broth and an off-gas is formed.

According to embodiments of the present disclosure, the oxygen concentration in the gas phase may be reduced by an oxygen removal scheme or an oxygen dilution scheme. In each of these processes, the oxygen concentration of the dispersed gas phase is reduced and then separated from the fermentation broth as off-gas in the headspace of the bioreactor. In particular, the oxygen concentration of the dispersed gas phase is reduced to below LOC or a safety margin below LOC, thereby preventing deflagration or explosion in the bioreactor when the individual gas phase mixture forms an exhaust gas. As shown in FIG. 1, the oxygen concentration is reduced by employing either an oxygen removal protocol (option 1) or an oxygen dilution protocol (option 2) prior to separation of the gas phase from the fermentation broth.

In some aspects, the method of reducing oxygen content may include an oxygen removal protocol (option 1). The oxygen removal scheme may include removing oxygen from the gas phase by adsorption or absorption. In some aspects, the oxygen reduction catalyst is fed to the fermentation broth prior to separation of the gas phase from the fermentation broth. In some aspects, the oxygen reduction catalyst is a fixed bed installed in a portion of the bioreactor. In some aspects, the fixed bed comprising the oxygen reduction catalyst is located within, directly above, or adjacent to the fermentation broth such that a separate bulk gas phase is not formed until above the oxygen reduction catalyst. In some aspects, the oxygen reduction catalyst is located at an interface between the fermentation broth and the headspace. In some aspects, the oxygen reduction catalyst is a solid oxygen reduction catalyst. The solid oxygen reduction catalyst can trap any unreacted oxygen which is then mixed with the off-gas in the headspace of the bioreactor.

In some aspects, the oxygen removal protocol includes a protective oxidant for reducing the oxygen content in the presence of potentially flammable gases or vapor mixtures. Protective oxidants are disclosed, for example, in U.S. patent nos. 6,888,034 and 9,221,737 and U.S. patent publication No. 2016/0176813, which are incorporated herein by reference. The guard oxidant may be used with, e.g., within, a bioreactor to reduce the oxygen concentration below the safety margin of LOC. In some aspects, the oxidant is shielded within, directly above, or adjacent to the fermentation broth to reduce oxygen in the gas phase, which then forms a bulk gas in the headspace of the bioreactor. In some aspects, shielding the oxidant can reduce the oxygen content in the gas phase of the fermentation broth, the off-gas mixture in the headspace, and/or the final off-gas during aerobic biosynthesis. Any unreacted oxygen (along with nitrogen present in the air) leaves the fermentor(s) as a gaseous effluent. In addition to reducing oxygen concentration, shielding the oxidizing agent also provides stability to the process.

Unfortunately, in the event that the oxygen breakthrough concentration exceeds the limiting oxygen concentration ("LOC"), unsafe flammable mixtures may form in the headspace and exhaust gas stream. Thus, as a safety margin, the oxygen leakage of the hydrogen-containing mixture is typically kept below 4 vol%. Higher oxygen leakage also means that the air fed to the fermenter is not fully utilized. In other words, the method requires more air, which results in increased compression costs. In addition, the increased volume of exhaust gas results in increased exhaust gas treatment costs. U.S. Pat. No. 3,957,876(Rapoport and White) teaches a method of reducing oxygen breakthrough from cyclohexane oxidation processes by using a so-called scavenging reaction zone.

Fig. 2 illustrates an oxygen removal scheme utilizing an oxygen reduction catalyst according to embodiments of the present disclosure. The oxygen reduction catalyst reduces the oxygen concentration from greater than 4.0 vol% in the dispersed gas phase to less than 4.0 vol% prior to separation of the gas phase from the fermentation broth. Figure 2 shows that the separated gas phase in the headspace of the bioreactor has an oxygen concentration of less than 4.0 vol%. In some aspects, the oxygen reduction catalyst reduces the oxygen concentration to a safety margin of less than 80% of LOC. In the case where air is used as the oxygen-containing gas, unreacted H will also be present in the off-gas₂Unreacted CO₂Nitrogen (from air) and water vapor (saturated concentration). The oxygen reduction catalyst can reduce the oxygen concentration to less than 4.0 vol%, which is less than 80% of LOC of flammability.

In the illustrated embodiment, an oxygen-containing gas stream (e.g., air) is added to the bioreactor at the highest possible oxygen concentration in order to maximize oxygen mass transfer and thus productivity. However, unreacted oxygen can be removed from the unreacted gas leaving the fermentor as a gaseous effluent using a solid oxygen reduction catalyst. A solid oxygen reduction catalyst may be located in the upper portion of the fermentation broth to remove excess oxygen before a separate off-gas phase is formed in the headspace. In some aspects, the solid oxygen reduction catalyst is located at an interface between the fermentation broth and the headspace of the bioreactor.

Fig. 3 illustrates an oxygen removal scheme that reduces the oxygen concentration in the gas phase of a fermentation broth below LOC using an oxygen-absorbing liquid that is separated from the fermentation broth using a liquid-impermeable gas membrane, according to an embodiment of the present disclosure. The liquid impermeable gas film reduces the oxygen concentration from greater than 4.0 vol% to less than 4.0 vol% in the dispersed gas phase. In some aspects, the liquid impermeable gas membrane reduces the oxygen concentration in the gas phase by a relative amount greater than 5%, such as greater than 10%, greater than 20%, greater than 40%, greater than 60%, greater than 80%, or greater than 90%. A liquid impermeable gas membrane may be located in the upper part of the fermentation directly above (adjacent to) the fermentation broth, e.g. a reaction zone for trapping oxygen in the membrane. In some aspects, a liquid impermeable gas membrane is located at the interface between the fermentation broth and the headspace of the bioreactor.

A liquid impermeable gas membrane provides a reaction zone containing an oxygen absorbing liquid to remove oxygen from the gas phase. The liquid impermeable gas membrane prevents the fermentation broth and the oxygen-absorbing liquid from blending, but allows gas (from collapsing bubbles in the fermentation broth) to escape from the fermentation broth into the liquid region.

Fig. 4 illustrates an oxygen dilution scheme according to embodiments of the present disclosure. In some aspects, the method of reducing oxygen content may include an oxygen dilution scheme. The oxygen dilution scheme dilutes the dispersed gas phase within the fermentation broth with nitrogen to reduce the oxygen concentration to less than the LOC of the dispersed gas phase. As shown in fig. 4, nitrogen is fed into the bioreactor to dilute the oxygen in the dispersed gas phase before the separate gas phase mixture is formed in the headspace and the off-gas. In some aspects, the nitrogen feed stream may be supplied to the bioreactor at an upper portion of the fermentation broth. Oxygen dilution scheme the dispersed gas phase in the fermentation broth is diluted with a suitable diluent gas stream (e.g., an oxygen depleted gas stream, an inert gas stream, or a gas stream having a high concentration of a flammable gas (e.g., pure hydrogen) prior to separating the dispersed gas into the headspace and the off-gas.

The oxygen dilution scheme involves diluting a dispersed gas phase containing unreacted oxygen with a suitable diluent. In some aspects, the diluent may be a stream depleted of oxygen or a stream of inert gas. In some aspects, the diluent may comprise nitrogen, hydrogen, carbon dioxide, or a combination thereof. In some aspects, the diluent may be a stream comprising less than 6.0 vol%, such as less than 5.9 vol%, less than 5.5 vol%, less than 5.0 vol%, less than 4.0 vol%, less than 3.0 vol%, less than 2.0 vol%, less than 1.0 vol%, less than 0.5 vol%, less than 0.1 vol%, less than.01 vol% oxygen, or no oxygen. For example, the diluent may consist of an inert gas.

By reducing the oxygen concentration to less than the flammable LOC, for example 6% by volume for a hydrogen/air mixture, the method reduces the flammability level of the gas mixture and reduces the risk of deflagration or explosion. In some aspects, the method of reducing the oxygen content comprises diluting the dispersed gas phase with an inert gas (e.g., nitrogen) before the separate bulk gas phase is formed in the headspace of the bioreactor. The process reduces the oxygen concentration within the safety margin to less than the LOC of flammability of the gas mixture.

In some aspects, the diluent is introduced into the bioreactor in the form of a recycle stream (e.g., a recycle stream comprising nitrogen or other gases from the fermentation process) entering the bioreactor. In some aspects, the oxygen concentration is diluted by adding hydrogen, nitrogen, carbon dioxide recycle streams at the upper portion of the fermentation broth. Unreacted hydrogen and carbon dioxide are recycled to the upper portion of the fermentation broth rather than to the bottom of the fermentor to effect recycle as well as dilution of oxygen in the gas phase of the fermentation broth. A diluent is fed to the reactor to reduce the oxygen concentration below the flammable LOC. Without the oxygen destruction (e.g., removal) and dilution schemes described above, it would be necessary to construct the bioreactor with thicker/stronger walls in order to safely contain potentially flammable mixtures, and such bioreactors would be more expensive.

In some aspects, an oxygen dilution scheme and an oxygen removal scheme may be used in combination to reduce the oxygen concentration.