Process intensification by means of immobilized acetogenic bacteria for gas fermentation on textiles

Abstract

The present invention relates to cell culture carriers comprising a three-dimensional textile carrier material and acetogenic bacteria for gas fermentation which form a biofilm on the carrier material. The present invention further relates to the use of corresponding carrier materials for the immobilization of acetogenic bacteria for gas fermentation, as well as to the use of the cell culture carrier according to the invention in gas fermentation for the production of organic compounds.

Description

[0001] Process intensification using immobilized acetogenic bacteria for gas fermentation on textiles

[0002] The present invention relates to cell culture carriers comprising a three-dimensionally formed, textile carrier material and acetogenic bacteria for gas fermentation, which form a biofilm on the carrier material. The present invention further relates to the use of corresponding carrier materials for immobilizing acetogenic bacteria for gas fermentation, as well as the use of the cell culture carrier according to the invention in gas fermentation for the production of organic substances.

[0003] To establish a circular (bio)economy, it is necessary to reduce the use of fossil resources and the associated CO2 emissions (greenhouse gas) into the atmosphere. Therefore, fossil resources should be replaced by sustainable technologies combined with CO2 emission reduction. In this context, gas fermentation is a very promising technology for the future. In this process, exhaust gases containing CO2, CO, and hydrogen are converted into, for example, organic acids and alcohols through microbial processes.

[0004] The gas fermentation of acetogenic bacteria is challenging, partly due to physical reasons such as the low solubility of gases in the liquid phase, and partly due to biological reasons, such as the limited metabolic efficiency of acetogenic bacteria, which leads to low product yields and rates. Consequently, the cells must be cultivated in large numbers or at high cell densities to produce significant quantities of ethanol and other organic molecules. This, in turn, makes the fermentation process more complex, as suitable means for mass transfer, medium exchange, and, in particular, for retaining the cells in the reactor must be employed. In a world-first commercial approach, now used in six production facilities in Asia and Europe, the anaerobic carboxytrophic acetogenic bacterium Clostridium autoethanogenum plays a crucial role.This bacterium is capable of utilizing chemolitho-autotrophic substrates such as H2, CO2, or CO as its sole carbon and energy source. With its help, exhaust gases from steel production can be directly converted into ethanol. It should be noted that the metabolic rates of anaerobic clostridia are relatively slow, which is also associated with slow growth rates. To nevertheless achieve high conversion rates (productivities) in the bioreactor, very high cell densities are required to allow for the overall conversion for production. The typically used method of choice is cell retention with membranes. These membranes are designed in such a way that the microorganisms are not released through their pores into the outflow from the bioreactor. The membranes used in this way require technically complex modifications (retention devices, etc.).Reflux methods) in an external circuit of the bioreactor, and are also susceptible to wear, long-term clogging, and degradation. Alternatively, approaches using in-situ installed membranes are known, which, for example, are perfused with gas on the side facing away from the medium, thus supplying the immobilized microorganisms on the liquid-side membrane surface with gas (e.g., CO2, CO, H2 mixtures). However, such approaches are very difficult and costly to scale in large bioreactors due to the unfavorable surface area-to-volume ratios. Furthermore, it is known that the immobilization of the industrially used microorganism Clostridium autoethanogenum outside of membranes only occurs under stress, which does not favor the use of this approach in industrial settings.

[0005] The present invention is therefore based on the objective of providing means that enable simple, technically feasible, and highly productive gas fermentation with acetogenic bacteria, such as Clostridium autoethanogenum. This objective is achieved by the embodiments characterized in the claims. In particular, the invention provides a cell culture carrier comprising a three-dimensionally formed textile carrier material and acetogenic bacteria for gas fermentation, which form a biofilm on the carrier material. Furthermore, the invention provides the use of corresponding carrier materials for immobilizing acetogenic bacteria for gas fermentation, as well as the use of the cell culture carrier according to the invention in gas fermentation for the production of organic substances.

[0006] Accordingly, one object of the present invention relates to a cell culture carrier comprising

[0007] (a) a three-dimensionally formed, textile support material and

[0008] (b) acetogenic bacteria for gas fermentation, which form a biofilm on the support material.

[0009] The term "cell culture carrier" as used herein refers to any carrier material suitable for being seeded with cells. This carrier material may be unseeded or seeded with cells. According to the present invention, the carrier material is a three-dimensionally formed, textile carrier material that supports a biofilm formed by acetogenic bacteria for gas fermentation.

[0010] The term "biofilm" as used herein refers to aggregates of microorganisms, according to the invention bacteria for gas fermentation, preferably Clostridium autoethanogenum, which are adhesively attached to the support material. The bacteria in question preferably immobilize themselves independently on the support material.

[0011] Preferably, the bacteria for gas fermentation are selected from the group consisting of Clostridium autoethanogenum, Clostridium carboxidivorans, Clostridium ijungdahlii, Acetobacterium ijungdahlii, Eubacterium callanderi, Acetobacterium wieringae, and Eubacterium limosum. Clostridium autoethanogenum and Clostridium carboxidivorans are particularly preferred, with Clostridium autoethanogenum being especially preferred. In preferred embodiments, the bacteria for gas fermentation are present as a monoculture. The bacteria mentioned are anaerobic, carboxytrophic, acetogenic bacteria capable of utilizing chemolitho-autotrophic substrates such as H₂, CO₂, or CO₂ as the sole source of carbon and energy. As such, these bacteria, especially C. autoethanogenum, are used in gas fermentation for the production of organic substances, for example, alcohols and organic acids. Suitable strains of the bacteria mentioned, especially C.autoethanogenum, are known in the prior art.

[0012] The carrier material used according to the invention is a three-dimensionally formed, textile carrier material (hereinafter also referred to as "3D textiles").

[0013] 3D textiles are essentially textile forms with a pronounced thickness of more than 1 mm. For use as a textile carrier for bacteria, it is advantageous if the 3D textile has a low overall fiber density and thus also a low material density, in order to offer the bacteria a surface for growth while simultaneously allowing good exchange of substances with the environment through a flow process. Spacer fabrics, spacer knits, spacer knitted fabrics, and nonwovens with a pronounced thickness are particularly suitable due to these properties. Accordingly, the three-dimensionally formed textile carrier material used according to the invention is preferably a spacer fabric, spacer knitted fabric, spacer knitted fabric, or a nonwoven with a pronounced thickness, with spacer knitted fabrics and nonwovens with a pronounced thickness being particularly preferred.

[0014] Polyester terephthalate (PET) has proven to be a suitable fiber material. Accordingly, the three-dimensionally formed textile carrier material used according to the invention preferably consists of PET. The textile structure is preferably formed in the form of continuous fibers. This avoids microplastics in the reactor and in wastewater. These continuous fibers can be processed into a 3D textile in the form of monofilaments, multifilaments, and mixtures, including those with other fiber materials.

[0015] Spacer textiles (spacer fabrics), in particular as growth carriers for microorganisms, with two planar textile layers (top layers) made of plastic threads and a pile layer of spacer threads connecting the textile layers, wherein textured multifilament yarns are incorporated into the top layers, are characterized by the fact that they form a rough, large-area growth zone through which air and liquids can flow without significant pressure loss.Such spacer textiles are characterized in that (i) the cover layers with textured multifilament yarns form large flow-through areas, (ii) the spacer threads consist, for example, of 50% monofilaments and 50% textured multifilament yarns, (iii) textured multifilament yarns are incorporated into both cover layers, acting as a porous, filamentous, and large-area growth carrier, and (iv) the cover layers, after a heat-setting process under tension, form a large open flow-through area as well as a large growth area. The three-dimensionally formed textile carrier material used according to the invention is preferably a spacer textile as described above.

[0016] A three-dimensionally formed textile carrier material used according to the invention can preferably be designed as a spacer fabric, consisting of at least 65 wt.% textured multifilament yarns and the remainder of monofilaments. The monofilaments serve to stabilize the 3D geometry and are incorporated in the pile yarn layer. The basis weight is at least 700 g / m². 2 and the thickness is between 15 and 20 mm. The specific surface area for immobilizing the bacteria is at least 1,000 m². 2 / m 3 .

[0017] In nonwovens, the individual fibers are less oriented than in woven or knitted fabrics and are mechanically stabilized through needling, chemical binders, or thermal processes. Coatings can influence surface properties, strength, and pore size.

[0018] For use as a carrier textile, nonwovens made of continuous filaments are suitable, as they have a relatively open structure to allow airflow. A three-dimensionally formed textile carrier material used according to the invention can preferably be a nonwoven fabric consisting of mechanically bonded PET filaments, fiber diameter between 17 and 20 pm, basis weight 150 g / m². 2 Thickness of 1.5 to 2.0 mm, density at least 80,000 g / m³ 3 The air permeability is approximately 2.0 m 3 / s*m 2 The pore size distribution averages 55 pm (min. 17 pm and max. 300 pm). The specific surface area is at least 10,000 m². 2 / m 3 .

[0019] In special embodiments, the carrier material used according to the invention is “3D spacer fabric” or “nonwoven fabric” from the German Institutes for Textile and Fiber Research Denkendorf.

[0020] Another aspect of the present invention relates to a bioreactor comprising the cell culture carrier according to the present invention. Suitable bioreactors, in particular bioreactors for gas fermentation, are known in the prior art.

[0021] In this context, all relevant definitions and limitations mentioned above for the cell culture carrier according to the invention also apply to the bioreactors according to the invention.

[0022] Another aspect of the present invention relates to the use of a three-dimensionally formed, textile support material for immobilizing bacteria for gas fermentation. In this context, all relevant definitions and limitations mentioned above for the cell culture support according to the invention also apply to the use according to the invention. In particular, the support material and the bacteria used are defined as above. Furthermore, as with the cell culture support according to the invention, the bacteria preferably form a biofilm on the support material.

[0023] Methods for cultivating bacteria for gas fermentation, in particular for cultivating C. autoethanogenum, and for immobilizing them on the support material are known in the prior art.

[0024] Finally, another object of the present invention relates to the use of a cell culture carrier according to the present invention in gas fermentation for the production of organic substances.

[0025] In this context, all relevant definitions and limitations mentioned above for the cell culture carrier according to the invention also apply to the use according to the invention.

[0026] Processes for gas fermentation to produce organic substances are known in the prior art.

[0027] The organic substances to be produced are not subject to any special restrictions, as long as they can be obtained by gas fermentation with the aforementioned bacteria. These organic substances include alcohols and / or organic acids, such as ethanol, butanol, and / or acetic acid.

[0028] For gas fermentation according to the present invention, gases comprising H2, CO, and / or CO2 are preferably used. These include, for example, industrial exhaust gases. The present invention focuses on bacteria for gas fermentation, in particular Clostridium autoethanogenum, which is considered industrially very promising, and provides for the immobilization of the cells on special textiles in the reactor instead of membrane-based cell retention. The establishment of this technology offers two advantages.

[0029] Firstly, the increase in biomass is achieved within the bioreactor itself with significantly fewer technical measures. Furthermore, the use of the textile cell culture supports according to the invention results in a greater retention of rising bubbles during gas fermentation, thereby enhancing mass transfer within the bioreactor. Thus, the alternative technology for membrane-supported biomass retention according to the present invention advantageously allows for an increase in bioreactor productivity by raising the active biomass concentrations during gas fermentation. Additionally, the design of the textiles ensures that biofilm formation is protected by the modified hydrodynamic flow pattern, which reduces stress and simultaneously enables good biomass growth under improved mass transfer conditions.

[0030] The aforementioned bacteria for gas fermentation, including Clostridium autoethanogenum, could not previously be immobilized outside of membranes without deliberately applying stress conditions. Furthermore, the prior art's use of membranes demonstrates that it was evidently not possible to sustainably use the cells for large-scale production. In this context, it should be noted that industrial bioreactors for gas fermentation can easily reach sizes exceeding 500,000 liters. The core of the present invention therefore lies in the combination of suitable bacteria for gas fermentation, particularly Clostridium autoethanogenum, with the textile support materials used according to the invention for immobilization and, secondly, for the application of these supports in a bioreactor, which also leads to increased mass transfer due to the influence on bubble residence times in the bioreactor.Thus, the present invention provides, for the first time, a successful method for immobilizing bacteria for gas fermentation, in particular Clostridium autoethanogenum. Within the Clostridia group, there are several strains that can be immobilized using alternative methods. However, it has never before been possible to immobilize the industrially significant strain Clostridium autoethanogenum under the aforementioned conditions, i.e., to induce it to form a biofilm on support materials. Only through the use of the textile support materials employed according to the invention has it been possible to demonstrate that Clostridium autoethanogenum colonizes these materials.

[0031] The present invention thus makes the immobilization of Clostridium autoethanogenum biomass technically feasible for the first time on a production scale for gas fermentation. This results in completely new and advantageous properties for flow control, mass transport, and an increase in the maximum production capacity in large-volume gas fermentation setups.

[0032] The figures show:

[0033] Figure 1 :

[0034] Cultivation of C. autoethanogenum on textile “fleece” with 1200x magnification.

[0035] Figure 2:

[0036] Cultivation of C. autoethanogenum on textile “3D spacer fabric”.

[0037] Figure 3:

[0038] Cultivation of C. carboxidivorans on 3D spacer fabric. Figure 4:

[0039] SEM images of the textile "fleece" at 300x magnification without / with dimensioning of the fiber diameters.

[0040] Figure 5:

[0041] Cultivation of C. autoethanogenum on 3D spacer fabric in a bubble column. Comparison of performance data: cell density in suspension (OD600), ethanol and acetate titers, and organic carbon components with (column 2) and without textile embedded materials (column 1).

[0042] Figure 6:

[0043] Schematic representation of textile 1 “3D spacer fabric” (left: cross-section, middle and right: structural analysis).

[0044] Figure 7:

[0045] Textile 1 consisting of '3D spacer fabric' before cultivation of C. autoethanogenum. 2000x magnification, no biofilms visible.

[0046] Figure 8:

[0047] Textile 1, consisting of '3D spacer fabric', after the cultivation of C. autoethanogenum. Biofilms are clearly visible.

[0048] Figure 9:

[0049] Schematic representation of textile 2 "nonwoven". Thermally-mechanically bonded fiber nonwoven. The fibers have a round cross-section with a diameter between 14 and 22 pm. The fine cavities offer optimal support for microorganisms.

[0050] Figure 10:

[0051] Textile 2 consisting of "fleece" before the cultivation of C. autoethanogenum. Fiqur 11 :

[0052] Textile 2 consisting of “fleece” after the cultivation of C. autoethanogenum.

[0053] The present invention is explained in more detail with reference to the following non-limiting examples.

[0054] Examples

[0055] C. autoethanogenum and C. carboxidivorans were cultured using several textiles in 250 mL bottles with a working volume of 50 mL in medium DSM879 at 37°C and 150 rpm. Biofilm formation after one week is shown in Figures 1 to 3.

[0056] Example 2:

[0057] Bioreactor cultivations with C. autoethanogenum were carried out in a 1 L bubble column in DSM879 medium (yeast extract content only 0.5 g / L; pH 5.5; 0.1 M MES as pH buffer). Textile layers were placed in the column. Figure 8 shows the biofilm formation after 8 days as documented by SEM.

[0058] Figure 5 shows the advantage of the invention through immobilization of the cells (column 2) compared to the results with microorganisms alone in suspension (column 1). Clearly recognizable are (a) the much greater increase in biomass (shown by the optical density ODeoo) and (b) the increased production of the (exemplary) target products ethanol and acetate, which is also reflected in the formation of all products containing organic carbon.

[0059] C. autoethanogenum was cultured with two different textiles in 250 mL bottles containing 50 mL of DSM879 medium. The bottles were shaken at 37°C and 150 U / rnin. After two weeks of cultivation, with four 80% medium changes on days 4, 8, and 12, the textiles were removed and used for SEM analysis. Figures 6 and 9 show schematic representations of the textiles used. Figures 7, 8, 10, and 11 show the textiles before and after cultivation.

[0060] Discussion:

[0061] Within the scope of the present investigations, a variety of different textiles from the German Institutes for Textile and Fiber Research Denkendorf were examined. Two of these, namely "3D spacer fabric" and "nonwoven fabric," showed the formation of a biofilm on the respective fibers (Figs. 1 and 2). When these fibers were used in gas fermentation (bubble column) in bioreactors, it was shown that, under comparative conditions, approximately twice the biomass concentration and associated activity in the bioreactor could be achieved by immobilizing the cells in the biofilm.

Claims

Claims 1. Cell culture carrier, comprising (a) a three-dimensionally formed, textile support material and (b) acetogenic bacteria for gas fermentation, which form a biofilm on the support material.

2. Cell culture carrier according to claim 1, wherein the acetogenic bacteria for gas fermentation are selected from the group consisting of Clostridium autoethanogenum, Clostridium carboxidivorans, Clostridium ijungdahlii, Acetobacteri um woodii, Eubacterium callanderi, Acetobacte ri um wieringae and Eubacterium limosum.

3. Cell culture carrier according to claim 1 or 2, wherein the acetogenic bacteria for gas fermentation are selected from the group consisting of Clostridium autoethanogenum and Clostridium carboxidivorans.

4. Cell culture carrier according to one of claims 1 to 3, wherein the acetogenic bacteria for gas fermentation are Clostridium autoethanogenum.

5. Cell culture carrier according to any one of claims 1 to 4, wherein the textile carrier material is a spacer fabric, spacer knit, spacer knitted or a nonwoven fabric of pronounced thickness.

6. Cell culture carrier according to one of claims 1 to 5, wherein the textile carrier material consists of polyester terephthalate (PET).

7. Cell culture carrier according to one of claims 1 to 6, wherein the textile presentation of the textile carrier material is formed in the form of continuous fibers.

8. Cell culture carrier according to claim 7, wherein the continuous fibers are processed into the textile carrier material in the form of monofilaments, in the form of multifilaments or in mixtures with other fiber materials thereof.

9. Cell culture carrier according to one of claims 1 to 8, wherein the textile The carrier material is designed in the form of a spacer fabric, wherein the spacer fabric consists of two flat textile layers made of plastic threads as The top layers comprise a pile layer of spacer threads connecting the textile layers, with textured multifilament yarns further incorporated into the top layers.

10. Cell culture carrier according to claim 9, wherein the textile carrier material consists of 65 wt.% or more textured multifilament yarns, the remainder consisting of monofilaments.

11. Cell culture carrier according to claim 10, wherein the monofilaments are processed in the polar layer.

12. Cell culture carrier according to one of claims 9 to 11, wherein the textile carrier material (i) a basis weight of 700 g / m² 2 or more (ii) a thickness of 15 to 20 mm, and / or (iii) a specific surface area for immobilizing the bacteria of 1,000 m² 2 / m 3 or more.

13. Cell culture carrier according to one of claims 1 to 8, wherein the textile carrier material is designed as a nonwoven fabric consisting of mechanically bonded PET filaments.

14. Cell culture carrier according to claim 13, wherein the textile carrier material (i) a fiber diameter of 17 to 20 pm, 15 (ii) a basis weight of 150 g / m² 2 , (iii) a thickness of 1.5 to 2.0 mm, (iv) a density of 80,000 g / m³ 3 or more (v) an air permeability of about 2.0 m 3 / s*m 2 , (vi) a mean pore size distribution of 55 pm, and / or (vii) a specific surface area of ​​10,000 m² 2 / m 3 or more.

15. Bioreactor comprising the cell culture carrier according to any one of claims 1 to 14.

16. Use of a three-dimensionally formed, textile support material for immobilizing acetogenic bacteria for gas fermentation.

17. Use according to claim 16, wherein the acetogenic bacteria for gas fermentation are defined according to any one of claims 2 to 4.

18. Use according to claim 16 or 17, wherein the acetogenic bacteria form a biofilm on the carrier material.

19. Use according to any one of claims 16 to 18, wherein the textile carrier material is defined according to any one of claims 5 to 14.

20. Use of a cell culture carrier according to any one of claims 1 to 14 in gas fermentation for the production of organic substances.

21. Use according to claim 20, wherein the organic substances comprise alcohols and / or organic acids.

22. Use according to claim 20 or 21, wherein the organic substances comprise ethanol, butanol and / or acetic acid. 16 23. Use according to any one of claims 20 to 22, wherein the gases used for gas fermentation comprise H2, CO and / or CO2.

24. Use according to any one of claims 20 to 23, wherein industrial exhaust gases are used for gas fermentation.

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