One-step process for the conversion of CO2 to organic molecules using a socialized microbial consortium

Abstract

A method for converting CO2 into one or more organic molecules selected from the group consisting of cadaverine, glutarate, 5-aminovalerate, and mixtures thereof, comprising the steps of: (a) providing a gas mixture containing CO2, (b) supplying this gas mixture to an associated microbial consortium in a liquid medium under anaerobic conditions, wherein the microbial consortium comprises acetogenic bacteria and genetically modified Corynebacterium glutamicum, wherein the Corynebacterium glutamicum is genetically modified such that it can thrive under anaerobic conditions without additives, and (c) incubating the microbial consortium under conditions and for a duration that enable (i) the acetogenic bacteria to convert the CO2 contained in the gas mixture into intermediate products selected from the group consisting of acetate, short-chain organic acids, short-chain alcohols, and mixtures thereof,and (ii) the genetically modified Corynebacterium glutamicum further converts said intermediates into organic molecules selected from the group consisting of cadaverine, glutarate, 5-aminovalerate and mixtures thereof.

Description

[0001] The present invention relates to processes for the conversion of CO2 to one or more organic molecules selected from the group consisting of cadaverine, glutarate, 5-amino-valerate and mixtures thereof by an associated microbial consortium in a liquid medium under anaerobic conditions, wherein the microbial consortium comprises acetogenic bacteria and genetically modified Corynebacterium glutamicum.

[0002] Global climate change is caused, among other things, by the release of greenhouse gases such as CO2. At the same time, geopolitical crises are leading to a situation where long-standing supply chains for raw materials, for example, for the chemical manufacturing industry, are no longer guaranteed. This leads to supply disruptions, which ultimately have a significant negative impact on Germany as a business and manufacturing location. It is therefore essential to make the manufacturing industry more resilient to these challenges. This requires establishing alternatives to the previously dominant fossil carbon sources.

[0003] One possibility is the use of CO2 and CO from point emissions. These can be, for example, exhaust gases from combustion processes, or emissions in which CO2 is intrinsically released due to chemical reactions, i.e. reactions in which the focus is not on heat generation but on material conversion.

[0004] Such processes can be found, for example, in the cement industry. About two-thirds of a cement plant's CO2 emissions are attributable to the oxidation of lime sand, which is necessary to achieve sufficiently high clinker qualities. Other examples are the CO2, CO, and H2 emissions in steel mills.

[0005] Current processes for producing raw materials for the chemical industry are dominated by the use of fossil resources. Alternative research approaches are known that exploit the production of these products from sugar or glycerin. While the latter are also sustainable, they require large areas of agricultural land to provide the substrate. For example, cadaverine can be combined with succinate to produce the thermoplastic polyamide P54, which is currently consumed at approximately 3.5 million tons annually. Assuming simply that sugar-based bioprocesses with a 50% yield are used for both cadaverine and succinate, completely replacing the fossil route would require 7 million tons of sugar annually, which would necessitate cultivation on approximately 546,000 hectares of agricultural land.This area exceeds the 397,000 hectares used for sugar beet cultivation in Germany in 2021 and therefore does not represent a viable alternative for establishing a resilient economy.

[0006] In this context, the publication Rohles et al. (2016) (Rohles, Ch. M. et al.; Microbial Cell Factories, 15:154; 2016) describes the biotechnological production of a Corynebacterium glutamicum strain for the production of 5-aminovalerate and glutarate. Furthermore, the publication Rohles et al. (2018) (Rohles, Ch. M. et al.; Green Chemistry, 20; 2018, pp. 4662-4674) describes the production of glutaric acid and bionylon using biotechnologically produced Corynebacterium glutamicum. The publication Kim et al. (2018) (Kim, HT et al.; ACS Sustainable Chemistry Engineering, 2018) describes the biotechnological production of a Corynebacterium glutamicum strain for the production of cadaverine and the subsequent synthesis of biopolyamide 510. The publication Kim et al. (2019) (Kim, HT et al.The publication by Baritugo et al. (Baritugo, K.-A.; ACS Sustainable Chemistry Engineering, 2019) also describes the biotechnological production of a Corynebacterium glutamicum strain for the production of cadaverine and the subsequent synthesis of biopolyamide 510. The publication by Baritugo et al. (Baritugo, K.-A.; Applied Microbiology and Biotechnology, 102; 2018; pp. 3915-3937) describes the biotechnological production of a Corynebacterium glutamicum strain for the fermentative production of chemicals in a biorefinery. Finally, the publication by Chae et al. (Chae, TU et al.; Metabolic Engineering; 2019) describes the biotechnological production of microorganisms for the production of dicarboxylic acids and diamines. The present invention is therefore based on the object of providing processes for the production of relevant raw materials for the chemical manufacturing industry that use CO2 and optionally CO2 as the sole carbon source.

[0007] This object is achieved by the embodiments characterized in the claims.

[0008] In particular, the invention provides a process for converting CO2 into one or more organic molecules selected from the group consisting of cadaverine, glutarate, 5-amino-valerate and mixtures thereof by an associated microbial consortium in a liquid medium under anaerobic conditions, wherein the microbial consortium comprises acetogenic bacteria and genetically modified Corynebacterium glutamicum.

[0009] Accordingly, the present invention relates to a process for converting CO2 into one or more organic molecules selected from the group consisting of cadaverine, glutarate, 5-aminovalerate and mixtures thereof, comprising the steps: (a) Providing a gas mixture containing CO2, (b) supplying said gas mixture to an associated microbial consortium in a liquid medium under anaerobic conditions, wherein the microbial consortium comprises acetogenic bacteria and genetically modified Corynebacterium glutamicum, wherein the Corynebacterium glutamicum is genetically modified such that it can thrive under anaerobic conditions without additives, and (c) incubating the microbial consortium under conditions and for a period that allow (i) the acetogenic bacteria convert the CO2 contained in the gas mixture into intermediate products selected from the group consisting of acetate, short-chain organic acids, short-chain alcohols and mixtures thereof, and (ii) the genetically modified Corynebacterium glutamicum further converts said intermediates into organic molecules selected from the group consisting of cadaverine, glutarate, 5-aminovalerate and mixtures thereof.

[0010] The gas mixture provided in step (a) of the process according to the invention can be a CO2-containing exhaust gas, for example from the cement, construction, or steel industries, from breweries, coal-fired power plants, or oil or gas production facilities. In addition to CO2, the gas mixture can also contain CO, which can also be converted by the acetogenic bacteria to the aforementioned intermediate products. In preferred embodiments, the CO2 contained in the gas mixture, or the CO2 and CO contained in the gas mixture, represent the only available carbon source. The gas mixture can also contain H2.

[0011] In preferred embodiments, the gas mixture provided in step (a) of the process according to the invention contains no O2. In particular, the O2 present could pose a problem for (strictly) anaerobic acetogenic microorganisms, since their metabolic performance at least ceases in the presence of O2, if not even inactivates essential enzymes. Accordingly, the process according to the invention can comprise a gas mixture preparation step before step (a). This can be the selective separation of CO2 from exhaust gas for further use in the process according to the invention. Alternatively, residual O2 contents can be converted, for example, by substoichiometric oxidation of methane to CO and H2. The thus pretreated exhaust gas is then fed to the process according to the invention. Corresponding processes are not subject to any particular restrictions and are known in the prior art.

[0012] In step (b) of the process according to the invention, the gas mixture provided in step (a) is fed under anaerobic conditions to an associated microbial consortium in a liquid medium. This consortium comprises acetogenic bacteria and genetically modified Corynebacterium glutamicum, wherein the Corynebacterium glutamicum is genetically modified such that it can thrive under anaerobic conditions without additives.

[0013] Suitable anaerobic acetogenic bacteria are not subject to any particular restrictions, are known in the art, and can be readily selected by a person skilled in the art. Preferably, the acetogenic bacteria are selected from the group consisting of Acetobacterium woodii, Clostridium spec., in particular Clostridium autoethanogenum, C. bovifaecis, C. carboxidivoras, C. dreakei, C. kluyveri, C. lungdahlii, C. luticellarii, C. muellerianum, C. ragsdalei, C. scatologenes, C. pfennigii, Archaeoglobus fulgidus, Eubaceterium limosum, Terrrisporobacter glycolicus, and mixtures thereof.

[0014] Furthermore, genetically modified Corynebacterium glutamicum, which are genetically modified in such a way that they can thrive under anaerobic conditions without additives, are known in the art. In preferred embodiments, the genetically modified Corynebacterium glutamicum is the strain Corynebacterium glutamicum GRLys1, or those C. glutamicum strains that have the same functionality. In a further preferred embodiment, the genetically modified Corynebacterium glutamicum is a cadaverin-producing strain, particularly preferably Corynebacterium glutamicum (pVWEx1-ldcC). Furthermore, C. glutamicum strains can be used that can utilize succinate and other C4-dicarboxylic acids by overexpression of the native genes dctA, dccT and optionally mctC. In addition, C. glutamicum strains known in the art that produce cadaverine (Prell, C. et al.; Metabolic engineering of Corynebacterium glutamicum for de novo production of 3-hydroxycadaverine; Current Research in Biotechnology, 4; 2022; pp. 32-46), Glutarat (Prell, C. et al.; Adaptive laboratory evolution accelerated glutarate production by Corynebacterium glutamicum; Microb Cell Fact, 20(1); 2021), oder 5-Amino-Valerat (Jorge, J. M. P. et al.; A new metabolic route for the fermentative production of 5-aminovalerate from glucose and alternative carbon sources; Bioresource Technology, 245, Part B; 2017; pp. 1701-1709) herstellen können.

[0015] In this context, the term "associated" means that all bacteria contained in the microbial consortium interact with each other. Indirect association occurs when, for example, the liquid or gaseous effluent or exhaust stream of one culture is used to co-feed the second culture. Direct association occurs when both microbial cultures are cultivated simultaneously in the same bioreactor.

[0016] In this context, suitable liquid media (culture media) that can be used in the process according to the invention are not subject to any particular restrictions and are known in the art.

[0017] In preferred embodiments, the gas mixture is supplied to the microbial consortium by passing the gas mixture through the liquid medium. This process is preferably continued during step (c) of the process according to the invention. Accordingly, the gas mixture is preferably passed through the liquid medium in step (b) and during step (c). Corresponding methods are known in the art and include, for example, the use of one or more injection tubes.

[0018] Furthermore, step (b) of the process according to the invention is preferably carried out in a bioreactor. Suitable bioreactors are not subject to any particular restrictions and are known in the art.

[0019] In step (c) of the process according to the invention, the microbial consortium is incubated under conditions and for a duration which enable (i) the acetogenic bacteria to convert the CO2 (and any CO present) contained in the gas mixture into intermediate products selected from the group consisting of acetate, short-chain organic acids, short-chain alcohols and mixtures thereof, and (ii) the genetically modified Corynebacterium glutamicum to further convert said intermediate products into organic molecules selected from the group consisting of cadaverine, glutarate, 5-amino-valerate and mixtures thereof.

[0020] The short-chain organic acids produced in step (c)(i) include, for example, formate, acetate, lactate, propionate, pyruvate, succinate, and other C4 dicarboxylic acids. Furthermore, the short-chain alcohols produced in step (c)(i) include, for example, ethanol, 2,3-butanediol, butanol, and hexanol.

[0021] Suitable conditions (e.g., temperature, pH) and incubation times that enable the processes mentioned above under (c)(1) are known in the art and can be readily determined and selected by a person skilled in the art. These include, for example, a pH in the range of pH 4 to pH 9, preferably pH 5 to pH 8, and a temperature in the range of 25 to 40°C, preferably 30 to 37°C.

[0022] The process according to the invention may further comprise a step (d) of purifying the produced organic molecules from the liquid medium containing the microbial consortium. Suitable purification methods are not subject to any particular restrictions and are known in the art.

[0023] The present invention is based on the object of producing products as raw materials for the chemical industry, such as cadaverine (1,5-pentanediamine, also known as diaminopentane, CAD), 5-aminovalerate, and / or glutarate, from CO2 (and optionally CO) as the sole carbon source. In addition to CO, H2 is also used as a reducing agent. The conversion takes place, for example, in a bioreactor.

[0024] The process presented here uses acetogenic microorganisms, but combines them with other specially produced recombinant microorganisms (here: Corynebacterium glutamicum) to produce the mentioned products in a single bioreactor.

[0025] In this context, only the production of biofuels in a single bioreactor has been described so far. The production of more complex molecules, such as those required as raw materials for the chemical industry, has so far only been known in multi-stage approaches, usually based on more highly reduced carbon sources (such as sugar, glycerol, etc.).

[0026] The process proposed here aims to produce valuable materials, particularly cadaverine, 5-aminovalerate, and / or glutarate, from CO2 and, where appropriate, CO and / or H2-containing exhaust gases. These substances are, among other things, starting materials for the production of plastics (CAD) and fine chemicals.

[0027] This assumes that either a CO2-containing exhaust air stream or one containing CO2, CO, and / or H2 is available from an emissions source. Residual O2 levels may still be present in the exhaust gas from combustion processes. These pose a problem for (strictly) anaerobic acetogenic microorganisms, as their metabolic performance at least ceases in the presence of O2, if not even inactivates essential enzymes.

[0028] It may therefore be necessary to provide an exhaust gas preparation step. This can involve the selective separation of CO2 from the exhaust air for further use in the process according to the invention. Techniques for this exist and are used particularly for CCS (Carbon Capture and Storage) processes. Alternatively, residual O2 contents can be converted to CO and H2, for example, by substoichiometric oxidation of methane. The thus pretreated exhaust gas is then fed to the process according to the invention.

[0029] If the exhaust air stream does not contain enough electrons for the necessary reductions, additional H2 and / or CO must be added as gases.

[0030] What is new about the process according to the invention is that the conversion of CO2 (and optionally CO) as the sole carbon source to the desired products is achieved in a single bioreactor.

[0031] The present invention makes CO2 and CO accessible as alternative carbon sources for the production of desired products. This is all the more important because the alternative access via sugar cannot be successfully implemented in Germany due to agricultural constraints. If the goal of self-sufficient production is pursued, CO2 must be considered as an oxidized carbon source.

[0032] The single-stage implementation also brings economic advantages for investment and operating expenses, which enables a promising realization.

[0033] At the same time, the need for very large quantities of CO2 solves a pressing problem for the domestic cement industry, as these CO2 emissions do not have to be offset by certificates. An average cement plant emits approximately 600,000 tons of CO2 per year, approximately two-thirds of which comes from mineral sand lime. Thus, the output of a single plant would theoretically be sufficient to cover, for example, more than 10% of the global P54 demand annually.

[0034] The figures show: Fig. 1: A. woodii was cultivated autotrophically in the bioreactor and, after 72 h, 40 mL portions were transferred into serum bottles. Simultaneously, C. glutamicum GLSA strains were cultivated aerobically, centrifuged, and transferred to batches 1-4 at OD4. Under anaerobic conditions, the conversion of acetate from A. woodii to CAD occurred. Batch 1 incorporates gassing with H2:CO2 in a medium without tryptone. This batch is closest to a large-scale, anaerobic conversion. The pH value in batches 1-4 was 7.2. Fig. 2: CAD formation after 72 h in Example 1: 1. Cadaverine standard 500 mM. 3. Medium sample (0 control), 2. Supernatant sample after 72 h.

[0035] The present invention is further explained using the following non-limiting example. Example:

[0036] Genetic modification of C. glutamicum was carried out in such a way that the resulting strain can, for example, produce cadaverine (CAD) anaerobically using acetate.

[0037] Furthermore, process engineering work was carried out in such a way that the microbial consortium consisting of Acetobacterium woodii (acetate producer) and C. glutamicum produces the target product CAD anaerobically from CO2, CO and H2.

[0038] For this purpose, the genetically modified C. glutamicum strain mentioned above was used alongside the wild-type A. woodii in shake flasks and bioreactors as part of the process engineering work. a) Preliminary tests:

[0039] The CAD producer C. glutamicum GSLA requires KNO3 under anaerobic conditions for nitrate respiration, i.e., for the disposal of excess electrons to avoid the undesirable formation of other byproducts (such as lactate). It was previously unknown whether the acetate producer A. woodii tolerates KNO3. Preliminary experiments in so-called 'hungate tubes' in DSMZ medium with and without KNO3 and using fructose as a carbon source showed that 30 mM KNO3 could be added. The growth phenotype in the comparison test was the same. b) Test series in shake flasks and bioreactor

[0040] Test series were conducted on the co-cultivation of C. glutamicum GSLA (pVWEx1-ldcC) and A. woodii. For the purposes of the present invention, the formation of CAD from CO2 + H2 should take place in a one-pot approach. This has obvious advantages by minimizing the necessary investment and operating costs. It should be noted that A. woodii can only be cultivated anaerobically, while C. glutamicum cannot grow anaerobically. Therefore, the approach envisages that previously aerobically cultivated C. glutamicum strains convert acetate, for example, into CAD in a dormant anaerobic co-culture.

[0041] Therefore, the Fig. 1 was carried out. All approaches showed CAD formation ( Fig. 2). c) Further experiments in the bioreactor

[0042] A. woodii was cultivated anaerobically in a 3.5 L bioreactor in the presence of KNO3 and gassed with a mixture of 80:20 v / v H2:CO2 at ambient pressure. In the batch setup, an OD600 of 1.15 and an acetate concentration of 3.4 g / L at pH 6.5 were achieved. No ethanol formation was observed, which is favorable for the subsequent uptake of acetate by C. glutamicum.

Claims

[1] A process for converting CO2 into one or more organic molecules selected from the group consisting of cadaverine, glutarate, 5-aminovalerate and mixtures thereof, comprising the steps: (a) Providing a gas mixture containing CO2, (b) supplying said gas mixture to an associated microbial consortium in a liquid medium under anaerobic conditions, wherein the microbial consortium comprises acetogenic bacteria and genetically modified Corynebacterium glutamicum, wherein the Corynebacterium glutamicum is genetically modified such that it can thrive under anaerobic conditions without additives, and (c) incubating the microbial consortium under conditions and for a period that allow (i) the acetogenic bacteria convert the CO2 contained in the gas mixture into intermediate products selected from the group consisting of acetate, short-chain organic acids, short-chain alcohols and mixtures thereof, and (ii) the genetically modified Corynebacterium glutamicum further converts said intermediates into organic molecules selected from the group consisting of cadaverine, glutarate, 5-aminovalerate and mixtures thereof. [2] Process according to claim 1, wherein the gas mixture provided in step (a) further contains CO and this CO is also converted by the acetogenic bacteria to said intermediate products. [3] A process according to claim 1 or claim 2, wherein the CO2 contained in the gas mixture or the CO2 and CO contained in the gas mixture are the only available carbon source. [4] A process according to any one of claims 1 to 3, wherein the gas mixture provided in step (a) further contains H2. [5] A process according to any one of claims 1 to 4, wherein the gas mixture provided in step (a) does not contain O2. [6] Method according to one of claims 1 to 5, wherein the acetogenic bacteria are selected from the group consisting of Acetobacterium woodii, Clostridium spec., and mixtures thereof. [7] A method according to any one of claims 1 to 6, wherein the genetically modified Corynebacterium glutamicum is a cadaverine-producing strain. [8] A process according to any one of claims 1 to 7, wherein the gas mixture is passed through the liquid medium in step (b) and during step (c). [9] A process according to any one of claims 1 to 8, wherein step (b) is carried out in a bioreactor. [10] A process according to any one of claims 1 to 9, wherein the short chain organic acids produced in step (c)(i) comprise lactate. [11] A process according to any one of claims 1 to 10, wherein the short chain alcohols produced in step (c)(i) comprise ethanol. [12] A method according to any one of claims 1 to 11, further comprising the step (d) purifying the produced organic molecules from the liquid medium containing the microbial consortium.

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