PROCESS FOR BIOTECHNOLOGICAL PRODUCTION OF A BIOPRODUCT

Abstract

The invention relates to a process and an apparatus for the biotechnological production of a bioproduct. The process involves a first and second bioprocess carried out in a first and second bioreactor (2, 3), and a vessel (4). The first bioprocess is a carbon dioxide producing bioprocess resulting in the formation of the bioproduct, the second bioprocess a chemoautotrophic bioprocess in which CO.sub.2 produced in the first bioprocess is consumed by hydrogen oxidizing bacteria oxidizing H.sub.2 with O.sub.2 as electron acceptor. Electrolytically generated H.sub.2 and O.sub.2 are separated, and the O.sub.2 is fed directly into the second and/or first bioprocess, while H.sub.2 is first dissolved in a medium contained in the vessel (4). H.sub.2 saturated medium is then fed into the second bioprocess. Biomass produced in the second bioprocess is fed into the first bioprocess as an additional C and N source.

Claims

1. A process for the biotechnological production of a bioproduct, the process comprising: a) a first bioprocess comprising cultivation of first microorganisms in a first bioreactor in a first medium at a first pressure (p1), the first bioprocess resulting in the formation of the bioproduct and carbon dioxide (CO.sub.2), b) a second bioprocess comprising cultivation of second microorganisms in a second bioreactor in a second medium at a second pressure (p2), the second bioprocess being a process producing biomass and consuming molecular oxygen (O.sub.2), molecular hydrogen H.sub.2, and at least part of the (CO.sub.2) produced in the first bioprocess of step a), c) electrolyzing water into O.sub.2 and H.sub.2, feeding at least part of the O.sub.2 produced by electrolysis into the second bioprocess and/or into the first bioprocess, and feeding at least part of the H.sub.2 produced by electrolysis into a vessel containing a head space and a third medium at a third pressure (p3), the third medium being identical to the second medium in the second bioreactor, and d) feeding at least part of the H.sub.2-containing third medium from the vessel to the second bioprocess, wherein at least part of the biomass produced in the second bioprocess of step b) is used as a C-source for the first bioprocess of step a), wherein the CO.sub.2 produced in the first bioprocess is fed into the second bioprocess, wherein the first microorganisms are yeast cells, and wherein the second bioprocess involves chemolithoautotrophic consumption of O.sub.2, H.sub.2 and CO.sub.2. wherein the first, second and third medium are in each case an aqueous medium that is suitable for supporting growth of the first microorganisms and the second microorganisms.

2. The process according to claim 1, wherein p3p2p1.

3. The process according to claim 1, wherein the first bioprocess is an aerobic bioprocess.

4. (canceled)

5. The process according to claim 1, wherein the second bioprocess is performed at a temperature between 40-55 C.

6. The process according to claim 1, wherein part of the second medium of the second bioreactor containing the second microorganisms is withdrawn from the second bioreactor, the biomass contained in the second medium is at least partly separated from the second medium, hydrolyzed and fed into the first bioprocess, and the second medium separated from the biomass is recycled into the second bioprocess.

7. The process according to claim 1, wherein part of the second medium from the second bioreactor is fed into the vessel and sprayed into the head space of the vessel, and the same amount of third medium is fed from the vessel to the second bioreactor.

8. The process according to claim 1, wherein part of the O.sub.2 produced by electrolysis is fed into the first bioprocess.

9. The process according to claim 1, wherein the first microorganisms are microorganisms of the genus Rhodosporidium.

10. The process according to claim 1, wherein p1 is greater than atmospheric pressure.

11. The process according to claim 1, wherein the process is a continuous process.

12. An apparatus for the biotechnological production of a bioproduct according to the process of one of the preceding claims, the apparatus comprising: a) a first bioreactor being configured for carrying out a first bioprocess, wherein the first bioprocess comprises the cultivation of first microorganisms in a first medium at a first pressure p1, and wherein the first bioprocess results in the formation of the bioproduct and carbon dioxide CO.sub.2, b) a second bioreactor being configured for carrying out a second bioprocess, wherein the second bioprocess comprises the cultivation of second microorganisms in a second medium at a second pressure p2, and wherein the second bioprocess is a process producing biomass and consuming molecular oxygen O.sub.2, molecular hydrogen H.sub.2, and at least part of the CO.sub.2 produced in the bioprocess of step a), c) a vessel being configured to contain a head space and a third medium at a third pressure p3, and d) an electrolyzer for electrolyzing water into O.sub.2 and H.sub.2, and wherein the apparatus further comprises a first fluid connection fluidically connecting a first outlet of the first bioreactor to a first inlet of the second bioreactor, for the transfer of CO.sub.2 produced in the first bioreactor to the second bioreactor, a second fluid connection fluidically connecting a first outlet of the second bioreactor to a first inlet of the first bioreactor, for the transfer of biomass produced in the second bioreactor to the first bioreactor, a third fluid connection fluidically connecting a second outlet of the second bioreactor to a first inlet of the vessel, for the transfer of second medium to the vessel, a fourth fluid connection fluidically connecting a first outlet of the vessel to a second inlet of the second bioreactor, for the transfer of third medium to the second bioreactor, a fifth fluid connection fluidically connecting a first outlet of the electrolyzer to a third inlet of the second bioreactor, for the transfer of oxygen O.sub.2 to the second bioreactor, a sixth fluid connection fluidically connecting a first outlet of the electrolyzer to a third inlet of the first bioreactor, for the transfer of oxygen O.sub.2 to the first bioreactor, and a seventh fluid connection fluidically connecting a second outlet of the electrolyzer to a second inlet of the vessel, for the transfer of hydrogen H2 to the vessel, wherein the first, second and third medium are in each case an aqueous medium that is suitable for supporting growth of the first microorganisms and the second microorganisms.

13. The apparatus according to claim 12, wherein, the second fluid connection comprises, between the first outlet of the second bioreactor and the first inlet of the first bioreactor, a separator for separating the second microorganisms from the second medium, and a hydrolyzer for hydrolyzing the second microorganisms separated from the second medium by the separator.

14. The apparatus according to claim 12, wherein the third fluid connection fluidically connecting the second outlet of the second bioreactor to the first inlet of the vessel, extends into an upper part of the vessel comprising the head space, the extension comprising a nozzle for atomizing second medium fed into the vessel.

15. The apparatus according to claim 12, wherein the first fluid connection fluidically connecting the first outlet of the first bioreactor to the first inlet of the second bioreactor, comprises a CO.sup.2 concentrator for concentrating the CO.sub.2 before its transfer to the second bioreactor.

16. The apparatus according to claim 12, wherein the first fluid connection fluidically connecting the first outlet of the first bioreactor to the first inlet for the second bioreactor, comprises a CO.sub.2 storage tank for intermediately storing CO.sub.2.

Description

[0065] In the following, the invention will be described in further detail by way of example only with reference to the accompanying figures.

[0066] FIG. 1. Schematic illustration depicting an embodiment of an apparatus according to the invention. Gas streams in dashed lines, liquid streams in solid lines.

[0067] FIG. 2. Simplified illustration of the embodiment of an apparatus according to the invention shown in FIG. 2 with gas and liquid streams. Gas streams in dashed lines, liquid streams in solid lines.

[0068] FIG. 1 shows a simplified schematic representation of an apparatus 1 of the invention. The apparatus comprises three sections A, B and C. Section A comprises a first bioreactor 2, section B a second bioreactor 3, and section C a vessel 4, which may be configured as a third bioreactor. The first bioreactor 2 and second bioreactor 3 are equipped with a first stirrer 21 and a second stirrer 31 for agitating the first medium 23 in the first bioreactor 2 or the second medium 33 in the second bioreactor 3, respectively. A first pressure p1 prevails in the first bioreactor 2, a second pressure p2 in the second bioreactor 3 and a third pressure p3 in the vessel 4.

[0069] In section A, comprising the first bioreactor 2, a first bioprocess is carried out, the first bioprocess being a bioprocess in which a desired biobased chemical is formed by first microorganisms cultivated in the first bioreactor 2, accompanied by the formation of carbon dioxide, CO.sub.2. An example of such a first bioprocess is an aerated yeast fermentation with Rhodosporidium toruloides for variable lipid production. Here, carbon-containing waste materials and by-products (biomass hydrolysates, molasses, etc.), for example, are used as the primary C source. In addition to lipids, R. toruloides produces carotenoid-containing biomass, which can be used as feed in aquaculture (shrimp, salmon farms; see, e.g., Blomqvist, J., Pickova, J., Tilami, S. K. et al., 2018, Oleaginous yeast as a component in fish feed, Sci Rep 8, 15945, doi:10.1038/s41598-018-34232-x) or as a coloring agent in the food industry (see, e.g., Igreja, W. S.; Maia, F. d. A.; Lopes, A. S.; Chist, R. C., 2021, Biotechnological Production of Carotenoids Using Low Cost-Substrates Is Influenced by Cultivation Parameters: A Review, Int. J. Mol. Sci. 22, 8819, doi:10.3390/ijms22168819). The lipid profile can be adjusted by genetic modifications of R. toruloides (Wen Z., Zhang S., Odoh C. K., Jin M., Zhao Z. K., 2020, Rhodosporidium toruloidesA potential red yeast chassis for lipids and beyond, FEMS Yeast Research, Volume 20, Issue 5, foaa038, doi:10.1093/femsyr/foaa038). The lipid profile produced can be variably adapted for the cosmetics/food industry so that the lipids can, for example, be classified as palm oil-like to cocoa butter-like.

[0070] The first bioreactor 2 containing a first medium 23 with first microorganisms, here, for example, cells of the oleaginous yeast Rhodosporidium toruloides, is aerated with air 100. Alternatively or additionally, the first bioreactor 2 can be supplied with oxygen generated in an electrolyzer 5 electrolyzing water. The exhaust gas containing CO.sub.2 produced in the first bioreactor 2 is withdrawn from the head space 32 of the first bioreactor 2 by means of a compressor 9 and fed via a first fluid connection 61 into the bioprocess carried out in a second bioreactor 3 in section B as a C source. The first fluid connection 61, which may, for example, be a pipe or tube, is fluidically connecting a first outlet 25 of the first bioreactor 2 to a first inlet 34 of the second bioreactor 3. On its way to the second bioreactor 3, the CO.sub.2 is concentrated by means of a CO.sub.2 concentrator 8, for example, a membrane-based CO.sub.2 concentrator 8, so that CO.sub.2-depleted air 102 containing mainly N.sub.2 is removed from the exhaust gas stream. The gassing rate vg2 (see FIG. 2) with which CO.sub.2, or, more precisely, air enriched with CO.sub.2, is fed into the second bioreactor 3 is thus set by the aeration rate, i.e. the rate with which air 100 is fed into the first bioreactor 2, and the compressor 9. In the embodiment shown, the first fluid connection 61 further comprises, here, in the direction of the flow of the CO.sub.2, after the CO.sub.2 concentrator 8, a storage tank 13 for intermediately storing CO.sub.2. A bioproduct 101, for example a biobased chemical, produced in section A, e.g. a chemical compound being excreted into the first medium 23 or accumulated in the cells grown in the first bioprocess, can be withdrawn from the bioprocess in section A.

[0071] In section B, second microorganisms, namely chemolithotrophic so-called Knallgas bacteria, are grown in a second bioreactor 3 on CO.sub.2, H.sub.2 and O.sub.2. The second microorganisms cultivated in section B can be any microorganisms with the specified gas requirements. An example of a suitable microorganism is Hydrogenophilus thermoluteolus, which has a particularly high growth rate (Arai H, Shomura Y, Higuchi Y, Ishii M., 2018, Complete Genome Sequence of a Moderately Thermophilic Facultative Chemolithoautotrophic Hydrogen-Oxidizing Bacterium, Hydrogenophilus thermoluteolus TH-1, Microbiol Resour Announc.7(6):e00857-18, doi: 10.1128/MRA.00857-18). The bioprocess in section B receives CO.sub.2 and residual O.sub.2 from the associated bioprocess in section A, as well as pure H.sub.2 and O.sub.2 from an electrolyzer 5, as explained in more detail below.

[0072] O.sub.2 and H.sub.2 used by the hydrogen oxidizing bacteria grown in the second bioreactor 3 are generated by an electrolyzer 5. O.sub.2 is directly fed into the second bioreactor via a fifth fluid connection 65 fluidly connecting a first outlet 55 of the electrolyzer 5 and a third inlet 38 of the second bioreactor 3. In the embodiment of an apparatus 1 of the invention shown in FIG. 1 an extension 651 of the fluid connection 65 fluidly connects a third inlet 28 of the first bioreactor 2 with the fluid connection 65 and thus with the first outlet 55 of the electrolyzer 5, such that O.sub.2 generated by the electrolyzer 5 can also be fed into the first bioreactor 2 via a sixth fluid connection 66, the sixth fluid connection 66 sharing part of the fifth fluid connection 65. A two-way valve 86 may, for example, be used to divide the O.sub.2 gas stream into a first part, vg4 (see FIG. 2), being fed into the second bioreactor 3, and a second part, vg5, being fed into the first bioreactor 2 via extension 651. The valve 86 may alternatively be used to direct the O.sub.2 stream exclusively into the first bioreactor 2, or to direct the O.sub.2 stream intermittently into the second or the first bioreactor 2. The O.sub.2 gas may be stored intermediary in a separate storage tank (not shown).

[0073] Due to the significantly poorer solubility of H.sub.2 in the aqueous medium used, and in order to separate gas phases containing O.sub.2 or H.sub.2 for avoiding flammable spaces and thus create explosion protection, H.sub.2 is not directly fed into the second bioreactor 3. Rather, the H.sub.2 entry is relocated to section C comprising the vessel 4 containing a third medium 43, which, however, corresponds to the second medium 33 in the second bioreactor 3. H.sub.2 gas is fed into the vessel 4 via a seventh fluid connection 67 fluidly connecting a second outlet 57 of the electrolyzer 5 with a second inlet 46 of the vessel 4. The H.sub.2 gas may also be stored intermediary in a separate storage tank (not shown). The electrolyzer 5 builds up an overpressure in the vessel 4. In this embodiment, the pressure p3 prevailing in the vessel 4 is the highest pressure, compared to the pressures p1 and p2 in the other sections, i.e. sections A and B. The increased pressure provides a higher solubility coefficient of H.sub.2. The pressure is adjusted via the electric current I applied to the electrolyzer 5 (proportional to the H.sub.2 gassing rate vg3, see FIG. 2) and the position (degree of closure) of the second valve 82. The medium cycling between sections B and C has an additional positive effect on the H.sub.2 gas entry. For this medium cycling, third and fourth fluid connections 63, 64 are arranged between the second bioreactor 3 and the vessel 4. The third fluid connection 63 fluidly connects a second outlet 37 of the second bioreactor 3 with a first inlet 44 of the vessel, the first inlet 44 being arranged in an upper part 41 of the vessel 4 in the height of the head space 42 of the vessel 4. A first pump 10 is arranged in the third fluid connection 63 for pumping second medium 33 from the second bioreactor 3 to the vessel 4. The fourth fluid connection 64 fluidly connects a first outlet 45 of the vessel 4 with a second inlet 36 of the second bioreactor 3, and allows reflow of third medium 43 from the vessel 4 to the second bioreactor 3. The flow of the third medium 43 to the second bioreactor 3 can, for example, be controlled with a second valve 82. Part of the second medium 33 is withdrawn from section B and brought to a pressure >p3 with the first pump 10 in order to finely spray it through a nozzle 12 into the head space 42 of the vessel 4 of section C. This fine distribution in the head space 42 of the vessel 4 with H.sub.2 atmosphere ensures further enrichment of H.sub.2 in the liquid phase. The inflow of H.sub.2-saturated medium into the second bioprocess in section B can be regulated by the circuit via the first pump 10 and the second valve 82 using vl1 and vl2 (see FIG. 2). There will also be second microorganisms, i.e. Knallgas bacteria, in this cycle that can benefit from the different dissolved gas concentrations.

[0074] In section B there is a pressure p2 that is preferably lower than the pressure p3, which allows the dissolved H.sub.2 to be better released into the surrounding medium. Here the CO.sub.2 and O.sub.2 input is increased by stirring with a second stirrer 31 and optional gas cycling between sections A and B via valve 81, arranged in an ninth fluid connection 69 fluidly connecting a third outlet 39 of the second bioreactor 3 arranged at the height of the head space 32 of the second bioreactor 3 and a second inlet 26 of the first bioreactor 2 arranged at the height of the head space 22 of the first bioreactor 2.

[0075] The pressure p1 in section A is preferably lower than in B and C, but preferably slightly above atmospheric pressure. In sections B and C, overpressures can be reduced or controlled with valves 83 and 84, if required.

[0076] The biomass generated in section B is preferably continuously extracted via a second pump 11 and a separator 6, once a sufficient cell density of the second microorganisms has been reached. In this process, second medium 33 containing the second microorganisms is withdrawn from the second bioreactor 3, the cells are separated from the second medium 33 using a separator 6, for example a continuous centrifuge, e.g. a disk stack centrifuge, or a hollow fiber membrane module, hydrolyzed in a hydrolyzer 7 and fed into the first bioreactor 2. For this purpose, a second fluid connection 62, e.g. a pipe or tube, fluidly connects a first outlet 35 of the second bioreactor 3 with a first inlet 24 of the first bioreactor 2. The separator 6 is, in relation to the flow direction of second medium 33, arranged after the pump 11, and the hydrolyser 7 is arranged after the separator 6. In this embodiment, second medium 33 separated from the cells is returned to the bioprocess of section B via an eighth fluid connection 68 fluidly connecting the part of the separator 6 with the separated second medium 33 to a fourth inlet 30 of the second bioreactor 3. Optionally, second medium can be removed from the circuit via a fifth valve 85 for reprocessing, for example, and reintroduced in section C. The extracted biomass is hydrolyzed with the hydrolyzer 7 and fed to the bioprocess in section A as an additional C and N source. At least part of the biomass can also be withdrawn and used as a separate bioproduct, e.g. fish food. Depending on the mode of operation of the bioprocess in section A (batch or continuous, for example), the biomass work-up can be carried out continuously or at intervals. In this manner, an estimated 20-60% of the expensive glucose can be saved in a lipid fermentation step and the C balance can be significantly shifted in the direction of higher lipid yields.

[0077] Fresh medium 103 can be introduced into the process in section C, e.g. at a third inlet 48 of the vessel 4. First microorganisms and/or first medium 23 can be withdrawn from the section A, for example via a third outlet 29 of the first bioreactor 2.

[0078] FIG. 2 shows a simplified scheme of the apparatus shown in FIG. 1, and shows gas flows (dashed lines) and liquid flows (solid lines) into and between sections A, B or C (see description to FIG. 1).