PL242678B1 - Method of separating alpha-ketoglutaric acid (AKG) from real digestate fluids using an integrated membrane system - Google Patents

Abstract

The subject of the application is a method of separation of alpha-ketoglutaric acid (AKG) from real post-fermentation fluids obtained as a result of bioconversion of glucose in an integrated system type CE - UF - NF - EDBM, in which the real post-fermentation liquid obtained as a result of bioconversion of glucose containing: alpha-ketoglutaric acid with a concentration of 5 to 25 g/dm3, lactic acid at a concentration of 8.5 g/dm3, acetic acid at a concentration of 1.1 g/dm3, 1,2-propanediol at a concentration of 0.2 g/dm3, ethanol at a concentration of 17.5 g /dm3, and pH values from 2.4 to 3.7 are subjected to centrifugation, ultrafiltration, nanofiltration and electrodialysis with a bipolar membrane.

Description

The subject of the invention is a method of separating alpha-ketoglutaric acid (AKG) from real post-fermentation fluids using an integrated membrane system.

Alpha-ketoglutaric acid occurs in living organisms as an intermediate in the cycle of tricarboxylic acids and in the biosynthesis of amino acids. This compound has many interesting chemical and functional properties, which allows it to be used in various areas (Blonde-Cynober F., Aussel C., Cynober L., Nutrition, 19, 73-75, 2003; Kamzolova S. V., Morgunov I. G., Appl. Microbiol . Biotechnol., 97, 5517-5525, 2013; Wu N., Yang M., Gaur U. Xu H., Yao Y., Li D., Biomol. Ther., 24, 1-8. 2016; Song Y. ., Li J., Shin H-D., Liu L., Du G., Chen J., Bioresour. Technol., 219, 716-724, 2016; Bayliak M. M., Lylyk M. P., Shmihel H. V., Sorohynska O. M., Semchyshyn O. I., Storey J.M., Storey K.B., Lushchak V., Comp. Biochem. Physiol., 204, 28-39, 2017). It is used in the production of food additives, dietary supplements, pharmaceuticals, biodegradable polymers and preparations used in agriculture. In addition, AKG acid is used as a substrate in the biochemical diagnostics of, for example, hepatitis, myocardial infarction, muscular dystrophy, and others. The proven health-promoting properties of AKG acid make it possible to use it as a food additive (often in complexes, e.g. with amino acids).

Alpha-ketoglutaric acid (AKG) is one of the organic acids, an example of the so-called. green chemicals that can be obtained by bioconversion as a result of microbial metabolism (Chernyavskaya O. G., Shishkanova N. V., Il'chenko A. P., Finogenova T. V., Appl. Microbiol. Biotechnol., 53, 152-158, 2000; Holz M., Otto C. , Kretzschmar A., Yovkova V., Aurich A., Potter M., Marx A., Barth G., Appl. Microbiol. Biotechnol., 89, 1519-1526, 2011; Yin X, Madzak C., Du G ., Zhou J., Chen J., Appl. Microbiol. Biotechnol., 96, 1527-1537, 2012; Rywińska A., Juszczyk P., Wojtanowicz M., Robak M., Lazar Z., Tomaszewska L., Rymowicz W., Biomass Bioenerg., 48, 148-166, 2013; Cybulski K., Rymowicz W., Tomszewska-Hetman L., Rywińska A., Eng. Ap. Chem., 54, 74-76, 2015; Zeng W. ., Du G., Chen J., Li J., Zhou J., Process Biochem., 50, 1516-1522, 2015; Guo H., Su S., Madzak C., Zhou J., Chen H., Chen G., Appl. Microbiol. Biotechnol. 100, 9875-9884, 2016).

Biotechnological conversion of carbon sources (glycerol, molasses, n-paraffins, whey, ethanol, etc.) with the use of microorganisms to low-molecular organic compounds is an environmentally friendly method of obtaining valuable raw materials. However, the post-fermentation fluid formed in the bioconversion process is a very complex mixture, which, apart from the main product, e.g. AKG acid, contains significant amounts of biomass and a number of impurities, such as: residues of unreacted substrates, non-ionic organic compounds and numerous inorganic salts. In addition, it should be emphasized that the carboxylic acid (e.g. AKG) formed as a result of bioconversion present in the post-fermentation medium is not in the acid form, but as a salt (usually sodium) of a given carboxylic acid.

Many separation methods are proposed that can be used to separate low molecular weight organic compounds from post-fermentation aqueous solutions, including: crystallization: Gebauer D., Kellermeier M., Gale J. D., Bergstrom L., Colfen H., Chem. Soc. Rev., 43, 2348-2371, 2014; Li Q., Wang D., Wu Y., Li W. L., Zhang Y. J., Xing J. M., Su Z. G., Sep. Purif. Technol, 72, 294-300, 2010, adsorption: Nam H-G., Park K-M., Lim S. S., Mun S., J. Chemi. Eng. Data., 56, 464-471, 2011, precipitation: Cao Y., Zhang R., Sun C., Cheng T., Liu Y., Xian M., BioMed Res. Int., 1-12, 2013, or extraction methods: de Souza Moraes L., de Araujo Kronemberger F., Conceiào Ferraz H., Habert A. C., J. Ind. Eng. Chem., 21,206-211,2015; Zhang W., Liu X., Fan H., Zhu D., Wu X., Huang X., Tang J., Ind. crop. Prod., 86, pp. 231-238, 2016: Huh Y. S., Jun Y-S., Hong Y. K., Song H., Lee S. Y., Hong W. H., Process. Biochem., 41, 1461-1465, 2006; CN 1887843A; US 5,168,055 A. However, due to the use of additional chemicals, e.g. organic solvents, these techniques may have adverse environmental impacts. Numerous literature reports indicate that the use of membrane techniques allows for the effective and waste-free acquisition of valuable bioconversion raw materials (Manttari M., Lahti J., Hatakka H., Louhi-Kultanen M., Kallioinen M., A Feasibility Study of a Novel Electro-Membrane Based Process to Acidify Kraft Black Liquor and Extract Lignin, J. Mem. Sci., 490, 84-91, 2015; Li Q-Z., Jiang X-L., Feng X-J., Wang J-M., Sun Ch., Zhang H-B., Xian M., Liu H-Z., Recovery Processes of Organic Acids from Fermentation Broths in the Biomass-Based Industry, J. Microbiol. Biotechnol., 26, 1-8, 2016; Jones R. J., Massanet-Nicolau J., Guwy A. , Premier G. C., Dinsdale R. M., Reilly M., Removal and recovery of inhibitors of volatile fatty acids from mixed acid fermentations by conventional electrodialysis, Bioresource Technol., 189, 279-284,

2015; Zhang L., Weng H-X., Chen H-L., Gao C-J., Remove volatile organic compounds (VOCs) with membrane separation techniques, J. Environ. sci. (China), 14, 181-187, 2002; US 20140371486 A1; US 20050003499 A1; US 8999171 B2.

Due to the large amount of components present in real post-fermentation solutions, effective and efficient separation of low molecular weight organic compounds is problematic, and sometimes even impossible to achieve using a single separation technique. The presence of biomass as well as many different organic compounds and inorganic salts, especially magnesium and calcium salts, induce separation processes in multi-stage integrated systems, e.g. microfiltration - nanofiltration; electrodialysis - nanofiltration - Donnan dialysis; ultrafiltration - bipolar electrodialysis - evaporation - distillation - vacuum crystallization; nanofiltration - bipolar electrodialysis - reactive extraction (T. K. Mai, S. Rodtong, Y. Baimark, J. Rarey, A. Boontawan, Membrane-based purification of optically pure D-lactic acid from fermentation broth to poly(D-lactide) polymer, Journal of Membrane Science, 551, 2018, 180-190 P. Khunnonkwao, K. Jantama, S. Kanchanatawee, S. Galier, H. Roux-de Balmann, A two steps membrane process for the recovery of succinic acid from fermentation broth , Separation and Purification Technology, 207, 2018, 451-460, N. Phanthumchinda, S. Thitiprasert, S. Tanasupawat, S. Assabumrungrat, N. Thongchul, Process and cost modeling of lactic acid recovery from fermentation broths by membrane-based process , Process Biochemistry, 68, 2018, 205-213;W. Cao, Y. Wang, J. Luo, J. Yin, Y. Wan, Improving α, ω-dodecanedioic acid productivity from n-dodecane and hydrolysate of Candida cells by membrane integrated repeated batch fermentation, Bioresource Technology, 260, 2018, 9-15 P. Pal, R. Kumar, J. Nayak, S. Banerjee, Fermentative production of gluconic acid in membrane-integrated hybrid reactor system: Analysis of process intensification , Chemical Engineering & Processing: Process Intensification 122, 2017, 258-268; N. Thi, H. Thuy, A. Boontawan, Production of very-high purity succinic acid from fermentation broth using microfiltration and nanofiItration-assisted crystallization, Journal of Membrane Science, 524, 2017, 470-481; C. Jiang, Y. Wang, T. Xu, Membranes for the recovery of organic acids from fermentation broths. Membrane Technologies for Biorefining, Book chapter, 2016, pp. 135-161; C.J. Davey, A. Havill, D. Leak, D.A. Patterson, Nanofiltration and reverse osmosis membranes for purification and concentration of a 2,3-butanediol producing gas fermentation broth, Journal of Membrane Science, 518, 2016, 150-158; M. J. Woźniak, K. Prochaska, Fumaric acid separation from fermentation broth using nanofiltration (NF) and bipolar electrodialysis (EDBM), Separation and Purification Technology, 125, 2014, 179-186; K. Prochaska, K. Staszak, M. J. Woźniak-Budych, M. Regel-Rosocka, M. Adamczak, M. Wiśniewski, J. Staniewski, Bioresource Technol., 167, 219-225, 2014).

So far, there have been no reports indicating the use of multi-stage integrated membrane systems for the separation of alpha-ketoglutaric acid from real fermentation liquors.

The subject of the invention is a method of separating alpha-ketoglutaric acid (AKG) from real post-fermentation fluids using an integrated membrane system.

The essence of the invention is a method of separating alpha-ketoglutaric acid from a real post-fermentation fluid obtained as a result of glucose bioconversion in an integrated system of the type: centrifugation (CE) - ultrafiltration (UF) - nanofiltration (NF) - electrodialysis with a bipolar membrane (EDBM). In the method according to the invention, in the first place, the actual post-fermentation liquid containing: alpha-ketoglutaric acid with a concentration of 5 to 25 g/dm ³ , lactic acid with a concentration of 8.5 g/dm ³ , acetic acid with a concentration of 1.1 g/dm ³ 1,2-propanediol at a concentration of 0.2 g/dm ³ , ethanol at a concentration of 17.5 g/dm ³ and pH values from 2.4 to 3.7 are centrifuged to remove biomass and solid particles.

The centrifugation process is carried out at a speed of 4,000 rpm, achieving separation of the fermentation fluid from biomass and solid particles.

Then, the biomass-free fermentation fluid solution obtained after centrifugation is subjected to the ultrafiltration process, which is carried out using ceramic membranes with a limiting molar mass of kDa and an active surface of 320 cm ² at a temperature of 30±2°C and an applied transmembrane pressure of 0.2 MPa and a flow rate of feed 350 dm ³ /h.

The purified and clear permeate solution obtained after the ultrafiltration process is then subjected to nanofiltration, which is carried out using ceramic membranes with a limiting molar mass of 1000 Da and an active surface of 123 ^cm2 at a temperature of 30±2°C and an applied transmembrane pressure of 1.2 MPa and feed flow rate of 350 dm ³ /h.

During the nanofiltration separation stage, the product is partially separated from macromolecular impurities (proteins, sugars) and inorganic ions that are the remains of the culture medium.

Then, the purified permeate solution obtained after the nanofiltration process is separated as a diluate in the process of electrodialysis with a bipolar membrane, which is carried out using an electrodialysis stack equipped with 10 bipolar membranes, 10 anion exchange membranes and 1 cation exchange membrane, each with an active surface of 207 cm ² in specific process conditions: the presence of a solution of alpha-ketoglutaric acid with a concentration of 1 g/dm ³ in the concentrate chamber, the flow rate of working solutions equal to 100 dm ³ /h, the value of the current density 115 A/m ² and at a temperature of 25±2°C. It is preferable that the concentrate solution obtained after the process of electrodialysis with a bipolar membrane is subjected to concentration and crystallization to the solid form of alpha-ketoglutaric acid.

Thanks to the method of separation and concentration according to the invention, the following technical and operational effects were obtained:

• preliminary purification of the actual post-fermentation fluid from the remains of biological material and macromolecular compounds after the fermentation process and complete reduction of the turbidity of the solution in the process of centrifugation and ultrafiltration;

• partial separation of alpha-ketoglutaric acid from macromolecular compounds (proteins, sugars) and inorganic salts in the solution in the nanofiltration process;

• complete separation of alpha-ketoglutaric acid from the actual post-fermentation fluid in the process of electrodialysis with a bipolar membrane;

• conversion of salts of alpha-ketoglutaric acid to alpha-ketoglutaric acid by using bipolar membranes;

• obtaining high purity alpha-ketoglutaric acid with high concentration in a multi-stage separation process: type CE - UF - NF - EDBM in an integrated system.

The invention in an exemplary embodiment is presented in the drawing where Fig. 1 shows a schematic diagram of an integrated system; Fig. 2 shows a graph of changes in the permeate stream during the ultrafiltration process of the post-fermentation fluid obtained as a result of glucose bioconversion containing: alpha-ketoglutaric acid at a concentration of 5 to 25 g/dm ³ , lactic acid at a concentration of 8.5 g/dm ³ , acetic acid with a concentration of 1.1 g/dm ³ , 1,2-propanediol with a concentration of 0.2 g/dm ³ , ethanol with a concentration of 17.5 g/dm ³ , and pH values from 2.4 to 3.7; Fig. 3 shows a graph of changes in the permeate stream during the nanofiltration process of the post-fermentation liquid obtained as a result of glucose bioconversion containing: alpha-ketoglutaric acid at a concentration of 5 to 25 g/dm ³ , lactic acid at a concentration of 8.5 g/dm ³ , acetic acid with a concentration of 1.1 g/dm ³ , 1,2-propanediol with a concentration of 0.2 g/dm ³ , ethanol with a concentration of 17.5 g/dm ³ , and pH values from 2.4 to 3.7; Figure 4 shows the construction and configuration of the electrodialytic stack used; Fig. 5 shows the graph of the change in the concentration of alpha-ketoglutaric acid in the concentrate chamber during the EDBM of the actual post-fermentation liquid obtained as a result of glucose bioconversion containing: alpha-ketoglutaric acid at a concentration of 15 g/dm ³ , lactic acid at a concentration of 8.5 g/dm ³ , acetic acid at a concentration of 1.1 g/ ^dm3 , 1,2-propanediol at a concentration of 0.2 g/ ^dm3 , ethanol at a concentration of 17.5 g/ ^dm3 (pH 2.8, current density 115 A/ ^m2 ); Fig. 6 shows the graph of the change in the concentration of alpha-ketoglutaric acid in the concentrate chamber during the EDBM of the actual post-fermentation liquid obtained as a result of glucose bioconversion containing: alpha-ketoglutaric acid at a concentration of 25 g/dm ³ , lactic acid at a concentration of 8.5 g/dm ³ , acetic acid at a concentration of 1.1 g/ ^dm3 , 1,2-propanediol at a concentration of 0.2 g/ ^dm3 , ethanol at a concentration of 17.5 g/ ^dm3 (pH 2.4, current density 115 A/ ^m2 ).

The essence of the invention is illustrated by the following examples:

Example 1

Level 1

Ultrafiltration of the real post-fermentation fluid obtained as a result of glucose bioconversion containing: alpha-ketoglutaric acid with a concentration of 5 to 25 g/dm ³ , lactic acid with a concentration of 8.5 g/dm ³ , acetic acid with a concentration of 1.1 g/dm ³ , 1,2-propanediol with a concentration of 0.2 g/dm ³ , ethanol with a concentration of 17.5 g/dm ³ and pH from 2.4 to 3.7 after the centrifugation process (purification and clarification of the actual post-fermentation fluid).

A solution of the actual post-fermentation fluid obtained as a result of glucose bioconversion, with a volume of 20 dm ³ , after prior centrifugation of the biomass constituting the waste after the microbiological conversion process, containing: alpha-ketoglutaric acid with a concentration of 5 to 25 g/dm ³ , lactic acid with a concentration of 8, 5 g/dm ³ , acetic acid at a concentration of 1.1 g/dm ³ , 1,2-propanediol at a concentration of 0.2 g/dm ³ , ethanol at a concentration of 17.5 g/dm ³ , and pH values from 2, 4 to 3.7 were separated by ultrafiltration. The ultrafiltration process was carried out in a system equipped with a ceramic membrane, at a transmembrane pressure of 0.2 MPa, with a feed flow rate of 350 dm ³ /h, at a temperature of 30±2°C for about 10 hours. As a result of ultrafiltration, the separated solution was separated into two fractions with a volume of 15 dm ³ - permeate and 5 dm ³ - concentrate. The obtained permeate after ultrafiltration was separated using the nanofiltration technique. The change in the permeate stream obtained during the ultrafiltration process is shown in Fig. 2.

Stage 2

Nanofiltration of the real post-fermentation fluid obtained as a result of glucose bioconversion containing: alpha-ketoglutaric acid with a concentration of 5 to 25 g/dm ³ , lactic acid with a concentration of 8.5 g/dm ³ , acetic acid with a concentration of 1.1 g/dm ³ , 1,2-propanediol with a concentration of 0.2 g/dm ³ , ethanol with a concentration of 17.5 g/dm ³ and pH from 2.4 to 3.7 (partial removal of proteins, sugars and inorganic ions).

A solution of the actual fermentation fluid obtained after the initial centrifugation and ultrafiltration process, with a volume of 15 dm ³ , containing: alpha-ketoglutaric acid at a concentration of 5 to 25 g/dm ³ , lactic acid at a concentration of 8.5 g/dm ³ , acetic acid at concentration of 1.1 g/dm ³ , 1,2-propanediol at a concentration of 0.2 g/dm ³ , ethanol at a concentration of 17.5 g/dm ³ , and pH values from 2.4 to 3.7 were separated in the process nanofiltration. The nanofiltration process was carried out in a system equipped with a ceramic membrane, at a transmembrane pressure of 1.2 MPa, with a feed flow rate of 350 dm ³ /h, at a temperature of 30±2°C for about 4 hours. As a result of nanofiltration, the separated solution was separated into two fractions with a volume of 7 dm ³ - permeate and 8 dm ³ - concentrate. The permeate obtained after nanofiltration was separated by electrodialysis with a bipolar membrane. The change in the permeate stream obtained during the nanofiltration process is shown in Fig. 3.

Stage 3

Electrodialysis with a bipolar membrane of a real post-fermentation fluid obtained as a result of glucose bioconversion containing: alpha-ketoglutaric acid at a concentration of 15 g/dm ³ , lactic acid at a concentration of 8.5 g/dm ³ , acetic acid at a concentration of 1.1 g/dm ³ , 1,2-propanediol at a concentration of 0.2 g/dm ³ , ethanol at a concentration of 17.5 g/dm ³ (pH 2.8, current density 115 A/m ² ) (testing the effect of the initial AKG concentration).

The actual post-fermentation fluid obtained after the initial purification by centrifugation, ultrafiltration and nanofiltration techniques was subjected to electrodialysis with a bipolar membrane, containing: alpha-ketoglutaric acid at a concentration of 15 g/dm ³ , lactic acid at a concentration of 8.5 g/dm ³ , acetic acid at a concentration of 1 .1 g/dm ³ , 1,2-propanediol with a concentration of 0.2 g/dm ³ , ethanol with a concentration of 17.5 g/dm ³ and pH 2.8. The solution separated in a volume of 1.5 dm ³ was introduced into the diluate chamber, while the solution introduced into the concentrate chamber in a volume of 1.5 dm ³ contained alpha-ketoglutaric acid with a concentration of 1 g/dm ³ and a pH of about 2.5. The EDBM process was carried out in an electrodialyser equipped with a membrane stack consisting of 10 anion exchange membranes, 10 bipolar membranes and one cation exchange membrane with an active area of 207 cm ² for each membrane, at a constant current density of 115 A/m ² . The process was carried out for 35 minutes, with a constant flow rate of working solutions equal to 100 dm ³ /h, at a temperature of 27°C. The obtained changes in the concentration of alpha-ketoglutaric acid during the EDBM process in the concentrate chamber are shown in Fig. 5.

Example 2

The example only applies to the variant EDBM step (Step 3). Electrodialysis with a bipolar membrane of a real post-fermentation fluid obtained as a result of glucose bioconversion containing: alpha-ketoglutaric acid at a concentration of 25 g/dm ³ , lactic acid at a concentration of 8.5 g/dm ³ , acetic acid at a concentration of 1.1 g/dm ³ , 1,2-propanediol at a concentration of 0.2 g/dm ³ , ethanol at a concentration of 17.5 g/dm ³ (pH 2.4, current density 135 A/m ² ) (testing the effect of the initial AKG concentration).

The actual post-fermentation fluid obtained after the initial purification by centrifugation, ultrafiltration and nanofiltration techniques was subjected to electrodialysis with a bipolar membrane, containing: alpha-ketoglutaric acid at a concentration of 25 g/dm ³ , lactic acid at a concentration of 8.5 g/dm ³ , acetic acid at a concentration of 1 .1 g/dm ³ , 1,2-propanediol with a concentration of 0.2 g/dm ³ , ethanol with a concentration of 17.5 g/dm ³ and pH 2.4. The separated solution with a volume of 1.5 dm ³ was introduced to the diluate chamber, while the solution introduced to the concentrate chamber (1.5 dm ³ ) contained alpha-ketoglutaric acid with a concentration of 1 g/dm ³ and a pH of about 2.5. The EDBM process was carried out in an electrodialyser equipped with a membrane stack consisting of 10 anion exchange membranes, 10 bipolar membranes and one cation exchange membrane with an active area of 207 cm ² for each membrane, at a constant current density of 115 A/m ² . The process was carried out for 60 minutes, with a constant flow rate of working solutions equal to 100 dm ³ /h, at a temperature of 25°C. The obtained changes in the concentration of alpha-ketoglutaric acid during the EDBM process in the concentrate chamber are shown in Fig. 6

Claims (2)

1. A method of separating alpha-ketoglutaric acid from a real post-fermentation fluid obtained as a result of glucose bioconversion in an integrated system, characterized in that the real post-fermentation liquid obtained as a result of glucose bioconversion containing: alpha-ketoglutaric acid with a concentration of 5 to 25 g/dm ³ , lactic acid at a concentration of 8.5 g/dm ³ , acetic acid at a concentration of 1.1 g/dm ³ , 1,2-propanediol at a concentration of 0.2 g/dm ³ , ethanol at a concentration of 17.5 g/dm ³ , and pH values from 2.4 to 3.7 are first subjected to centrifugation at a speed of 4,000 rpm, obtaining separation of the post-fermentation liquid from biomass and solid particles, then the solution of post-fermentation liquid without biomass obtained after centrifugation is subjected to the ultrafiltration process, which is carried out using ceramic membranes with a critical molar mass of 50 kDa and an active surface of 320 cm ² at a temperature of 30±2°C and an applied transmembrane pressure of 0.2 MPa and a feed flow rate of 350 dm ³ /h, after which the purified permeate is subjected to nanofiltration which is carried out using ceramic membranes with a limiting molar mass of 1000 Da and an active surface of 123 cm ² at a temperature of 30±2°C and an applied transmembrane pressure of 1.2 MPa and a feed flow rate of 350 dm ³ /h, followed by a purified solution of the permeate obtained after the nanofiltration process is separated as a diluate in the process of electrodialysis with a bipolar membrane, which is carried out using an electrodialytic stack equipped with 10 bipolar membranes, 10 anion exchange membranes and 1 cation exchange membrane, each with an active surface of 207 cm ² under specific process conditions: the presence of a solution of alpha-ketoglutaric acid with a concentration of 1 g/dm ³ in the concentrate chamber, the flow rate of working solutions equal to 100 dm ³ /h, the value of the current density 115 A/m ² and at a temperature of 25±2°C. 2. The method according to claim, characterized in that the concentrate solution obtained after the process of electrodialysis with a bipolar membrane is subjected to concentration and crystallization to the solid form of alpha-ketoglutaric acid.