Extracellular polymeric substance iron-based nanocomposite and its application in separating and purifying lactic acid from fermentation broth

Abstract

The application discloses an extracellular polymer iron-based nanocomposite and application of the extracellular polymer iron-based nanocomposite in separation and purification of lactic acid in fermentation liquor. The extracellular polymer iron-based nanocomposite is prepared by using a complex high polymer compound synthesized and secreted to the extracellular by microorganisms in a growth process, has a large surface area and a porous structure, can be efficiently adsorbed, and has reproductivity and good expansibility, and has great industrial application potential. The extracellular polymer iron-based nanocomposite can be used for adsorbing lactic acid to separate and purify lactic acid in fermentation liquor.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of lactic acid separation and purification, and particularly relates to an extracellular polymeric substance iron-based nanocomposite and application thereof in separation and purification of lactic acid from fermentation broth. BACKGROUND

[0002] Lactic acid is a carboxylic acid with a molecular formula of C3H6O3 and contains a hydroxyl group, and belongs to alpha-hydroxy acid (AHA). Lactic acid has optical activity and is divided into L-lactic acid, D-lactic acid and DL-lactic acid, and is one of basic structural units of green chemistry. Lactic acid has a wide range of downstream products and is widely used in food, medicine and chemical industry. In recent years, degradable polymeric material polylactic acid (PLA) has attracted extensive attention. At present, the main method for producing lactic acid in industry is microbial fermentation, and L-lactic acid with an optical purity of about 99% can be obtained by using modified Bacillus coagulans (Food Industry Science and Technology, 2011, 10:5), which overcomes the shortcomings of small yield and low optical purity of L-lactic acid in traditional methods. However, the fermentation broth system is complex, and impurities include cells, proteins, residual sugars, organic acids, inorganic salts and other impurities, which are derived from raw materials, consumed nutrients or fermentation intermediates, so it is difficult to extract lactic acid from lactic acid fermentation broth.

[0003] At present, the method for extracting lactic acid from fermentation broth in industry is as follows: first, biological macromolecules such as bacteria in the fermentation broth are removed by plate and frame filtration, then lactic acid salt in the fermentation broth is converted into lactic acid by sulfuric acid acidification, and then plate and frame filtration is performed to remove calcium sulfate and other precipitates, followed by active carbon adsorption decolorization to remove pigments, and finally lactic acid product is obtained by crystallization and other means. This method has many unit operations, high labor intensity, and produces a large amount of calcium sulfate waste residue in the production process, and the product purity is low.

[0004] With the increasing demand for lactic acid, separation and purification methods have gradually diversified, including reactive distillation, molecular distillation, membrane separation, extraction, ion exchange resin method, etc. Reactive distillation process is complex, and the use of homogeneous catalysts can cause corrosion problems of equipment; molecular distillation requires high vacuum conditions, high equipment investment, and is difficult to expand production; membrane separation has high membrane cost and may cause membrane pollution; the product obtained by extraction has low purity, and the extractant contains toxicity, which can cause pollution problems. In recent years, ion exchange resin method has stood out due to its high selectivity, high efficiency, controllability, reusability, simple process and other advantages. Specifically: it can selectively adsorb and release target substances, and can effectively separate lactic acid from mixtures; it has a large surface area and pore structure, which can provide a large number of adsorption sites, thereby increasing the adsorption capacity and purification efficiency, making it a feasible method for handling large amounts of fermentation broth; the adsorption and desorption process of lactic acid can be controlled by adjusting the operating parameters (such as pH, temperature, salt concentration, etc.), which makes the purification process more controllable and can optimize the purification effect; ion exchange resin can be regenerated to restore its adsorption performance, so it can be reused, which reduces the cost of the purification process and reduces waste generation; compared with other separation and purification methods, ion exchange resin method is relatively simple to operate, it can be operated in batch or continuous mode, and has high applicability and operational flexibility. For example: Wang Peng et al. used anion exchange resin to extract lactic acid from corn starch wastewater fermentation broth. After centrifugal filtration, activated carbon decolorization and cation exchange resin column decalcification treatment of the fermentation broth, the lactic acid extraction was completed by using 315 type anion exchange resin adsorption and deionized water elution. The operation is simple, the selectivity and static exchange capacity are high, the lactic acid extraction rate is 78%-80%, the purity is 85-86%, and the eluent is non-toxic and easy to separate. For example: Yuya et al. used 001*7 strong acid ion exchange resin to remove iron and calcium ions from the pretreated crude lactic acid solution, and used D315 weak base ion exchange resin to remove sulfate and chloride ions. After ion exchange, the concentration of ions in the filtrate can be reduced to: Fe 3+ 0.1-5ppm, Ca 2 + 2-10ppm, Cl - 10-30ppm, SO4 2- 2-10ppm.

[0005] Although there are many polymer materials used to adsorb lactic acid in fermentation broth, there is no report on the use of complex high molecular compounds (Extracellular Polymeric Substances, EPS) synthesized and secreted by microorganisms during growth to adsorb lactic acid in fermentation broth. SUMMARY

[0006] To solve the above problems, the present application provides an extracellular polymeric substance iron-based nanocomposite and its application in separating and purifying lactic acid in fermentation broth.

[0007] In the first aspect of the present application, the embodiments of the present application provide a preparation method of an extracellular polymeric substance iron-based nanocomposite, comprising the following steps:

[0008] (1) Centrifuging a fermentation broth of Bacillus licheniformis CGMCC 2876, alcohol precipitating and freeze-drying the supernatant to obtain an extracellular polymeric substance;

[0009] (2) Dissolving the extracellular polymeric substance in distilled water, mixing uniformly, pouring into a reactor, introducing nitrogen into the reactor, exhausting oxygen in the system, heating in a water bath at 30-70℃, under the conditions of continuously introducing nitrogen and stirring, adding a potassium persulfate solution, stirring, then adding sodium sulfite, sealing the reactor after adding FeSO4·7H2O, stopping the introduction of nitrogen, reacting for 1-3h, cooling to room temperature after the reaction is completed, adding excess ethanol and stirring to make the reaction product precipitate, centrifuging, washing the precipitate with acetone three times, and vacuum drying to obtain an extracellular polymeric substance iron-based nanocomposite.

[0010] According to the preparation method of the extracellular polymeric substance iron-based nanocomposite provided by the embodiments of the present application, the extracellular polymeric substance iron-based nanocomposite is prepared by using the complex high molecular compound synthesized and secreted by microorganisms during growth, which has a large surface area and a porous structure for efficient adsorption, and has reproducibility and good expandability, and has great potential for industrial application.

[0011] Alternatively, in step (2), the mass ratio of the extracellular polymeric substance to FeSO4·7H2O is 1:2-8, and the amount of potassium persulfate is 0.4%-0.8% of FeSO4·7H2O.

[0012] In the second aspect of the present application, an extracellular polymeric substance iron-based nanocomposite prepared by the above preparation method is provided. The composite material can be used for adsorbing lactic acid in fermentation broth to separate and purify lactic acid in fermentation broth.

[0013] In the third aspect of the present application, the above extracellular polymeric substance iron-based nanocomposite is used for separating and purifying lactic acid in fermentation broth.

[0014] Specifically, it comprises: soaking the extracellular polymeric substance iron-based nanocomposite in ethanol for 4-6h, adjusting the initial pH value of the pretreated lactic acid fermentation broth to 1.5-3, column chromatographing at a flow rate of 1.3-1.8BV / h, using deionized water as an eluent, eluting the column at a flow rate of 0.7-1.4BV / h, and collecting the eluate to obtain a lactic acid solution.

[0015] Further, the pretreated lactic acid fermentation liquor is the lactic acid fermentation liquor after centrifugal filtration, activated carbon decolorization and cation exchange resin decalcification.

[0016] Additional aspects and advantages of the application will be set forth in part in the description that follows, and in part will become apparent to those skilled in the art upon examination of the following and / or can be learned by practice of the application. BRIEF DESCRIPTION OF DRAWINGS

[0017] Figure 1 Ion chromatogram of monosaccharide mixture standard (A) and B. licheniformis CGMCC 2876 extracellular polymer monosaccharide component (B).

[0018] Figure 2 Adsorbed lactic acid purity chart after four times reuse of the extracellular polymer iron-based nanocomposite under the same adsorption conditions DETAILED DESCRIPTION

[0019] The technical solutions of the present application will be described below by specific specific examples. It should be understood that the one or more method steps mentioned in the present application do not exclude the existence of other method steps before and after the combination steps or the insertion of other method steps between the explicitly mentioned steps; it should also be understood that these examples are only used to illustrate the present application and not to limit the scope of the present application. Moreover, unless otherwise stated, the numbering of each method step is only a convenient tool to identify each method step, and is not a limitation on the arrangement order of each method step or a limitation on the scope of the present application. Changes or adjustments of the relative relationship without substantial changes in technical content are also considered as the scope of the present application.

[0020] In order to better understand the above technical solutions, the exemplary embodiments of the present application will be described in more detail below. Although exemplary embodiments of the present application are shown, it should be understood that the present application can be implemented in various forms and should not be limited by the embodiments described herein. On the contrary, these embodiments are provided to enable a more thorough and complete understanding of the present application and to convey the full scope of the present application to those skilled in the art.

[0021] The test materials used in the present application are all ordinary commercially available products and can be purchased in the market.

[0022] The present application will be described below with reference to specific examples, and it should be noted that these examples are merely descriptive and do not limit the present application in any way.

[0023] Example 1 Preparation of extracellular polymer iron-based nanocomposite

[0024] (1) Extracellular polymer preparation method

[0025] Remove 1 mL of Bacillus licheniformis CGMCC 2876, an extracellular polymeric substance (EPS) strain, from the freezer (-80°C). Using an inoculation loop, transfer the bacterial culture from the glycerol tube onto LB agar and streak it onto a medium. Incubate at 37°C for 16 h. Pick a single colony of activated B. licheniformis from the LB agar and incubate it in 50 mL of seed culture medium for 16 h at 35°C and 100 rpm. Transfer the seed culture to fermentation medium (50 mL / 250 mL Erlenmeyer flask) at a 4% inoculation rate (v / v). Incubate the flask at 35°C and 100 rpm on a shaker for 48 h. Both the seed culture medium and the fermentation medium are LB agar.

[0026] After fermentation, the fermentation broth was centrifuged at 8000 rpm for 10 min, the supernatant was collected, anhydrous ethanol was added and mixed thoroughly, and the mixture was allowed to stand overnight at 4°C. The product after standing was centrifuged at 8000 rpm for 10 min, the supernatant was discarded, and the precipitate was freeze-dried in a freeze dryer for 24 h to obtain the extracellular polymer product.

[0027] Extracellular polymeric component analysis: After acid hydrolysis, the extracellular polymeric components were analyzed using ion chromatography. A mixed standard of 16 monosaccharide standards was used as a control. The 16 monosaccharide standards included fucose (Fuc), rhamnose (Rha), galacturonic acid (GalA), arabinose (Ara), glucose (Glc), xylose (Xyl), galactose (Gal), mannose (Man), fructose (Fru), ribose (Rib), glucuronic acid (GlcA), glucosamine hydrochloride (GlcN), galactosamine hydrochloride (GalN), N-acetyl-D-glucosamine (GlcNAc), mannuronic acid (ManA), and guluronic acid (GulA).

[0028] The results are as follows Figure 1 As shown, this provides a relatively intuitive representation of the types and amounts of monosaccharides contained in the extracellular polymer. Specifically, the proportion of monosaccharides in the extracellular polymer is Glc:GlcN:Gal:Man:Rha:Ara:GalN:GalA:Xyl:Fuc = 26.1:24.1:20.3:11.1:7.9:3.6:3.5:1.4:1.2:1. It can be seen that Glc, GlcN, and Gal are present in large quantities and in similar proportions in the extracellular polymer, while Man and Rha are present in smaller quantities.

[0029] (2) Preparation method of extracellular polymeric iron-based nanocomposites

[0030] Extracellular polymer 2g was dissolved in 50ml distilled water, mixed well, poured into the reactor, the reactor was purged with nitrogen, the oxygen in the system was exhausted, the water bath was heated to 70℃, under the condition of continuous purging with nitrogen and stirring, 10g / L potassium persulfate solution was added, stirred, then 10g / L sodium sulfite was added (the molar ratio of potassium persulfate to sodium sulfite was 1:1). After adding a certain amount of FeSO4·7H2O, the reactor was sealed, the nitrogen was stopped, and the reaction was carried out for 1h. The mass ratio of extracellular polymer to FeSO4·7H2O was 1:2, and the amount of potassium persulfate was 0.8% of FeSO4·7H2O. After the reaction was completed, the product was cooled to room temperature, an excess of ethanol was added and stirred to precipitate the reaction product, centrifuged, the precipitate was washed with acetone three times, and vacuum dried to obtain the product (extracellular polymer iron-based nanocomposite) as a gray-green powder, which was insoluble in water.

[0031] Example 2 Adsorption of extracellular polymer iron-based nanocomposite in lactic acid fermentation broth

[0032] The extracellular polymer iron-based nanocomposite was soaked in ethanol for 5h, then loaded into a column, and residual ethanol was washed with deionized water until the colorless solution became transparent. The pH of the lactic acid fermentation broth after centrifugal filtration, activated carbon decolorization, and cation exchange resin decalcification was adjusted to 2, and then loaded onto the column at a flow rate of 1.5BV / h. Deionized water was used as the eluent, and the column was eluted at a flow rate of 1.0BV / h. The eluate was collected to obtain a lactic acid solution, which was the completion of lactic acid extraction from the lactic acid fermentation broth. The purity reached 81% (the definition of purity is the percentage of lactic acid in the mass of all substances in the fermentation broth except water), and the total yield after lactic acid purification was 82% (the total yield of lactic acid is defined as the ratio of actual yield to theoretical yield).

[0033] Example 3

[0034] The same adsorption conditions as in Example 2 were used to investigate the effect of ethanol soaking time on lactic acid separation and purification. The difference is that the soaking time is 4h. The purity of lactic acid in this example reached 78%, and the total yield was 77%.

[0035] Example 4

[0036] The same adsorption conditions as in Example 2 were used to investigate the effect of ethanol soaking time on lactic acid separation and purification. The difference is that the soaking time is 6h. The purity of lactic acid in this example reached 75%, and the total yield was 74%.

[0037] Example 5

[0038] The same adsorption conditions as in Example 2 were used to investigate the effect of crude lactic acid initial pH on lactic acid separation and purification. The difference is that the initial pH is 1.5. The purity of lactic acid in this example reached 79%, and the total yield was 80%.

[0039] Example 6

[0040] The same adsorption conditions as in Example 2 were used to investigate the effect of the initial pH of the crude lactic acid on the separation and purification of lactic acid. The difference is that the initial pH is 3. In this example, the purity of lactic acid reaches 80%, and the total yield is 79%.

[0041] Example 7

[0042] The same adsorption conditions as in Example 2 were used to investigate the effect of the column flow rate on the separation and purification of lactic acid. The difference is that the column flow rate is 1.4 BV / h. In this example, the purity of lactic acid reaches 73%, and the total yield is 72%.

[0043] Example 8

[0044] The same adsorption conditions as in Example 2 were used to investigate the effect of the column flow rate on the separation and purification of lactic acid. The difference is that the column flow rate is 1.7 BV / h. In this example, the purity of lactic acid reaches 77%, and the total yield is 78%.

[0045] Example 9

[0046] The same adsorption conditions as in Example 2 were used to investigate the effect of the column flow rate on the separation and purification of lactic acid. The difference is that the column flow rate is 0.8 BV / h. In this example, the purity of lactic acid reaches 70%, and the total yield is 69%.

[0047] Example 10

[0048] The same adsorption conditions as in Example 2 were used to investigate the effect of the column flow rate on the separation and purification of lactic acid. The difference is that the column flow rate is 1.2 BV / h. The purity reaches 79%, and the total yield is 77%.

[0049] Test Example

[0050] The same adsorption conditions as in Example 2 were used to investigate the adsorption effect of extracellular polymeric substance iron-based nanocomposites and macroporous adsorption resins on lactic acid, acetic acid, and pyruvic acid, respectively. The specific data are shown in Table 1. It can be seen that the extracellular polymeric substance iron-based nanocomposites have higher adsorption performance compared to other materials.

[0051] Table 1: High-efficiency adsorption experimental data table of extracellular polymeric substance iron-based nanocomposites

[0052]

[0053] The same adsorption conditions as in Example 2 were used to investigate the adsorption performance of extracellular polymeric substance iron-based nanocomposites after four repeated adsorptions, and to explore their recyclability. From the results, it can be seen that the extracellular polymeric substance iron-based nanocomposites have good recyclability. Figure 2It can be seen that the extracellular polymeric substance iron-based nanocomposite still has good adsorption performance after four repeated adsorptions, and the purity of lactic acid is 81%, 78%, 75% and 73% in turn, indicating that the reusability is good.

[0054] In the description of the present specification, the description referring to the terms "one embodiment", "some embodiments", "an example", "a specific example", or "some examples" and the like means that the specific features, structures, materials or characteristics described in connection with the embodiment or example are included in at least one embodiment or example of the present application. In the present specification, the illustrative description of the above terms should not be understood as necessarily referring to the same embodiment or example. Moreover, the specific features, structures, materials or characteristics described can be combined in any one or more embodiments or examples in a suitable manner. Furthermore, those skilled in the art can combine and integrate different embodiments or examples described in the present specification.

[0055] Although the embodiments of the present application have been shown and described above, it is understood that the above-described embodiments are exemplary and are not to be construed as limiting the present application, and those skilled in the art can make changes, modifications, replacements and variations to the above-described embodiments within the scope of the present application.

Claims

1. Use of extracellular polymeric substance-iron based nanocomposite for separation and purification of lactic acid from fermentation broth, characterized in that, The preparation of the extracellular polymer iron-based nanocomposite comprises the following steps: (1) centrifuging the fermentation liquor of Bacillus licheniformis CGMCC 2876, alcohol precipitating the supernatant and freeze-drying to obtain extracellular polymer; (2) dissolving the extracellular polymer in distilled water, mixing uniformly, pouring into a reactor, introducing nitrogen into the reactor, exhausting oxygen in the system, heating in a water bath at 30-70℃, under the conditions of continuously introducing nitrogen and stirring, adding potassium persulfate solution, stirring, then adding sodium sulfite, adding FeSO4·7H2O, sealing the reactor, stopping the introduction of nitrogen, reacting for 1-3h, cooling to room temperature after the reaction is completed, adding excess ethanol and stirring, precipitating the reaction product, centrifuging, washing the precipitate with acetone three times, vacuum drying to obtain the extracellular polymer iron-based nanocomposite.

2. Use according to claim 1, wherein In step (2), the mass ratio of the extracellular polymer to FeSO4·7H2O is 1:2-8, and the amount of potassium persulfate is 0.4%-0.8% of FeSO4·7H2O.

3. The use according to claim 1, wherein the compound is ###0002### It comprises: Soaking the extracellular polymer iron-based nanocomposite in ethanol for 4-6h, adjusting the initial pH value of the pretreated lactic acid fermentation liquor to 1.5-3, column chromatographing at a flow rate of 1.3-1.8BV / h, using deionized water as eluent, eluting the column at a flow rate of 0.7-1.4BV / h, collecting the eluent to obtain a lactic acid solution.

4. The use according to claim 3, wherein the compound is ###0002### The pretreated lactic acid fermentation liquor is lactic acid fermentation liquor after centrifugal filtration, activated carbon decolorization and cation exchange resin decalcification.

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