Method for extracting polyhydroxyalkanoate by using natural eutectic solvent
By using a natural eutectic solvent to treat fresh bacterial suspensions, the problem of toxicity and harmfulness of traditional extraction solvents is solved, achieving efficient and environmentally friendly PHA extraction and reducing production costs.
Patent Information
- Application Number
- CN202511474021.1
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-10-15
- Publication Date
- 2026-01-09
AI Technical Summary
Traditional extraction solvents are toxic and harmful, and it is difficult to extract PHA from fresh microbial communities, resulting in high production costs. Existing methods are time-consuming and energy-intensive.
Fresh bacterial suspensions were treated with two natural eutectic solvents (CA-NADES and Ac-NADES), and PHA was extracted through stirring, centrifugation and precipitation steps, avoiding the use of toxic solvents, simplifying the operation and reducing energy consumption.
This method enables the extraction of PHA with high purity and high yield, reducing production costs and minimizing adverse environmental impacts.
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Figure CN121294564A_ABST
Abstract
Description
Technical Field
[0001] This invention relates to the field of extraction and separation of polyhydroxy fatty acid esters, and more particularly to a method for extracting polyhydroxy fatty acid esters (PHA) from mixed bacterial cultures using two natural eutectic solvents (NADES). Background Technology
[0002] Polyhydroxyalkanoates (PHAs) are intracellular polymers synthesized by microorganisms. PHAs possess properties similar to petroleum-based plastics and are biodegradable and biocompatible, making them an ideal alternative to traditional plastics. However, despite their many advantages, PHAs are significantly more expensive to produce than traditional petroleum-based plastics, limiting their industrial production. The high price of PHAs is primarily due to the fermentation process itself and the downstream recycling costs. Pure bacterial synthesis of PHA is currently the mainstream method for industrial PHA production, but its high production costs still restrict its widespread application. Furthermore, municipal wastewater treatment plants generate large amounts of excess sludge daily, resulting in high sludge disposal costs. Using inexpensive and widely available waste as substrates for PHA synthesis and utilizing mixed microbial communities in activated sludge as inoculum can significantly reduce PHA production costs and achieve sludge reduction and resource recovery. Therefore, the synthesis of PHA using mixed microbial communities (MMCs) has become a research hotspot in recent years. Compared to pure bacteria, the challenge in MMC extraction is obtaining high molecular weight and high purity extractable polymers in an inexpensive and environmentally friendly manner.
[0003] PHA extraction typically employs two methods: NPCM digestion (for non-PHA bacterial components) and solvent extraction. Solvent-based extraction is more efficient and widely applicable due to its simplicity, higher recovery efficiency and purity, and minimal polymer degradation. Chloroform is commonly used as the standard extractant in solvent extraction, but it is harmful to both humans and the environment. Furthermore, current extraction methods often require PHA extraction experiments on freeze-dried biomass, a time-consuming and energy-intensive process. Compared to freeze-dried activated sludge, fresh activated sludge contains more bound water, flocs, and extracellular polymeric substances (EPS). Therefore, extracting PHA from fresh bacterial communities is more challenging. Summary of the Invention
[0004] The technical problem to be solved by this invention is to address the issues of toxic and harmful traditional extraction solvents and the difficulty in extracting PHA from fresh microbial communities. This invention provides a method for extracting polyhydroxy fatty acid esters (PHA) using two natural low co-solvents (NADES). This method has the advantages of high purity and yield of extracted PHA, environmental and human-friendly properties, and reduced PHA production costs.
[0005] To achieve the above objectives, the present invention provides a method for extracting polyhydroxy fatty acid esters (PHA), wherein the method includes the following steps:
[0006] (1) Two types of NADES were prepared;
[0007] (2) Concentrate the bacterial culture containing polyhydroxy fatty acid esters to obtain a bacterial suspension containing polyhydroxy fatty acid esters;
[0008] (3) Add CA-NADES to the bacterial suspension of polyhydroxy fatty acid esters and stir during pretreatment to obtain a mixture containing polyhydroxy fatty acid esters.
[0009] (4) The obtained mixture containing polyhydroxy fatty acid esters was subjected to solid-liquid separation to obtain a precipitate containing polyhydroxy fatty acid esters.
[0010] (5) Add Ac-NADES to the precipitate containing polyhydroxy fatty acid esters and stir during extraction;
[0011] (6) Remove residual cell debris to obtain a solution rich in PHA;
[0012] (7) Add methanol to the above PHA-rich solution and allow it to precipitate for a period of time to obtain PHA precipitate;
[0013] (8) The obtained solution containing PHA precipitate is subjected to solid-liquid separation, and then the solid is dried to obtain the PHA product.
[0014] In this invention, the two methods for preparing NADES in step (1) are as follows: solid choline chloride is mixed with citric acid at a molar ratio of 1:1, and the mixture is heated at 80-90℃ for 25-30 min to synthesize. The synthesized solution is added to distilled water at a volume ratio of 1:9 and mixed well, and is denoted as CA-NADES; solid menthol is mixed with acetic acid at a molar ratio of 1:3, and the mixture is heated at 80-90℃ for 15-20 min to synthesize, and is denoted as Ac-NADES.
[0015] In this invention, in step (2), the microbial cells and supernatant are separated by a centrifuge at a speed of 4000 to 4500 revolutions per minute for 5-10 minutes, and then distilled water is added to concentrate to 50g / L.
[0016] In this invention, in step (3), 5-20 mL of CA-NADES is added to the bacterial suspension (50 g / L, 2 mL), more preferably 10-12 mL of CA-NADES; the temperature during pretreatment is set at 25℃-70℃, preferably 25-30℃; the pretreatment time is set at 15-90 min, preferably 50-70 min.
[0017] In this invention, in step (4), the microbial cells and supernatant are separated by a centrifuge at a speed of 4000 to 4500 revolutions per minute for 5 to 10 minutes.
[0018] In this invention, in step (5), the amount of Ac-NADES added to the bacterial cell precipitate containing polyhydroxy fatty acid ester is: 5 ml-15 mL of CA-NADES, more preferably 10-12 mL of CA-NADES; the extraction temperature is set at 25℃-105℃, preferably 80-85℃; the extraction time is set at 50 min-150 min, preferably 50-70 min.
[0019] In this invention, in step (6), cell residue is filtered out by a polypropylene membrane filter with a porosity of 0.45 μm.
[0020] In this invention, in step (7), methanol and Ac-NADES solution containing PHA are added at volume ratios of 3:1 and 2:1, respectively, preferably 3:1; the reaction temperature is set to -4℃ and -20℃, preferably -20℃.
[0021] In this invention, in step (8), the PHA precipitate and solution are separated by a centrifuge at a speed of 10,000 to 12,000 revolutions per minute for 5-10 minutes. The supernatant is discarded to obtain the PHA precipitate, which is then dried in a 50°C oven for 24 hours.
[0022] In this scheme, on the one hand, the natural eutectic solvent CA-NADES can destroy the bacterial cell wall and remove EPS, which is conducive to the subsequent release of PHA. On the other hand, the natural eutectic solvent Ac-NADES can dissolve PHA and extract it. Therefore, the whole process is simple, energy-efficient, and low-cost, and does not require the use of toxic and harmful organic solvents, which greatly reduces the adverse impact on the environment. Attached Figure Description
[0023] Figure 1 This is a schematic diagram of a preferred system for extracting polyhydroxy fatty acid esters according to the present invention.
[0024] Figure 2 The finished product PHA is obtained after solid-liquid separation and drying. Detailed Implementation
[0025] The technical solution described in this invention will be further illustrated with specific examples, but this invention is not limited thereto. Unless otherwise specified in the examples, the experimental methods used are conventional methods, and the reagents and instruments used are commercially available. The bacterial culture used in the examples was enriched in a laboratory-scale sequencing batch reactor (SBR) with a working volume of 3L. The inoculum used was waste activated sludge (WAS) from the secondary sedimentation tank of the Gaobeidian Wastewater Treatment Plant in Beijing, China. The substrate used for MMC enrichment was simulated wastewater fermentation broth. The carbon source consisted of acetic acid, propionic acid, butyric acid, and valeric acid in a volume ratio of 6:2:1:1. The nitrogen and phosphorus sources were (NH4)2SO4 and KH2PO4, respectively, with a constant COD:N:P ratio of 100:2:0.2. The dosage of trace elements was 1 mL / L. In addition, 20 mg / L thiourea was added to inhibit nitrification. The SBR was operated in a rich-starvation mode with a cycle length of 2 days and a rich-starvation ratio of 1:3.
[0026] All experiments were performed in triplicate. Data are expressed as the mean ± standard deviation of three parallel tests. One-way ANOVA was performed using SPSS 27.0 statistical software to evaluate statistical significance.
[0027] The yield and purity of PHA products are calculated using the following formula:
[0028] PHA yield (%) = (mass of PHA in the dried isolated sample / mass of PHA in the fermentation broth) × 100%
[0029] PHA purity (%) = (mass of PHA in the dried separated sample / mass of the dried separated sample) × 100%
[0030] Example 1: Effect of CA-NADES pretreatment on PHA extraction efficiency
[0031] A certain amount of bacterial culture was taken from the SBR reactor, washed twice with distilled water, and concentrated to a 50 g / L bacterial suspension. The PHA content was determined. Two mL of the bacterial suspension (50 g / L) was taken from each sample and 5 mL, 10 mL, 15 mL, and 20 mL of CA-NADES were added, respectively. Each condition was repeated in triplicate. The samples were placed in a constant-temperature magnetically stirred water bath at 40°C and stirred for 60 min. Then, solid-liquid separation was performed by centrifugation at 4000 rpm for 5 min. The supernatant was discarded, and then 10 mL of Ac-NADES was added. The mixture was placed in a constant-temperature magnetically stirred water bath at 55°C and stirred for 120 min to extract PHA. The mixture was then filtered through a 0.45 μm porosity polypropylene membrane to obtain a mixed solution of PHA and Ac-NADES. Methanol was then added at a 1:3 volume ratio, and the mixture was placed in a 4°C refrigerator for 24 h. Solid-liquid separation was then performed by centrifugation at 10000 rpm for 5 min. Discard the supernatant to obtain PHA precipitate, and dry the obtained PHA precipitate in an oven at 50℃ for 24 hours.
[0032] The PHA content in the dried sample was determined by gas chromatography, and the yield and purity of the product were calculated as shown in Table 1.
[0033] Table 1: Effect of pretreatment reagent (CA-NADES) dosage on pretreatment effect
[0034]
[0035] As shown in Table 1, adding CA-NADES reagent to the mixed bacterial culture PHA extraction process can improve the PHA extraction yield and purity. With increasing reagent volume, both the extraction rate and purity gradually increase, reaching a maximum at 10 mL and then ceasing to increase further. Therefore, the optimal amount of CA-NADES reagent is 10 mL.
[0036] Secondly, after determining the optimal dosage of CA-NADES reagent, further optimization experiments were conducted on the pretreatment temperature. Regarding the determination of the optimal reaction temperature for CA-NADES pretreatment, the experiment used a pretreatment time of 60 min and the already determined optimal reagent dosage, i.e., treating 2 mL of 50 g / L fresh bacterial culture with 10 mL of CA-NADES, while keeping other conditions constant, and adjusting the pretreatment temperature to achieve the desired result. In this batch of experiments, four treatment methods were set at temperatures of 25℃, 40℃, 55℃, and 70℃.
[0037] The PHA content in the dried sample was determined by gas chromatography, and the yield and purity of the product were calculated as shown in Table 2.
[0038] Table 2: Effect of Pretreatment Temperature on Pretreatment Efficacy
[0039]
[0040] As shown in Table 2, temperature has no effect on extraction rate and purity, therefore the preferred temperature is room temperature (25℃).
[0041] In addition, after determining the optimal dosage of CA-NADES reagent and the pretreatment temperature, further optimization experiments were conducted on the pretreatment time. Regarding the determination of the optimal contact time for CA-NADES pretreatment, the experiment used a pretreatment temperature of 25℃ and the already determined optimal reagent dosage, i.e., treating 2 mL of 50 g / L fresh bacterial culture with 10 mL of CA-NADES, while keeping other conditions constant, and adjusting the pretreatment time to achieve the desired result. In this batch of experiments, four treatment methods were set up with temperatures of 15 min, 30 min, 60 min, and 90 min.
[0042] The PHA content in the dried sample was determined by gas chromatography, and the yield and purity of the product were calculated as shown in Table 3.
[0043] Table 3: Effect of pretreatment time on pretreatment effect
[0044]
[0045]
[0046] As shown in Table 3, the extraction rate and purity gradually increase with the increase of pretreatment time, reaching a maximum at 60 min and then no longer increasing. Therefore, the optimal pretreatment time is 60 min.
[0047] Example 2: Factors affecting PHA extraction by Ac-NADES
[0048] The bacterial population was pretreated according to the optimized conditions of Example 1. 2 mL of bacterial suspension (50 g / L) was added to 10 mL of CA-NADES, and the mixture was stirred in a constant-temperature magnetically stirred water bath at 25°C for 60 min. Then, solid-liquid separation was performed by centrifugation at 4000 rpm for 5 min. The supernatant was discarded, and the treated bacterial cells were obtained. Different doses of extraction reagent (Ac-NADES) were added: 5 mL, 8 mL, 10 mL, and 15 mL. PHA extraction was then performed by stirring in a constant-temperature magnetically stirred water bath at 55°C for 120 min, with other conditions remaining unchanged, to investigate the effect of the amount of extraction reagent on the extraction efficiency.
[0049] The PHA content in the dried sample was determined by gas chromatography, and the yield and purity of the product were calculated as shown in Table 4.
[0050] Table 4: Effect of extractant (Ac-NADES) dosage on extraction efficiency
[0051]
[0052] As shown in Table 4, the amount of Ac-NADES has a significant impact on extraction. The extraction rate gradually increases with increasing dosage, reaching a maximum at 10 mL and then ceasing to increase further. Therefore, the optimal amount of Ac-NADES is 10 mL.
[0053] After determining the optimal dosage of Ac-NADES reagent, further optimization experiments were conducted on the extraction temperature. The experiment used the determined optimal reagent dosage, i.e., adding 10 mL of Ac-NADES extractant to the pretreated bacterial cells. Different extraction temperatures (25℃, 55℃, 85℃, 105℃) were controlled, and PHA was extracted by stirring for 120 min, with other conditions remaining constant. The effect of extraction temperature on the extraction efficiency was investigated.
[0054] The PHA content in the dried sample was determined by gas chromatography, and the yield and purity of the product were calculated as shown in Table 5.
[0055] Table 5: Effect of extraction temperature on extraction efficiency
[0056]
[0057] As shown in Table 5, the purity remains basically unchanged with increasing extraction temperature, but the extraction temperature gradually increases initially, reaching a maximum at 85℃ and then ceasing to rise. Therefore, the optimal extraction temperature is 85℃.
[0058] After determining the optimal dosage and extraction temperature of Ac-NADES reagent, further optimization experiments were conducted on the extraction time. Using the established optimal reagent dosage and temperature, 10 mL of Ac-NADES extractant was added to the pretreated bacterial cells and stirred on an 85℃ constant-temperature magnetic stirrer. Different extraction times (60 min, 90 min, 120 min, and 150 min) were controlled while keeping other conditions constant to investigate the effect of extraction time on the extraction efficiency.
[0059] The PHA content in the dried sample was determined by gas chromatography, and the yield and purity of the product were calculated as shown in Table 6.
[0060] Table 6: Effect of extraction time on extraction efficiency
[0061]
[0062] As shown in Table 6, the purity remained relatively constant with increasing extraction time, but the extraction rate initially remained relatively constant for about 120 minutes before rapidly decreasing. Therefore, the optimal extraction time is 60 minutes.
[0063] Example 3: The effect of precipitation method on PHA recovery
[0064] The bacterial community was pretreated and extracted according to the optimized conditions of Examples 1 and 2. Specifically, 2 mL of bacterial suspension (50 g / L) was added to 10 mL of CA-NADES, and the mixture was stirred in a constant-temperature magnetically stirred water bath at 25°C for 60 min. Then, solid-liquid separation was performed by centrifugation at 4000 rpm for 5 min. The supernatant was discarded, and the treated bacterial cells were obtained. 10 mL of extraction reagent (Ac-NADES) was added, and the mixture was stirred in a constant-temperature magnetically stirred water bath at 85°C for 60 min to extract PHA. The extract was then filtered through a polypropylene membrane with a porosity of 0.45 μm to obtain a mixed solution of PHA and Ac-NADES. Different precipitation methods were set up: (1) 2 volumes of methanol, overnight in a 4°C refrigerator (2V, -4°C); (2) 3 volumes of methanol, overnight in a 4°C refrigerator (3V, -4°C); (3) 2 volumes of methanol, overnight in a -20°C refrigerator (2V, -20°C); (4) 3 volumes of methanol, overnight in a -20°C refrigerator (3V, -20°C). All other conditions remained unchanged to investigate the effect of the precipitation method on the extraction efficiency.
[0065] The PHA content in the dried sample was determined by gas chromatography, and the yield and purity of the product were calculated as shown in Table 7.
[0066] Table 7: Effects of different precipitation methods on extraction efficiency
[0067]
[0068] As shown in Table 7, the precipitation process has a significant impact on polymer recovery. Cold methanol (-20℃) precipitates PHA better than methanol at higher temperatures (-4℃), and the optimal ratio of 3 times the volume of ethanol to 1 volume of the PHA-containing mixture is found to be the best. Therefore, the precipitation method using 3 times the volume of methanol and a temperature of -20℃ was chosen.
[0069] After optimization, this invention ultimately achieved an extraction rate of 91.4% and a purity of 97.2%.
[0070] The preferred embodiments of the present invention have been described in detail above; however, the present invention is not limited thereto. Within the scope of the inventive concept, various simple modifications can be made to the technical solutions of the present invention, including combinations of various technical features in any other suitable manner. These simple modifications and combinations should also be considered as the content disclosed in the present invention and are all within the protection scope of the present invention.
Claims
1. A method for extracting polyhydroxy fatty acid esters using a natural eutectic solvent, characterized in that, Includes the following steps: (1) Two types of NADES were prepared; (2) Concentrate the bacterial culture containing polyhydroxy fatty acid esters to obtain a bacterial suspension containing polyhydroxy fatty acid esters; (3) Add CA-NADES to the bacterial suspension of polyhydroxy fatty acid esters and stir during pretreatment to obtain a mixture containing polyhydroxy fatty acid esters. (4) The obtained mixture containing polyhydroxy fatty acid esters was subjected to solid-liquid separation to obtain a precipitate containing polyhydroxy fatty acid esters. (5) Add Ac-NADES to the precipitate containing polyhydroxy fatty acid esters and stir during extraction; (6) Remove residual cell debris to obtain a solution rich in PHA; (7) Add methanol to the above PHA-rich solution to precipitate PHA precipitate; (8) The obtained solution containing PHA precipitate is subjected to solid-liquid separation, and then the solid is dried to obtain the PHA product.
2. According to the method of claim 1, the two methods for preparing NADES in step (1) are as follows: solid choline chloride is mixed with citric acid at a molar ratio of 1:1, and the mixture is heated at 80-90℃ for 25-30 min to synthesize; the synthesized solution is added to distilled water at a volume ratio of 1:9 and mixed well, and is denoted as CA-NADES; solid menthol is mixed with acetic acid at a molar ratio of 1:3, and the mixture is heated at 80-90℃ for 15-20 min to synthesize, and is denoted as Ac-NADES.
3. According to the method of claim 1, the concentration of the concentrated bacterial suspension in step (2) is 50 g / L.
4. According to the method of claim 1, in step (3), 10-12 mL of CA-NADES is added to 2 mL of 50 g / L bacterial suspension; the temperature is set at 25-30℃ during pretreatment; and the pretreatment time is set at 50-70 min.
5. According to the method of claim 1, in step (5), 10-12 mL of CA-NADES is added to the bacterial precipitate after CA-NADES pretreatment; the extraction temperature is set to 80-85℃; and the extraction time is set to 50-70 min.
6. According to the method of claim 1, the precipitation conditions in step (7) are as follows: methanol and Ac-NADES solution containing PHA are mixed at a volume ratio of 3:1, placed at -20°C, and precipitated for 20-24 hours.
7. According to the method of claim 1, the solid-liquid separation is performed by centrifugation or filtration. The centrifugation conditions are as follows: in steps (2) and (4), the rotation speed is 4000 to 4500 rpm and the centrifugation time is 5-10 minutes; in step (8), the rotation speed is 10000 to 12000 rpm and the centrifugation time is 5-10 minutes. The filtration conditions are as follows: in step (6), cell residues are filtered out by a polypropylene membrane filter with a porosity of 0.45 μm.