A method for directly extracting polyhydroxy fatty acid esters from fermentation broth using plasticizers.

Abstract

This invention relates to the field of downstream product processing technology in bioengineering, specifically disclosing a method for directly extracting polyhydroxyalkanoates (PHA) from fermentation broth using plasticizers. The method includes: first, adding a certain amount of plasticizer to the fermentation broth producing PHA, mixing thoroughly to form a turbid liquid for cell disruption and extraction; then, performing solid-liquid separation to remove the aqueous phase and plasticizer, retaining the PHA-containing solid phase; second, repeatedly washing with detergent to remove residual plasticizer and other impurities, obtaining a solid after solid-liquid separation; finally, drying the solid to obtain the PHA; the plasticizer and detergent used are recycled. This method integrates cell disruption and PHA extraction, featuring a simple process, environmental friendliness, low investment cost, low energy consumption, high production efficiency, high product purity, and stable quality, making it suitable for industrial production.

Description

Technical Field

[0001] This invention relates to the field of downstream product processing technology in bioengineering, and specifically discloses a method for directly extracting polyhydroxy fatty acid esters from fermentation broth by breaking down the cell walls of a plasticizer. Background Technology

[0002] Polyhydroxyalkanoates (PHAs) are a collective term for a class of high-molecular-weight polyesters synthesized entirely by microorganisms. PHAs are biodegradable and biocompatible, thus considered environmentally friendly materials that can help solve the increasingly serious problem of environmental pollution and have great potential for application.

[0003] Although extensive basic and applied research has been conducted on PHA in recent years, large-scale industrial production of PHA is still lacking in China. Therefore, research on industrial production processes and methods for PHA is urgently needed. The main reason for the lack of large-scale industrial production is that its production cost is much higher than that of petrochemical resins. The production cost of PHA mainly includes raw material costs and separation and purification costs. The raw material cost depends on the production efficiency of the strain and the fermentation process used, while the separation and purification cost mainly depends on the process used. Currently, the production scale of PHA mainly depends on extraction and separation purification. PHA extraction usually requires collecting cells from the fermentation broth, then breaking down the cell walls to release PHA, followed by extraction with organic solvents, and finally washing and drying. Cell wall breaking is a key operational step, generally using physical methods (high-pressure homogenization, bead milling, ultrasound, etc.), chemical methods (surfactants, acids or alkalis, organic solvents, etc.), enzymatic methods, or a combination of these methods. Extraction methods are usually organic solvent extraction, and washing agents often include chemical reagents, surfactants, and water.

[0004] Chinese patent application CN1844185A discloses a method for extracting intracellular PHA from microorganisms. The method involves first collecting bacterial cells, then adding an ester-based organic solvent (including one or more of the following: n-butyl acetate, isobutyl acetate, tert-butyl acetate, amyl acetate, methyl butyrate, ethyl butyrate, propyl butyrate, and methyl hexanoate) with a structure similar to that of PHA monomers. Finally, the PHA is obtained through alcohol precipitation, washing, and drying. This method consumes a large amount of solvent, and these organic solvents are volatile, flammable, and explosive, making it unsuitable for large-scale industrial production.

[0005] The invention patent application with publication number CN1328160A discloses a one-step separation extraction method for directly extracting polyhydroxyalkanoates from cell fermentation broth containing polyhydroxyalkanoates. However, this method requires the use of a large amount of sodium hypochlorite, resulting in a harsh operating environment, severe pollution, and a heavy burden of wastewater treatment.

[0006] Chinese patent application CN115058461A discloses a method for adjusting the pH value of fermentation broth using alkali solution and extracting PHA from the fermentation broth using sodium salt and surfactant. This method requires the use of a large amount of alkali solution to adjust the pH value, which easily pollutes the environment. Furthermore, the use of large amounts of alkali solution and surfactant increases the difficulty of wastewater treatment, resulting in high wastewater treatment costs. Additionally, the alkali solution has a shearing and degradation effect on the molecular weight of the extracted PHA product, affecting product quality.

[0007] Existing extraction and separation methods suffer from significant drawbacks such as numerous steps, low yields, and excessive wastewater generation, leading to high production costs, unstable product quality, lack of market competitiveness, and difficulty in large-scale industrial production. This invention aims to develop a highly efficient new technology coupling cell disruption and salting-out extraction to directly extract intracellular PHA from fermentation broth. Summary of the Invention

[0008] To overcome the problems of high cost, high energy consumption, high wastewater treatment cost, and alkaline degradation affecting quality in existing technologies, which are unfavorable for industrial production, this invention provides a method for directly extracting polyhydroxy fatty acid esters from fermentation broth by breaking down the cell walls of plasticizers. This method employs a simple, efficient, economical, and green extraction process, which can obtain polyhydroxy fatty acid esters with high yield and purity. This method is suitable for large-scale industrial production.

[0009] The technical solution adopted by the present invention to achieve the above-mentioned objective is: a method for directly extracting polyhydroxy fatty acid esters from fermentation broth using plasticizers, comprising the following steps:

[0010] S1: Add a certain amount of plasticizer to the fermentation broth that produces polyhydroxy fatty acid esters (PHA), mix evenly to form a turbid liquid for cell wall disruption and extraction, and then perform solid-liquid separation to remove the aqueous phase and plasticizer, retaining the solid phase containing PHA.

[0011] S2: Add a certain amount of detergent to the solid phase containing PHA, mix well, and then perform solid-liquid separation to remove residual plasticizers and other impurities.

[0012] S3: Dry the solid after solid-liquid separation to constant weight to obtain the polyhydroxy fatty acid ester.

[0013] Furthermore, in step S1, before adding the plasticizer to the fermentation broth that produces polyhydroxy fatty acid esters (PHA), a solid salt is added and dissolved in the fermentation broth before adding the plasticizer, or a concentrated salt solution is added after the plasticizer is added to the fermentation broth to form a turbid liquid, and then a salt precipitation extraction system is formed for solid-liquid separation.

[0014] Furthermore, the salt is water-soluble (NH4)2SO4, K2HPO4, or Na2CO3, and the salt concentration in the salting-out extraction system is 9-25 wt%.

[0015] Further, in step S1, the plasticizer includes hydrophobic plasticizer and hydrophilic plasticizer, and the plasticizer is at least one of triethyl citrate (TEC), tributyl citrate (TBC), trimethyl phosphate (TMP), tricresyl phosphate (TCP), or triethyl phosphate (TEP).

[0016] Further, in step S1, the volume ratio of the added plasticizer to the fermentation liquid is (0.5-3):1, the PHA content of the fermentation liquid is 43.09-60.64 g / L, and the mixing and cell wall breaking extraction temperature is 15-70℃.

[0017] Furthermore, solid-liquid separation is performed using centrifugation or filtration. Centrifugation operating conditions: centrifugal force of 2000-5000g and centrifugation time of 1-10min.

[0018] Further, in step S2, when the plasticizer is hydrophilic, the detergent used is water; when the plasticizer is hydrophobic, the detergent used is one or a combination of methanol or ethanol. The volume ratio of detergent to PHA fermentation liquid is (1-3):1, the washing time is 2-10 min, the washing temperature is 15-35℃, and the number of washing cycles is 1-10.

[0019] Furthermore, in step S3, the solid drying is carried out using atmospheric pressure drying or vacuum drying.

[0020] Furthermore, the plasticizer and detergent removed by solid-liquid separation are recycled and reused. The plasticizer is recovered by vacuum rotary evaporation at a distillation temperature of 60-80℃, a pressure of 0.060-0.095MPa, and a rotation speed of 60-90rpm.

[0021] Compared with the prior art, the beneficial effects of this invention are as follows:

[0022] 1. This invention ingeniously utilizes the property that plasticizers can combine with PHA and swell, integrating cell wall disruption and PHA extraction into one process. This simplifies the PHA extraction process, eliminating the need for prior separation and washing of bacterial cells and avoiding the use of complex equipment such as high-speed centrifuges. Polyhydroxyalkanoates are directly separated and purified from the fermentation broth, reducing investment in production equipment, lowering energy consumption, shortening production time, and reducing wastewater discharge, making it more suitable for large-scale industrial production.

[0023] 2. The present invention offers high production efficiency, product purity, and stable quality. The unsalted plasticizer treatment process of the present invention can achieve a maximum PHA yield of 86.72% and a purity of 95.52%; the salting-out extraction process with added salt can further increase the PHA yield to 96.31% and the purity to 98.86%. None of the raw materials added in the present invention cause shear degradation of PHA, thus ensuring very stable product quality.

[0024] 3. Existing technologies use large amounts of alkaline solutions and surfactants, which increases the difficulty of wastewater treatment and thus the cost of wastewater treatment is high. This invention uses only plasticizers and alcohol detergents, which generates less wastewater, and the plasticizers can be recycled and reused. This reduces both the cost of environmental treatment and the cost of production.

[0025] 4. The plasticizer used in this invention is a commonly used industrial reagent, which is not only relatively inexpensive, but also non-toxic or low-toxic. The entire process is more green and environmentally friendly than existing technologies. Attached Figure Description

[0026] Figure 1 The images show the experimental results of extracting PHA from the fermentation broth using three plasticizers in Example 1. (A) shows the experimental phenomena after centrifugation of the fermentation broth and plasticizer; (B) shows the addition of ethanol to elute the plasticizer and some water-insoluble impurities; (C) shows the addition of water to elute residual ethanol and purify the solid; and (D) shows the finished PHA product after solid-liquid separation and drying.

[0027] Figure 2 The images show experimental results of adding different concentrations of K2HPO4 to the PHA process of triethyl phosphate cell disruption and salting-out extraction in Example 2. (A)-(F) show the experimental phenomena after centrifugation of fermentation broths containing 9, 12, 15, 18, 20, and 25 wt% K2HPO4 with triethyl phosphate, respectively.

[0028] Figure 3 The images show experimental results under different washing conditions during the extraction of PHA from the fermentation broth using plasticizer in Example 3. (A) shows the experimental phenomena after the first washing and centrifugation; (B) shows the experimental phenomena after the second washing and centrifugation; and (C) shows the finished PHA product after solid-liquid separation and drying. Detailed Implementation

[0029] The present invention will be further described in detail below with reference to embodiments and accompanying drawings, but the embodiments of the present invention are not limited thereto. Unless otherwise specified in the embodiments, the experimental methods used are conventional methods, and the reagents or instruments used are all commercially available conventional products.

[0030] The fermentation broth used in the examples was prepared by fermenting *Haloxylon ammodendron* under conventional fermentation conditions in the art. The PHA content in the unbroken fermentation broth was 43.09-60.64 g / L; the PHA content in the cell-broken broth treated with surfactant (0.03% SDS) and heated (70°C) after cell collection was 45.50 g / L; and the PHA content in the cell-broken broth after three cycles of high-pressure homogenization was 69.06 g / L.

[0031] The product detection method of this invention is gas chromatography. The gas chromatograph is a Shimadzu GC-2010, and the column is an Agilent HP-5-MS UI. The temperature program settings are as follows: injection port (SPL1) 180℃; detector (FID) 200℃; initial column temperature 50℃, hold for 1 min, then increase to 180℃ at 15℃ / min, hold for 2 min; split ratio is set to 10:1.

[0032] Preparation of standard curve: Weigh 2, 5, 10, 15, and 20 mg of pure PHA powder, respectively, add 2 mL of chloroform and 2 mL of methanol solution (containing 1 g / L benzoic acid internal standard and 3% (v / v) concentrated sulfuric acid), mix well, and heat to 100℃ for esterification for 4 h. Cool to room temperature, add 1 mL of pure water, vortex for 30 s, and let stand for 2 h until complete separation. Take the lower phase (chloroform phase) into a sample vial containing 150 mg of anhydrous sodium sulfate to remove any small amount of water. Then take 1 mL of the sample, filter it through a 0.22 μm membrane, and perform gas chromatography detection.

[0033] Quantitative analysis of samples: Weigh 10 mg of the extracted and purified solid PHA, process it under the same conditions as the standard curve, and perform gas chromatography detection.

[0034] The yield and purity of PHA products are calculated using the following formula:

[0035] PHA yield (%) = (mass of PHA in the dried isolated sample / mass of PHA in the fermentation broth) × 100%

[0036] PHA purity (%) = (mass of PHA in the dried separated sample / mass of the dried separated sample) × 100%

[0037] Comparative Example 1: Effect of Different Cell Disruption Methods on PHA Extraction Efficiency

[0038] Take 5 mL each of the high-pressure homogenized liquid and the cell-wall-breaking liquid obtained by combined heating with surfactant into different centrifuge tubes. Add 5 mL of different extraction solvent or detergent to each tube at room temperature, vortex for 2 min, and centrifuge at 3000 g for 5 min. Take the precipitate, add 10 mL of water, vortex and centrifuge under the same conditions. Place the solid in a 60℃ oven and dry to constant weight.

[0039] 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.

[0040] Table 1. Results of PHA preparation using existing cell wall disruption and extraction processes.

[0041]

[0042] Example 1: Combined effect of plasticizer on cell wall disruption and extraction of PHA fermentation broth

[0043] Take 5 mL of untreated Halomonas fermentation broth and add different volume ratios of plasticizer. Vortex for 2 min at different cell wall breaking extraction temperatures to mix the plasticizer and fermentation broth evenly and form a turbid liquid. Then centrifuge at 3000g for 5 min. Take the precipitate and add 10 mL of washing solution. Vortex and centrifuge under the same conditions. Take the precipitate and add 10 mL of water. Vortex and centrifuge under the same conditions. Collect the solid precipitate and dry it in a 70℃ oven to constant weight.

[0044] 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.

[0045] Table 2. Results of PHA extraction from fermentation broth using different plasticizers after cell wall disruption.

[0046]

[0047] Note: TEC: Triethyl citrate; TMP: Trimethyl phosphate; TEP: Triethyl phosphate; TBC: Tributyl citrate; TCP: Trimethylbenzene phosphate

[0048] As shown in Table 2, the use of plasticizers has a combined effect of cell wall disruption and extraction of PHA-producing fermentation broth, with high yield and purity. The highest PHA yield in the comparative example was 57.77%, while the PHA yield in this example was generally greater than 60%. The PHA extracted using triethyl citrate had a purity greater than 70%, and the PHA extracted using triethyl phosphate had a purity greater than 80%.

[0049] Example 2: Effects of Salt Type and Concentration on Plasticizer Cell Disruption and Salting-out Extraction in PHA Process

[0050] Take 5 mL of unbroken *Haloxylon ammodendron* fermentation broth. Before or after adding 5 mL of plasticizer, add solid salt or concentrated salt solution, respectively. Vortex at 25°C for 2 min to form a salting-out extraction system. Centrifuge at 2000 g for 10 min to separate solid and liquid. For the triethyl citrate (TEC) system, add 10 mL of anhydrous ethanol to the precipitate, vortex and centrifuge under the same conditions, then add 10 mL of water to the precipitate, vortex and centrifuge under the same conditions to obtain the solid. For the triethyl phosphate (TEP) system, add 10 mL of water to the precipitate, repeat the vortex and centrifugation process multiple times under the same conditions to obtain the solid. Collect the solid and dry it in an 80°C oven to constant weight.

[0051] 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.

[0052] Table 3. Results of plasticizer cell disruption and salting-out extraction of PHA

[0053] Types of plasticizers Salt addition time Salt types Salt concentration (wt%) Yield (%) purity(%) TEC forward <![CDATA[Na2CO3]]> 9 76.93 78.46 TEC forward <![CDATA[Na2CO3]]> 18 79.69 82.45 TEC back <![CDATA[Na2CO3]]> 20 80.54 80.43 TEC back <![CDATA[Na2CO3]]> 25 86.02 85.98 TEC forward <![CDATA[(NH4)2SO4]]> 9 78.75 74.93 TEC forward <![CDATA[(NH4)2SO4]]> 18 79.39 76.89 TEC back <![CDATA[(NH4)2SO4]]> 25 78.38 77.43 TEC back <![CDATA[K2HPO4]]> 15 87.62 79.38 TEC back <![CDATA[K2HPO4]]> 18 86.51 77.68 TEC forward <![CDATA[K2HPO4]]> 20 88.15 86.79 TEP forward <![CDATA[Na2CO3]]> 15 82.45 86.27 TEP forward <![CDATA[Na2CO3]]> 18 80.29 87.06 TEP back <![CDATA[Na2CO3]]> 25 81.69 88.41 TEP forward <![CDATA[(NH4)2SO4]]> 15 78.88 90.49 TEP forward <![CDATA[(NH4)2SO4]]> 18 79.28 88.70 TEP back <![CDATA[K2HPO4]]> 15 84.58 92.94 TEP back <![CDATA[K2HPO4]]> 18 86.75 93.71 TEP back <![CDATA[K2HPO4]]> 25 96.31 98.86

[0054] As shown in Table 3, adding a high concentration of salt in the plasticizer cell disruption extraction process can improve the extraction yield and purity of PHA. Under high salt concentration (salt concentration ≥15wt%), the mixture forms a salting-out extraction system. After centrifugation, the system separates into two phases: the upper phase is the plasticizer phase, and the lower phase is the salt solution phase. PHA is distributed at the interface between the two phases, which is beneficial for the separation of PHA and cellular impurities.

[0055] Example 3: Effect of different washing conditions on PHA yield and purity

[0056] Take 5 mL of unbroken Halomonas fermentation broth and add triethyl citrate (TEC), triethyl phosphate (TEP), or trimethyl phosphate (TMP) at a 1:1 ratio. Vortex at 20 °C for 2 min and centrifuge at 5000 g for 1 min. Take the precipitate and wash it with different amounts of detergent. Vortex and centrifuge under the same conditions. Take the precipitate and add 10 mL of water. Vortex and centrifuge under the same conditions. Collect the solid and dry it in a 55 °C oven to constant weight.

[0057] 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.

[0058] Table 4. Effects of different washing conditions on PHA yield and purity

[0059]

[0060] As shown in Table 4, in this embodiment, the parameter with the greatest impact on PHA yield and purity is the number of washes, followed by the type of detergent. Increasing the number of washes can improve PHA purity, but it will lead to a decrease in yield.

[0061] Example 4: The effect of plasticizer recycling on PHA extraction by cell wall disruption

[0062] The supernatant and the first washing liquid after centrifugation of triethyl phosphate and fermentation broth were rotary evaporated at 60℃, 70 rpm and 0.075 MPa until the water was completely evaporated to recover triethyl phosphate. At room temperature, 5 mL of the recovered plasticizer was added to 5 mL of unbroken Halomonas fermentation broth, mixed, and centrifuged at 3000 g for 5 min. The precipitate was then added to 10 mL of water, mixed and centrifuged under the same conditions. The precipitate was then added to 10 mL of water, mixed and centrifuged under the same conditions. The solid was collected and dried in a 60℃ oven to constant weight. Triethyl phosphate was recovered again using the above steps and reused for the cell-wall breaking extraction of PHA in the fermentation broth. The plasticizer recovery rates in the three experiments were 94.62%, 82.51%, and 76.31%, respectively.

[0063] 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.

[0064] Table 5. Results of triethyl phosphate recovery and reuse and extraction of PHA from fermentation broth by cell wall disruption.

[0065] Plasticizer recycling times Plasticizer recovery rate (%) Yield (%) purity(%) first 94.62 76.07 86.58 The second 82.51 72.54 81.99 The third 76.31 70.03 79.40

[0066] As shown in Table 5, the plasticizer recovered by rotary evaporation can still be used to extract PHA from the fermentation broth by cell wall disruption. The plasticizer recovery rate, PHA yield, and purity gradually decrease with the increase of the number of plasticizer recovery cycles.

[0067] The embodiments of the present invention are given for illustrative and descriptive purposes only, and are not intended to be exhaustive or to limit the invention to the forms disclosed. Many modifications and variations will be apparent to those skilled in the art. The embodiments were chosen and described in order to better illustrate the principles and practical application of the invention, and to enable those skilled in the art to understand the invention and to design various embodiments with various modifications suitable for a particular purpose.

Claims

1. A method for directly extracting polyhydroxy fatty acid esters from fermentation broth using a plasticizer, characterized in that, Includes the following steps: S1: A certain amount of plasticizer is added to the fermentation broth that produces polyhydroxyalkanoates (PHA). The plasticizer is selected from at least one of triethyl citrate (TEC), tributyl citrate (TBC), trimethyl phosphate (TMP), tricresyl phosphate (TCP), or triethyl phosphate (TEP). The volume ratio of the added plasticizer to the fermentation broth is (0.5-3):

1. The PHA content of the fermentation broth is 43.09-60.64 g / L. The mixing and cell wall breaking extraction temperature is 15-70 ℃. After mixing evenly to form a turbid liquid, cell wall breaking and extraction are performed. Then, the turbid liquid is subjected to solid-liquid separation to remove the aqueous phase and plasticizer, and the solid phase containing PHA is retained. S2: Add a certain amount of detergent to the solid phase containing PHA. When the plasticizer is hydrophilic, the detergent used is water. When the plasticizer is hydrophobic, the detergent used is one or a combination of methanol or ethanol. The volume ratio of detergent to PHA fermentation liquid is (1-3):

1. The washing time is 2-10 min. The washing temperature is 15-35℃. The number of washing times is 1-10. After mixing, perform solid-liquid separation to remove residual plasticizer and other impurities. S3: Dry the solid after solid-liquid separation to constant weight to obtain the polyhydroxy fatty acid ester.

2. The method for directly extracting polyhydroxy fatty acid esters from fermentation broth using plasticizers according to claim 1, characterized in that, In step S1, before adding the plasticizer to the fermentation broth that produces polyhydroxy fatty acid esters (PHA), a solid salt is added and dissolved in the fermentation broth before adding the plasticizer, or a concentrated salt solution is added after the plasticizer is added to the fermentation broth to form a turbid liquid, and then a salt precipitation extraction system is formed for solid-liquid separation.

3. The method for directly extracting polyhydroxy fatty acid esters from fermentation broth using plasticizers according to claim 2, characterized in that, The salt is water-soluble (NH4)2SO4, K2HPO4 or Na2CO3, and the salt concentration in the salting-out extraction system is 9-25 wt%.

4. The method for directly extracting polyhydroxy fatty acid esters from fermentation broth using plasticizers according to any one of claims 1-3, characterized in that, Solid-liquid separation is performed by centrifugation or filtration. Centrifugation conditions: centrifugal force of 2000-5000g and centrifugation time of 1-10 min.

5. The method for directly extracting polyhydroxy fatty acid esters from fermentation broth using plasticizers according to any one of claims 1-3, characterized in that, In step S3, the solid is dried using either atmospheric pressure drying or vacuum drying.

6. The method for directly extracting polyhydroxy fatty acid esters from fermentation broth using plasticizers according to any one of claims 1-3, characterized in that, Plasticizers and detergents removed by solid-liquid separation are recovered and reused. Plasticizers are recovered by vacuum rotary evaporation at a distillation temperature of 60-80℃, a pressure of 0.060-0.095 MPa, and a rotation speed of 60-90 rpm.

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