Method for extracting polyhydroxyalkanoate from fermentation liquor through direct wall breaking of plasticizer

Abstract

The invention relates to the technical field of post-treatment of bioengineering downstream products, and particularly discloses a method for extracting polyhydroxyalkanoate from fermentation liquor through direct wall breaking of a plasticizer. The method comprises the following steps: firstly, adding a certain amount of plasticizer into a zymophyte solution for producing PHA, uniformly mixing to form a turbid solution, carrying out cell wall breaking and extraction, then carrying out solid-liquid separation on the turbid solution to remove a water phase and the plasticizer, and reserving a solid phase containing PHA; secondly, washing with a detergent for multiple times to remove residual plasticizer and other impurities, and carrying out solid-liquid separation to obtain a solid; and finally, drying the solid to obtain the polyhydroxyalkanoate. The used plasticizer and detergent are recycled. The method integrates cell wall breaking and PHA extraction, is simple in process, green and environment-friendly, low in investment cost, low in energy consumption, high in production efficiency and product purity and stable in quality, and is suitable for industrial production.

Description

Technical Field

[0001] The invention relates to the technical field of post-processing of downstream products of bioengineering, and specifically discloses a method for extracting polyhydroxyalkanoate from fermentation broth by directly breaking the wall of a plasticizer. Background Art

[0002] Polyhydroxyalkanoates (PHA) are a class of high molecular weight polyesters that are completely synthesized by microorganisms. PHA is biodegradable and biocompatible, so it is considered to be an environmentally friendly material that helps solve the increasingly serious environmental pollution problem and has a good future.

[0003] Although extensive basic and applied research has been conducted on PHA in recent years, there is currently no large-scale industrial production of PHA in China, so it is urgent to study the industrial production process and method of PHA. The main reason why large-scale industrial production has not been achieved is that its production cost is much higher than that of petrochemical resins, and the production cost of PHA mainly includes raw material cost and separation and purification cost. The raw material cost depends on the production efficiency of the strain and the fermentation process adopted, while the separation and purification cost mainly depends on the process adopted. At present, the production scale of PHA mainly depends on extraction and separation and purification. PHA extraction usually requires collecting bacteria from the fermentation broth first, then breaking the cell wall to release PHA, then extracting with an organic solvent, and finally washing and drying. Cell wall breaking is a key operation step, generally using physical wall breaking method (high-pressure homogenization, bead milling, ultrasound, etc.), chemical wall breaking method (surfactant, acid or alkali, organic solvent, etc.), enzymatic method or combined wall breaking; the extraction method is usually organic solvent extraction, and detergents are mostly chemical reagents, surfactants, water, etc.

[0004] The invention patent with publication number CN1844185A discloses a method for extracting intracellular PHA from microorganisms, firstly collecting bacterial cells, then adding an ester organic solvent similar to the PHA monomer structure (including one or more mixed solvents of n-butyl acetate, isobutyl acetate, tert-butyl acetate, amyl acetate, methyl butyrate, ethyl butyrate, propyl butyrate, and methyl hexanoate), and finally obtaining the PHA through alcohol precipitation, washing, and drying. This method consumes a large amount of solvents, and these organic solvents are volatile, flammable, and explosive, so they are not suitable for large-scale industrial production.

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

[0006] The invention patent with publication number CN115058461A discloses a method for extracting PHA from fermentation broth by adjusting the pH value of fermentation broth with alkali solution and combining sodium salt and surfactant. This method requires the use of a large amount of alkali solution to adjust the pH value, which is easy to pollute the environment. The use of a large amount of alkali solution and surfactant increases the difficulty of sewage treatment, so the cost of wastewater treatment is high. In addition, the alkali solution has a shear degradation effect on the molecular weight of the extracted product PHA, affecting the product quality.

[0007] Existing extraction and separation methods have prominent problems such as many steps, low yield, and a lot of wastewater, which leads to high production costs, unstable product quality, lack of market competitiveness, and difficulty in large-scale industrial production. The present invention application aims to develop a new technology for coupling efficient cell wall disruption and salting-out extraction to directly extract intracellular PHA from fermentation broth. Summary of the invention

[0008] In order to overcome the problems in the prior art that are not conducive to industrial production, such as high cost, high energy consumption, high wastewater treatment cost, and alkaline degradation affecting quality, the present invention provides a method for directly breaking the wall of a plasticizer to extract polyhydroxyalkanoates from a fermentation broth. A simple, efficient, economical and green extraction process is adopted to obtain polyhydroxyalkanoates with a higher yield and purity. The method is suitable for large-scale industrial production.

[0009] The technical solution adopted by the present invention to achieve the above-mentioned purpose is: a method for extracting polyhydroxyalkanoates from fermentation broth by directly breaking the wall of a plasticizer, comprising the following steps:

[0010] S1: adding a certain amount of plasticizer to the fermentation liquid producing polyhydroxyalkanoate (PHA), mixing evenly to form a turbid liquid for cell wall breaking and extraction, and then performing solid-liquid separation on the turbid liquid to remove the water phase and the plasticizer, and retain the solid phase containing PHA;

[0011] S2: adding a certain amount of detergent to the solid phase containing PHA, mixing and then performing solid-liquid separation to remove residual plasticizer and other impurities;

[0012] S3: Drying the solid after solid-liquid separation to a constant weight to obtain the polyhydroxyalkanoate.

[0013] Furthermore, in step S1, before adding the plasticizer to the fermentation broth producing polyhydroxyalkanoate (PHA), solid salt is added and dissolved in the fermentation broth before adding the plasticizer, or after adding the plasticizer to the fermentation broth to form a turbid liquid, a concentrated salt solution is added to form a salting-out extraction system and then solid-liquid separation is performed.

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

[0015] Further, in step S1, the plasticizer includes a hydrophobic plasticizer and a 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] Furthermore, in step S1, 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, and the mixing cell wall breaking extraction temperature is 15-70°C.

[0017] Furthermore, the solid-liquid separation adopts centrifugation or filtration, and the centrifugal operation conditions are: centrifugal force is 2000-5000g, and the centrifugal time is 1-10min.

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

[0019] Furthermore, in step S3, the solid is dried by normal pressure drying or vacuum drying.

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

[0021] Compared with the prior art, the present invention has the following beneficial effects:

[0022] 1. The present invention ingeniously utilizes the property that plasticizers can combine with PHA to swell, integrates cell wall breaking and PHA extraction, simplifies the PHA extraction process, eliminates the need for prior separation and washing of bacteria, avoids the use of complex equipment such as high-speed centrifuges, and directly separates and purifies polyhydroxyalkanoate from fermentation broth, thereby reducing investment in production equipment, reducing production energy consumption, shortening production time, and reducing wastewater discharge, making the process more suitable for large-scale industrial production.

[0023] 2. The production efficiency and product purity of the present invention are high and the quality is stable. The PHA yield obtained by the plasticizer treatment process without adding salt can reach up to 86.72% and the purity can reach 95.52%; the salting-out extraction process with salt can further increase the PHA yield to 96.31% and the purity to 98.86%. The raw materials added by the present invention will not produce shear degradation effect on PHA, so the product quality is very stable.

[0024] 3. The prior art uses a large amount of alkali solution and surfactant, which increases the difficulty of sewage treatment, so the cost of wastewater treatment is relatively high. The present invention only uses plasticizers and alcohol detergents, generates less sewage, and the plasticizer can be recycled and reused, which reduces the cost of environmental protection treatment and the production cost.

[0025] 4. The plasticizer used in the present invention is a commonly used industrial reagent, which is not only relatively cheap, but also non-toxic or low-toxic. The entire process is more environmentally friendly than the existing technology. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 The experimental pictures of extracting PHA from fermented bacterial broth by breaking the cell wall of three plasticizers in Example 1. (A) shows the experimental phenomenon after the fermented bacterial broth and plasticizers are mixed and centrifuged; (B) shows the addition of ethanol to elute the plasticizer and some impurities that are insoluble in water; (C) shows the addition of water to elute the residual ethanol and purify the solid; (D) shows the finished PHA after solid-liquid separation and drying.

[0027] Figure 2 The experimental actual pictures of adding different concentrations of K2HPO4 in the process of triethyl phosphate cell wall breaking and salting out extraction of PHA in Example 2. (A)-(F) are the experimental phenomena after the fermentation liquid with 9, 12, 15, 18, 20, and 25 wt% K2HPO4 was mixed with triethyl phosphate and centrifuged.

[0028] Figure 3 The experimental actual pictures of different washing conditions when the plasticizer was broken to extract PHA from the fermented bacterial broth in Example 3. (A) shows the experimental phenomenon after the first washing and centrifugation; (B) shows the experimental phenomenon after the second washing and centrifugation; (C) shows the finished PHA after solid-liquid separation and drying. DETAILED DESCRIPTION

[0029] The present invention is further described in detail below in conjunction with the examples and drawings, but the embodiments of the present invention are not limited thereto. Where specific conditions are not specified in the examples, the experimental methods used are all conventional methods, and the reagents or instruments used can be conventional products purchased from the market.

[0030] The fermentation broth used in the embodiment was prepared by fermentation culture of Halomonas under conventional fermentation culture conditions in the art. The PHA content in the unbroken fermentation broth was 43.09-60.64 g / L; the PHA content in the broken broth treated with surfactant (0.03% SDS) and heating (70°C) after the cells were collected was 45.50 g / L; the PHA content in the broken broth after three cycles of high-pressure homogenization was 69.06 g / L.

[0031] The product detection method of the present invention is gas chromatography. The gas chromatograph model is Shimadzu GC-2010, and the chromatographic column is Agilent HP-5-MS UI. The temperature program is set as follows: injection port (SPL1) 180°C; detector (FID) 200°C; column temperature is initially 50°C, retained for 1 minute, then increased to 180°C at 15°C / min, retained for 2 minutes; split ratio is set to 10:1.

[0032] Preparation of standard curve: Weigh 2, 5, 10, 15, and 20 mg of pure PHA powder, 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, heat to 100°C, and esterify for 4 hours. Cool to room temperature, add 1 mL of pure water, vortex for 30 seconds, stand for 2 hours until complete stratification, take the lower phase (chloroform phase) into a sample bottle containing 150 mg of anhydrous sodium sulfate to remove a small amount of water, and then take 1 mL of sample and filter through a 0.22 μm membrane for gas chromatography detection.

[0033] Sample quantitative analysis: Weigh 10 mg of solid PHA after extraction and purification, treat it under the same conditions as those for making the standard curve, and perform gas chromatography detection.

[0034] The yield and purity of PHA product are calculated according to the following formula:

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

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

[0037] Comparative Example 1 Effect of different cell wall breaking methods on PHA extraction

[0038] Take 5 mL of high-pressure homogenate and 5 mL of surfactant-heated cell wall breaking solution in different centrifuge tubes, add 5 mL of different extractants or detergents at room temperature, vortex for 2 minutes, and centrifuge at 3000g for 5 minutes; take the precipitate and add 10 mL of water, vortex and centrifuge under the same conditions. Take the solid and place it in a 60℃ oven to dry to constant weight.

[0039] The PHA content in the dried sample was detected by gas chromatography, and the yield and purity of the product were calculated as shown in Table 1.

[0040] Table 1. Results of PHA produced by existing cell wall breaking and extraction processes

[0041]

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

[0043] Take 5 mL of untreated Halomonas fermentation broth, add plasticizers in different volume ratios, vortex and oscillate for 2 minutes at different wall-breaking extraction temperatures to mix the plasticizer and fermentation broth evenly to form a turbid liquid, and then centrifuge at 3000g for 5 minutes; take the precipitate and add 10 mL of washing solution, vortex and oscillate under the same conditions, and then centrifuge; take the precipitate and add 10 mL of water, vortex and oscillate under the same conditions, and then centrifuge, collect the solid precipitate and place it in a 70°C oven to dry to constant weight.

[0044] The PHA content in the dried sample was detected 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

[0046]

[0047] Note: TEC: triethyl citrate; TMP: trimethyl phosphate; TEP: triethyl phosphate; TBC: tributyl citrate; TCP: tricresyl phosphate

[0048] As can be seen from Table 2, the use of plasticizers has a combined effect of cell wall breaking and extraction on the PHA fermentation broth, and the yield and purity are relatively high. The highest yield of PHA in the comparative example is 57.77%, while the PHA yield in this example is generally greater than 60%. The purity of PHA extracted with triethyl citrate is greater than 70%, and the purity of PHA extracted with triethyl phosphate is greater than 80%.

[0049] Example 2 Effect of salt type and concentration in plasticizer wall breaking and salting out extraction of PHA

[0050] Take 5mL of unbroken Halomonas fermentation broth, add solid salt or concentrated salt solution before or after adding 5mL of plasticizer, vortex and oscillate at 25℃ for 2min, form a salting-out extraction system, and centrifuge at 2000g for 10min for solid-liquid separation; for triethyl citrate (TEC) system, take the precipitate and add 10mL of anhydrous ethanol, vortex and oscillate under the same conditions and centrifuge, then take the precipitate and add 10mL of water, vortex and oscillate for 2min, vortex and oscillate under the same conditions and centrifuge to obtain solid; for triethyl phosphate (TEP) system, take the precipitate and add 10mL of water, repeat the vortex and oscillate under the same conditions and centrifuge to obtain solid. Collect the solid and place it in an oven at 80℃ to dry to constant weight.

[0051] The PHA content in the dried sample was detected 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 wall breaking and salting out extraction of PHA

[0053] Plasticizer Type Salt addition time Salt Type 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 can be seen from Table 3, adding high concentration of salt in the plasticizer wall-breaking extraction process can improve the PHA extraction yield and purity. Under high salt concentration (salt concentration ≥ 15wt%), the mixed solution forms a salting-out extraction system, and the system is phase-separated after centrifugation, with the upper phase being the plasticizer phase and the lower phase being the salt solution phase. PHA is distributed at the interface between the two phases, which is beneficial to the separation of PHA and cell impurities.

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

[0056] Take 5 mL of unbroken Halomonas fermentation broth, add 1:1 triethyl citrate (TEC), triethyl phosphate (TEP) or trimethyl phosphate (TMP), vortex at 20°C for 2 min, and centrifuge at 5000g for 1 min; take the precipitate and add different amounts of detergent for washing, 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 place it in a 55°C oven to dry to constant weight.

[0057] The PHA content in the dried sample was detected by gas chromatography, and the yield and purity of the product were calculated as shown in Table 4.

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

[0059]

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

[0061] Example 4 Effect of plasticizer recycling on cell wall extraction of PHA

[0062] Take the upper phase liquid and the first washing liquid after centrifugation of triethyl phosphate and fermentation broth, and revolute at 60°C, 70rpm and 0.075MPa until the water is completely evaporated to recover triethyl phosphate. Add 5mL of the recovered plasticizer to 5mL of unbroken Halomonas fermentation broth at room temperature, mix and centrifuge at 3000g for 5min; take the precipitate and add 10mL of water, mix and centrifuge under the same conditions; take the precipitate again and add 10mL of water, mix and centrifuge under the same conditions; collect the solid and place it in a 60°C oven to dry to constant weight. According to the above steps, triethyl phosphate is recovered again and reused for the broken wall extraction of PHA in the fermentation broth. The plasticizer recovery rates of the three experiments are 94.62%, 82.51% and 76.31% respectively.

[0063] The PHA content in the dried sample was detected by gas chromatography, and the yield and purity of the product were calculated as shown in Table 5.

[0064] Table 5. Results of recycling and reusing triethyl phosphate and extracting PHA from fermentation broth by breaking the cell wall

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

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

[0067] The embodiments of the present invention are given for the purpose of illustration and description, and are not intended to be exhaustive or to limit the invention to the disclosed forms. Many modifications and variations will be apparent to those of ordinary skill in the art. The embodiments are selected and described in order to better illustrate the principles and practical applications of the present invention and to enable those of ordinary skill in the art to understand the present invention and thereby design various embodiments with various modifications suitable for specific uses.

Claims

1. A method for extracting polyhydroxyalkanoates from fermentation broth by directly breaking the cell wall of a plasticizer, characterized in that: The following steps are involved: S1: adding a certain amount of plasticizer to the fermentation liquid producing polyhydroxyalkanoate (PHA), mixing evenly to form a turbid liquid for cell wall breaking and extraction, and then performing solid-liquid separation on the turbid liquid to remove the water phase and the plasticizer, and retain the solid phase containing PHA; S2: adding a certain amount of detergent to the solid phase containing PHA, mixing and then performing solid-liquid separation to remove residual plasticizer and other impurities; S3: Drying the solid after solid-liquid separation to a constant weight to obtain the polyhydroxyalkanoate.

2. The method for extracting polyhydroxyalkanoates from fermentation broth by directly breaking the plasticizer according to claim 1, characterized in that: In step S1, before adding a plasticizer to the fermentation broth producing polyhydroxyalkanoate (PHA), solid salt is added and dissolved in the fermentation broth before adding the plasticizer, or after adding the plasticizer to the fermentation broth to form a turbid liquid, a concentrated salt solution is added to form a salting-out extraction system and then solid-liquid separation is performed.

3. The method for extracting polyhydroxyalkanoates from fermentation broth by directly breaking the plasticizer according to claim 2, characterized in that: The salt is (NH4)2SO4, K2HPO4 or Na2CO3 which is soluble in water, and the concentration of the salt in the salting-out extraction system is 9-25wt%.

4. The method for extracting polyhydroxyalkanoate from fermentation broth by directly breaking the wall of the plasticizer according to any one of claims 1 to 3, characterized in that: In step S1, the plasticizer includes a hydrophobic plasticizer and a 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).

5. The method for extracting polyhydroxyalkanoate from fermentation broth by directly breaking the wall of plasticizer according to any one of claims 1 to 3, characterized in that: In step S1, 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, and the mixing cell wall breaking extraction temperature is 15-70°C.

6. The method for extracting polyhydroxyalkanoate from fermentation broth by directly breaking the cell wall of a plasticizer according to any one of claims 1 to 3, characterized in that: Solid-liquid separation is carried out by centrifugation or filtration. The centrifugal operation conditions are: centrifugal force is 2000-5000g, and centrifugal time is 1-10min.

7. The method for extracting polyhydroxyalkanoate from fermentation broth by directly breaking the cell wall of a plasticizer according to any one of claims 1 to 3, characterized in that: In step S2, when the plasticizer is hydrophilic, the detergent used is water, and when the plasticizer is hydrophobic, the detergent used is one or a combination of methanol or ethanol, the volume ratio of the detergent to the PHA fermentation liquid is (1-3):1, the washing time is 2-10 minutes, the washing temperature is 15-35°C, and the number of washing times is 1-10 times.

8. The method for extracting polyhydroxyalkanoate from fermentation broth by directly breaking the wall of a plasticizer according to any one of claims 1 to 3, characterized in that: In step S3, the solid is dried by normal pressure drying or vacuum drying.

9. The method for extracting polyhydroxyalkanoate from fermentation broth by directly breaking the cell wall of a plasticizer according to any one of claims 1 to 3, characterized in that: The plasticizer and detergent removed by solid-liquid separation are recovered and reused, and the plasticizer is recovered by vacuum rotary evaporation, with a distillation temperature of 60-80°C, a pressure of 0.060-0.095MPa, and a rotation speed of 60-90rpm.

Images