Extraction and purification method for intracellular polymer polyhydroxyalkanoate (PHA)

Abstract

The invention discloses an extraction and purification method for an intracellular polymer polyhydroxyalkanoate (PHA). The extraction and purification method comprises the following steps: 1) carrying out flora separation after completing maximized PHA synthesis on a mixed flora; 2) separating the PHA product and the mixed flora by using a chemical digestion method, and 3) extracting and purifying the PHA product. According to the method, the SDS chemical digestion method is carried out on the PHA in the mixed flora cells for PHA extraction, then the organic extraction solvent is used for purification treatment, and the extraction efficiency and purity of the PHA product are remarkably improved. Meanwhile, the PHA extraction cost is reduced by using an organic solvent recovery device and repeatedly using a chemical solvent, and the secondary pollution in the extraction process is reduced by using an SDS (Sodium Dodecyl Sulfate) recovery method, so that the whole extraction process is more environment-friendly and low in cost.

Description

Technical Field

[0001] The present invention relates to a production process of green bio-based degradable plastics, namely polyhydroxyalkanoates (PHA), and in particular to a method for extracting and purifying intracellular PHA. Background Art

[0002] Polyhydroxyalkanoates (PHA) are the only type of high-molecular-weight polymer currently under study that is synthesized intracellularly by microorganisms from precursors to products. PHA's fully biosynthesized degradation properties can meet both full biodegradability requirements and the performance requirements of plastic products, and are therefore widely studied. Furthermore, existing PHA extraction methods primarily target pure bacterial PHA synthesis processes, and require the addition of large amounts of oxidants and toxic, volatile organic solvents, which can easily cause secondary pollution during the extraction process and lead to high PHA extraction costs. Currently, the most widely used PHA extraction method is the NaClO+chloroform extraction technique, but this technique can produce secondary pollution and destroy the molecular structure of the extracted PHA.

[0003] Therefore, there is insufficient research on green, efficient and low-cost extraction methods based on the characteristics of mixed bacterial communities, and it is necessary to develop an extraction and purification method and recovery device for intracellular PHA. Summary of the Invention

[0004] The present invention aims to provide a method for extracting and purifying intracellular PHA with high extraction efficiency and high purity. The method comprises four main steps: 1) digestion of non-PHA structures, 2) purification of PHA, 3) recovery of chemical digestion agents, and 4) recovery and reuse of organic solvents.

[0005] The present method extracts PHA from the cells of a mixed bacterial community using SDS chemical digestion, followed by purification using an organic extraction solvent. This significantly improves the extraction efficiency and purity of the PHA product. Furthermore, the reuse of chemical solvents using an organic solvent recovery method reduces PHA extraction costs, while the SDS recovery device reduces secondary contamination during the extraction process, making the entire extraction process more environmentally friendly and cost-effective.

[0006] In order to achieve this object, the technical solution of the present invention is as follows:

[0007] A method for extracting and purifying an intracellular polymer polyhydroxyalkanoate (PHA), characterized in that the method based on separating the PHA product from the bacterial flora and purifying the product comprises the following main steps:

[0008] 1) After the mixed bacterial population has achieved maximum PHA synthesis, the bacterial population is separated by high-speed centrifugation at 10,000 rpm for 30 min to obtain the mixed bacterial population, which is then disrupted using an ultra-high pressure cell disruptor (JN-Mini, Guangzhou Juneng) and proceeds to the next step;

[0009] 2) The cell wall-broken bacterial community described in step (1) is subjected to a chemical digestion method to separate the PHA product and the mixed bacterial community, using sodium dodecyl sulfate (SDS) at a concentration of 5 to 10 g / L for 30 to 60 minutes to dissolve the non-PHA structures in the biomass in the SDS, and the PHA product precipitate is separated from the SDS, and the PHA product is precipitated and enters the next step;

[0010] 3) extracting and purifying the precipitated PHA product described in step (2), adding an organic solvent to the PHA particles for extraction and purification, dissolving the PHA in the organic solvent to cause stratification, removing non-PHA impurities, and heating the organic solvent to volatilize to obtain the PHA product;

[0011] The cell wall breaking treatment method in the mixed bacterial population of the present invention is characterized in that the cell disruption is carried out at a pressure of 20 to 80 MPa using the low-temperature ultrahigh pressure cell disruptor;

[0012] After the chemical digestion described in the present invention, stratification occurs, with the upper layer being non-PHA bacterial impurities and the lower layer being the PHA product, while the solution in the middle layer flows out and the SDS digestion solvent is recovered by precipitation;

[0013] The organic extraction solvents for PHA described in the present invention are chloroform, acetone and dimethyl carbonate, etc., all of which have boiling points below 100°C and can be recycled and extracted using a heating method;

[0014] After the PHA is extracted and purified, the upper layer is composed of non-PHA impurities and the lower layer is composed of an organic solvent phase. The organic solvent is heated and volatilized at 100°C. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1 Schematic diagram of the process for extracting and purifying the intracellular polymer polyhydroxyalkanoate (PHA) described in Example 1 of the present application. DETAILED DESCRIPTION

[0016] To make the objectives, technical solutions, and advantages of the present invention more clear, the technical solutions of the present invention will be clearly and completely described below in conjunction with the accompanying drawings. Obviously, the embodiments described are only some of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts shall fall within the scope of protection of the present invention.

[0017] Example 1

[0018] This embodiment provides a method for extracting and purifying intracellular polymer polyhydroxyalkanoate (PHA), such as Figure 1 As shown, it includes:

[0019] The SDS recovery step 1 is used to recover the SDS solution after chemical digestion in the PHA extraction and separation device 2 by adding an equal volume of KCl solution (5-10 g / L) to precipitate SDS. The inlet of the SDS recovery device 1 is connected to the outlet of the PHA extraction and separation device 2.

[0020] PHA extraction and separation 2 separates the PHA mixed flora and PHA products; the outlet of the PHA extraction and separation device 2 is connected to the inlet of the SDS recovery device 1, and the organic solvent outlet of the PHA extraction and separation device 2 is connected to the inlet of the centrifugal system 3.

[0021] Centrifugal system 3 is used to separate the mixed liquid after organic solvent extraction from bacterial impurities. The inlet of centrifugal system 3 is connected to the organic solvent outlet of PHA extraction and separation device 2. After centrifugation, the upper layer contains bacterial impurities, and the lower layer contains PHA organic solvent. A discharge valve is provided at the bottom of centrifugal system 3 to separate the impurities in the upper layer. The outlet of centrifugal system 3 is connected to the inlet of organic solvent heating device 4.

[0022] The organic solvent heating device 4 is used to separate PHA from the organic solvent. The organic solvent heating device 4 is externally heated in a water bath with adjustable heating temperature, which is controlled to be above the boiling point of the organic solvent to volatilize the extraction solvent. After evaporation, the purified PHA solid remains. A removable base is provided at the bottom for collecting the purified PHA sample. The organic solvent gas outlet of the organic solvent heating device 5 is connected to the inlet of the organic solvent condensation recovery device 5.

[0023] The organic solvent condensation and recovery unit 5 is used to recover the organic solvent extractant. The organic solvent condensation and recovery system is equipped with a low-temperature water bath set at a temperature below the boiling point of the recovered organic solvent to condense and collect the organic solvent. The recovered organic solvent is used for the next batch of PHA purification in the PHA extraction and separation unit 2. The outlet of the organic solvent condensation and recovery unit 5 is connected to the inlet of the PHA extraction and separation unit 2.

Claims

1. A method for extracting and purifying intracellular polymer polyhydroxyalkanoate (PHA), characterized in that: The method for separating and purifying PHA products from bacterial flora includes the following main steps: 1) After the mixed bacterial population has achieved maximum PHA synthesis, the bacterial population is separated by high-speed centrifugation at 10,000 rpm for 30 min to obtain the mixed bacterial population, which is then disrupted using an ultra-high pressure cell disruptor (JN-Mini, Guangzhou Juneng) and proceeds to the next step; 2) The cell wall-broken bacterial community described in step (1) is subjected to a chemical digestion method to separate the PHA product and the mixed bacterial community, using sodium dodecyl sulfate (SDS) at a concentration of 5 to 10 g / L for 30 to 60 minutes to dissolve the non-PHA structures in the biomass in the SDS, and the PHA product precipitate is separated from the SDS, and the PHA product is precipitated and enters the next step; 3) extracting and purifying the precipitated PHA product described in step (2), adding an organic solvent to the PHA particles for extraction and purification, dissolving the PHA in the organic solvent to cause stratification, removing non-PHA impurities, and heating the organic solvent to volatilize to obtain the PHA product.

2. The method for breaking the cell wall of a mixed bacterial community according to claim 1, wherein: The low-temperature ultra-high-pressure cell disruptor process parameters are to perform disruption at a pressure of 20 to 80 MPa.

3. After the chemical digestion according to claim 1, stratification occurs, the upper layer is non-PHA bacterial impurities, the lower layer is PHA product, and the middle layer solution flows out and uses precipitation method to recover SDS digestion solvent.

4. The organic extraction solvent for PHA according to claim 1 is chloroform, acetone, dimethyl carbonate, etc., all of which have boiling points below 100° C. and can be recycled and extracted using a heating method.

5. The PHA according to claim 1 is separated into layers after extraction and purification, wherein the upper layer is non-PHA impurities and the lower layer is an organic solvent phase, and the organic solvent is heated and volatilized at 100°C.

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