阅读说明：本技术 用于检测高分子材料在海洋环境中的好氧生物降解性能的方法 (Method for detecting aerobic biodegradation performance of high polymer material in marine environment ) 是由 王格侠 季君晖 黄丹 李飞 卢波 甄志超 于 2020-05-15 设计创作，主要内容包括：本发明公开了一种用于检测高分子材料在海洋环境中的好氧生物降解性能的方法,其包括如下步骤：(i)提供待检测的高分子材料样品和海水样本；(ii)将所述样品置于所述海水样本中,在好氧环境下进行降解试验；(iii)吸收降解产生的CO-(2)气体,使其反应生成碳酸根离子；(iv)定期取样测定碳酸根离子含量,基于碳酸根离子含量推算降解产生的CO-(2)气体释放量,根据CO-(2)气体释放量随降解时间的变化评价高分子材料的生物降解性能,其中,碳酸根离子含量可采用离子色谱仪测定,因此,本发明的方法相比于现有的检测方法具有更高的灵敏度和检测限,并且,便于实现检测自动化。(The invention discloses a method for detecting the aerobic biodegradation performance of a high polymer material in a marine environment, which comprises the following steps: (i) providing a high polymer material sample to be detected and a seawater sample; (ii) placing the sample in the seawater sample, and performing a degradation test in an aerobic environment; (iii) absorption of CO produced by degradation 2 Reacting the gas to produce carbonate ions; (iv) periodically sampling to determine the content of carbonate ions, and calculating CO generated by degradation based on the content of the carbonate ions 2 Amount of gas released according to CO 2 The biodegradation performance of the high polymer material is evaluated according to the change of the gas release amount along with the degradation time, wherein the content of carbonate ions can be measured by an ion chromatograph, so that compared with the existing detection method, the method disclosed by the invention has higher sensitivity and detection limit, and is convenient for realizing detection automation.)

1. A method for detecting the aerobic biodegradation performance of a high molecular material in a marine environment comprises the following steps:

(i) providing a high molecular material sample to be detected and a seawater sample, wherein the seawater sample contains microorganisms and inorganic nutrients;

(ii) placing the sample in the seawater sample, and performing a degradation test in an aerobic environment for a preset time;

(iii) absorbing carbon dioxide gas generated by degradation, and reacting to generate carbonate ions;

(iv) and (3) periodically sampling and determining the content of carbonate ions, calculating the release amount of carbon dioxide gas generated by degradation based on the content of the carbonate ions, and evaluating the biodegradation performance of the high polymer material according to the change of the release amount of the carbon dioxide gas along with the degradation time.

2. The method of claim 1, wherein at least one alkali solution is used in step (iii) to absorb hydrazine to absorb carbon dioxide gas and generate carbonate ions.

3. The method of claim 1 or 2, wherein step (iv) detects the carbonate ion content using anion chromatography.

4. The method of claim 2, wherein the alkali solution is capable of forming water soluble carbonate ions with carbon dioxide.

5. The method of claim 4, wherein the alkali solution is a NaOH solution or a KOH solution.

6. A process according to claim 2 wherein in step (iii) at least two alkali solutions are used to absorb hydrazine, each absorption being in series with the other absorption.

7. The method of claim 1, further comprising: and (3) converting the mineralization rate of the high polymer material according to the release amount of carbon dioxide gas, and evaluating the biodegradation performance based on the mineralization rate, wherein the conversion formula of the mineralization rate is as follows:

wherein (CO)₂)_TFor the accumulated CO generated in the detection process of the sample to be detected₂The amount of (c); (CO)₂)_BCO produced for blank control₂The amount of (c); ThCO₂For CO theoretically produced by the sample₂The amount of (c).

8. The method of claim 1, wherein the seawater sample comprises 10% of microorganisms⁴-10⁷CPU/mL, the inorganic nutrient comprises NH₄Cl and KH₂(PO₄)。

9. The method of claim 1, wherein the predetermined period of time is 3 to 6 months.

10. The method of claim 1, wherein the periodic sampling determines carbonate ion content as frequently as every 5 to 20 days.

Technical Field

The invention relates to a method for detecting biodegradability of a high polymer material, in particular to a method for detecting biodegradability of a high polymer material in a marine environment.

Background

The degradation of the polymer material is classified into photodegradation, oxidative degradation, hydrolysis, biodegradation, light/oxygen degradation and the like according to a degradation mechanism, wherein biodegradation is the most concerned degradation mode and the most widely applied degradation mode, and biodegradation generally takes carbon dioxide gas finally generated by the polymer material in a medium as a core evaluation standard. Therefore, in the prior art, the biodegradation performance of the material is evaluated by detecting the release amount of carbon dioxide in the degradation process of the high polymer material.

In a real marine degradation environment, the highest temperature does not exceed 30 ℃, the seawater temperature gradually decreases with the increase of the water depth, and the seabed temperature is lower than 10 ℃. Because the temperature of the marine environment is lower, the degradation of the high polymer material in the seawater environment is slower, and the amount of carbon dioxide released by degradation is less. For detecting the biodegradation performance of the high polymer material, the existing detection method usually adopts a respirometer, an infrared spectrometer or an acid-base titration method to detect the carbon dioxide generated in the degradation process, wherein, the detection limits of the respirometer and the infrared spectrometer are both above 1ppm, the detection limits of the two detection instruments are higher, and the two detection instruments are only suitable for testing a large amount of carbon dioxide generated by the degradation of high molecular materials under the conditions of high microorganism content and high temperature such as compost, however, in a real marine degradation environment, because the temperature of the degradation environment is lower and the degradation of the high polymer material is slower, the amount of carbon dioxide released by degradation is lower, the detection limits of the respirometer and the infrared spectrometer are high, and when the amount of the carbon dioxide released by degradation is lower than the detection limit, the carbon dioxide cannot be detected, so that the degradation performance is easily subjected to wrong evaluation. In addition, the acid-base titration method needs to frequently prepare and replace alkali liquor and titrate with standard solution, so that experimental error sources are many, errors are easily caused by more human factors, in addition, manpower is consumed, the detection efficiency is low, and the degradation of the high-molecular material in the marine environment cannot be efficiently, accurately and sensitively evaluated.

Therefore, a method with higher sensitivity suitable for detecting the biodegradability of the polymer material in the marine environment is needed.

Disclosure of Invention

In view of the above problems in the prior art, the present invention aims to provide an improved method for detecting the aerobic biodegradation performance of a high molecular material in a marine environment, which can more truly reflect the degradation performance of the material in the marine environment.

The method for detecting the aerobic biodegradation performance of the high polymer material in the marine environment comprises the following steps:

(i) providing a high molecular material sample to be detected and a seawater sample, wherein the seawater sample contains microorganisms and inorganic nutrients;

(ii) placing the sample in the seawater sample, and performing a degradation test in an aerobic environment;

(iii) absorbing carbon dioxide gas generated by degradation, and reacting to generate carbonate ions;

(iv) and (3) periodically sampling and determining the content of carbonate ions, calculating the release amount of carbon dioxide gas generated by degradation based on the content of the carbonate ions, and evaluating the biodegradation performance of the high polymer material according to the change of the release amount of the carbon dioxide gas along with the degradation time.

In some embodiments of the present invention, at least one alkali solution may be used in step (iii) to absorb hydrazine to absorb carbon dioxide gas and generate carbonate ions. When the number of the alkali solution absorbing hydrazines is at least two, the absorbing hydrazines are connected in series, and the absorption efficiency of the carbon dioxide is further improved.

In some embodiments of the present invention, step (iv) uses an anion chromatograph to detect the carbonate ion content, and due to the low detection limit of the ion chromatograph, the carbonate ion content in the absorbed hydrazine can be accurately and sensitively detected even in the case of a small amount of carbon dioxide released in the early stage of degradation.

In some embodiments of the present invention, the alkali solution in the carbon dioxide absorption hydrazine can generate water-soluble carbonate ions with carbon dioxide, preferably an alkali solution that can react rapidly with carbon dioxide to generate water-soluble carbonate ions, such as a strong alkali solution like NaOH solution or KOH solution.

In some embodiments of the present invention, the detection method of the present invention may further convert the mineralization rate of the polymer material according to the amount of released carbon dioxide gas, and evaluate the biodegradation performance based on the mineralization rate, wherein the conversion formula of the mineralization rate is as follows:

wherein (CO)₂)_TFor the accumulated CO generated in the detection process of the sample to be detected₂The amount of (c); (CO)₂)_BCO produced for blank control₂The amount of (c); ThCO₂For CO theoretically produced by the sample₂The amount of (c).

In some embodiments of the invention, the seawater sample comprises 10% of microorganisms⁴-10⁷CPU/mL, inorganic nutrients including NH₄Cl and KH₂(PO₄)。

In some embodiments of the present invention, for detecting the degradation performance of the polymer material in the marine environment, the predetermined time period for degradation is 3 to 6 months, and the time period can be appropriately adjusted according to the property of the polymer material to be detected and the detection condition.

In some embodiments of the present invention, the frequency of periodically sampling the absorption liquid in the carbon dioxide absorption hydrazine to measure the content of carbonate ions is once every 5 to 20 days, and can be adjusted according to the specific situation of the sample to be measured.

Compared with the existing method for detecting the degradation performance of the high polymer material in the marine environment, the method provided by the invention absorbs carbon dioxide released by degradation to generate carbonate ions, and calculates the accumulated release amount of the carbon dioxide based on the content of the carbonate ions, wherein the content of the carbonate ions is determined by an ion chromatograph.

Drawings

Fig. 1A shows the cumulative release of carbon dioxide over a degradation period of 90 days for four test samples obtained by the test method according to the present invention.

Fig. 1B shows the calculated mineralization rate versus time for the cumulative release of carbon dioxide over the 90 day degradation period for the four test samples shown in fig. 1A.

Detailed Description

The present invention will be described in detail with reference to specific examples, which are provided for illustration only and are not intended to limit the scope of the present invention.

In an exemplary embodiment of the invention, samples in the form of thin films were selected, with Thin Layer Chromatography (TLC) cellulose as a reference control, and a reactor containing only a sample of seawater as a blank control.

In an exemplary embodiment of the present invention, the seawater sample may be extracted from natural seawater or prepared by itself according to the requirement, and may be referred to the seawater sample in the existing standard (for example, ASTM D6691-2009), wherein inorganic salts, microorganisms and inorganic nutrients required by microorganisms are contained, and the components and contents thereof may be referred to the existing standard. Wherein the number of microorganisms in the seawater sample can be adjusted by enrichment, cultivation or dilution, and in order to more truly simulate the seawater environment, in an exemplary embodiment of the present invention, the number of microorganisms in the seawater sample is adjusted to 10⁴-10⁷CPU/ML. The microorganism count can be carried out by a conventional method known in the art, for example, plate count method.

In an exemplary embodiment of the invention, the degradation test temperature is chosen to be 30 ℃ and also to float within a suitable range, which degradation temperature on the one hand can simulate a seawater environment and on the other hand can control the degradation detection time duration within a reasonable range, for example within 6 months. In addition, temperature conditions for accelerating the degradation test may also be set.

In an exemplary embodiment of the invention, oxygen may be introduced directly into the degradation vessel or CO may be removed for the purpose of conducting the degradation test in an aerobic environment₂Gaseous air, e.g. air can be blown into the CO using an air compressor₂Absorption of hydrazine to remove CO₂Gas, CO removal₂The air of the gas is slowly introduced into the degradation container, and a gas buffer tank and an air flow meter may be provided in the gas flow path as necessary in order to appropriately control the gas flow rate. In addition, CO₂The amount of hydrazine absorbed can be set according to the requirement and can be single CO₂Absorbing hydrazine, optionally more CO₂The absorption hydrazine is used in series.

In an exemplary embodiment of the invention, the CO generated by the degradation of the polymeric material₂Collecting gas by absorbing hydrazine with at least one alkali solution with a certain concentration, and optionally adding CO for improving absorption efficiency₂The contact area of the gas and the absorption solution, or a plurality of absorption hydrazines in series. Absorbing alkali solution in hydrazine for absorbing CO₂The gas generates carbonate ions, and the alkali solution is preferably a solution which can easily and rapidly generate water-soluble carbonate ions with carbon dioxide, for example, a NaOH solution or a KOH solution with a proper concentration is selected.

The content of carbonate ions in the solution in the absorbed hydrazine is periodically measured by adopting an anion chromatograph, and the CO generated by degradation is calculated₂When a plurality of absorption hydrazines are used in series, the sum of the absorption amounts of the absorption hydrazines in the same degradation period is calculated. Cumulative generation of CO from degradation₂The percentage of the gas quantity in the theoretical carbon dioxide generation quantity of the seawater degraded plastics is the degradation rate or mineralization rate.

In an exemplary embodiment of the present invention, the concentration of carbonate ions is measured by an anion chromatograph, and in order to analyze the change relationship of the degradation performance with time, the present invention periodically measures the content of the accumulated carbonate ions generated in the degradation process, further calculates the corresponding accumulated release amount of carbon dioxide, and finally calculates the mineralization rate (i.e., degradation rate) of the detected polymer material to evaluate the biodegradation degree.

The extent of biodegradation of the sample can be assessed by calculating the mineralization rate (see equation 1):

wherein (CO)₂)_TCumulative CO production for the sample₂Content of (g), (CO)₂)_BCumulative CO production for blank set₂Content (mg) and ThCO₂Theoretical generation of CO for a sample₂Total content (mg) (see equation 2).

Wherein M is_TOTTo test the total dry solids (mg) of the material, C_TOTThe total organic carbon content (%) of the test material was determined.

Example 1

In this example, PBS (poly butylene succinate), PBSG30 and PBSG50 were used as polymer material test samples, cellulose was used as reference control, and the molecular weight, carbon content and theoretical CO of the test samples and the reference control samples₂The contents are shown in the following table. The structural formulas of the test samples PBSG30 and PBSG50 are shown below.

Respectively placing 1.5g of the four film-like samples into a 5L degradation reaction tank containing 3L of seawater samples, wherein the number of microorganisms is 3489CPU/mL, and the inorganic nutrients comprise 0.5g/L NH₄Cl and 0.1g/L KH₂(PO)₄Placing the sample in the seawater sample, degrading for 3 months at 30 ℃, introducing carbon dioxide generated by degradation into carbon dioxide absorption hydrazine containing 0.1mol/L NaOH aqueous solution, collecting, extracting solution from the absorption hydrazine every 10 days, and measuring the content of carbonate ions in the extracted NaOH solution by an IC940 type ion chromatograph. The ion chromatograph uses a MetroSep Organic Acids-250/7.8 chromatographic column, and the eluent is 0.5mM H₂SO₄The flow rate is 0.5mL/min, the column temperature is 30 ℃, the quantitative loop is 100 mu L, a 858 autosampler is used for sample injection, and the analysis is carried out by a conductivity detector. Three replicates of each sample were averaged. The cumulative amount of carbon dioxide produced at each sampling time point was estimated from the measurement data of the carbonate ion concentration. Fig. 1A shows the cumulative release of carbon dioxide over a degradation period of 90 days for the four test samples, and fig. 1B shows the cumulative release of carbon dioxide over time for the mineralization rate (i.e., degradation rate) corresponding to the four samples shown in fig. 1A, and the calculation formula for calculating the degradation rate or mineralization rate based on carbon dioxide is as follows:

wherein (CO)₂)_TFor the accumulated CO generated in the detection process of the sample to be detected₂The amount of (c); (CO)₂)_BCO produced for blank control₂The amount of (c); ThCO₂For CO theoretically produced by the sample₂Amounts (listed in the table above).

FIGS. 1A and 1B show that the cellulose, PBSG30, PBSG50 samples all biodegraded with significant CO in natural seawater samples with an initial microbial content of 3489CPU/ML at 30 ℃ for 3 months₂The biological degradation rates of the cellulose, PBSG30 and PBSG50 samples are respectively 65.8%, 32.5% and 19.3% as known by measurement and calculation. The biodegradation process of PBS is not obvious, and only trace CO is present₂The biological degradation rate is less than 2%.

Comparative example 1(measurement of Oxidation of Hydrogen dioxide Using Infrared SpectroscopyCarbon)

In the same manner as in example 1, 1.5g of the above four polymer material samples were placed in 5L reaction tanks containing 3L seawater samples, respectively, wherein the content of microorganisms was 3489CPU/mL, the seawater samples were degraded at 30 ℃ for 3 months, the carbon dioxide concentration was detected in real time from the gas output from the degradation reaction tanks by an infrared spectrometer every morning and evening, and the concentration of the carbon dioxide gas generated on the day was calculated. No carbon dioxide generated by the degradation of the sample is detected in the experimental result.

Theoretically, taking the reference control cellulose sample as an example, the theoretical amount of carbon dioxide production is 2300mg, and assuming 100% mineralization rate is achieved over 60 days, the average amount of carbon dioxide production per minute is 2300mg/60 days/24 hours/60 minutes, which is 0.00266 mg. The detection limit of the current carbon dioxide infrared detector is 10⁴-10⁶ppm, if the gas flow is 100ml/min, the gas passing through the detector per minute should be at least 0.1 mg. It is clear that the carbon dioxide released by the degradation test cannot be detected using an infrared spectrometer. Therefore, the infrared spectrometer cannot meet the requirement of detecting carbon dioxide generated by degraded high polymer materials in the marine environment.

Comparative example 2(measuring carbon dioxide by gas chromatograph)

In the same manner as in example 1, 1.5g of each of the four polymer material samples was placed in a 5L reaction tank containing 3L of a seawater sample, wherein the content of microorganisms was 3489CPU/mL, the seawater sample was degraded at 30 ℃ for 3 months, and the gas discharged from the 5L reaction tank for degradation was collected every morning and evening and the gas was sampled by a microsyringe and introduced into a gas chromatograph to detect the concentration of carbon dioxide. The experimental results are as follows: no carbon dioxide was detected for all samples. Due to common CO₂The detection limit of the gas chromatography detector is 1ppm, so that the requirement of detecting carbon dioxide generated by high molecular materials degraded in the marine environment cannot be met.

Comparative example 3(carbon dioxide measurement by acid-base titration)

In the same manner as in example 1, 1.5g of the above four samples to be detected were placed in 5L reaction tanks containing 3L of simulated seawater (in which the microorganism content was 3489CPU/mL), degraded in the simulated seawater environment at 30 ℃ for 3 months, the carbon dioxide generated by the degradation was introduced into a container containing a barium hydroxide solution, a new barium hydroxide solution was periodically prepared for replacement, the supernatant was titrated by an acid-base titration method, and the amount of carbon dioxide generated was detected.

The method needs to frequently prepare and replace alkali liquor and titrate by using standard solution, so that the method has the disadvantages of many experimental error sources, labor waste and low efficiency.

As can be seen from a comparison between the above specific examples and the comparative examples, in the degradation detection method of the present invention, an alkali solution is used to absorb hydrazine to collect carbon dioxide generated by degradation of the polymer material, and the carbon dioxide is converted into carbonate ions, and an ion chromatograph is used to measure the content of the carbonate ions in the absorbed hydrazine solution, and the cumulative amount of carbon dioxide released within the degradation duration is obtained by conversion based on the content of the carbonate ions, and the detection limit is significantly lower than the detection limits of an infrared spectrometer and a gas chromatograph used in the prior art, so that the detection sensitivity is high, and the method is particularly suitable for detecting the degradation performance of the polymer material in a seawater environment. For an infrared spectrometer and a gas chromatograph, carbon dioxide gas is directly measured, and the measurement of the carbon dioxide gas generated cumulatively within a long degradation period is not only restricted by a high detection limit, but also limited by the volume of a gas container, so that the degradation performance of the high polymer material in a seawater environment with slow degradation is not easy to accurately measure. The acid-base titration method is influenced by various human factors, has a plurality of detection result errors and can not reflect the degradation performance of the real marine environment.

The present invention has been described in detail with reference to the specific embodiments, which are exemplary only, and are not intended to limit the scope of the present invention, and those skilled in the art may make various modifications, changes, or alterations to the present invention without departing from the spirit and scope of the present invention. Therefore, various equivalent changes made in accordance with the present invention are also within the scope of the present invention.