CN113671056A - Method for detecting aerobic biodegradation performance of high polymer material in marine environment - Google Patents

Method for detecting aerobic biodegradation performance of high polymer material in marine environment Download PDF

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CN113671056A
CN113671056A CN202010411449.2A CN202010411449A CN113671056A CN 113671056 A CN113671056 A CN 113671056A CN 202010411449 A CN202010411449 A CN 202010411449A CN 113671056 A CN113671056 A CN 113671056A
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王格侠
季君晖
卢波
李飞
黄丹
甄志超
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Technical Institute of Physics and Chemistry of CAS
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    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
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Abstract

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) regulating and controlling microorganisms in the seawater sample, wherein the quantity of the microorganisms is regulated and controlled to be 103‑107CPU/mL; (iii) placing a sample to be detected in the seawater sample regulated and controlled in the step (ii), and performing a degradation test in an aerobic environment for a preset time; (iv) periodic determination of CO produced by degradation2Cumulative amount released according to CO2And evaluating the biodegradation performance of the high polymer material according to the change of the gas release amount with the degradation time. The invention can better simulate the real seawater degradation environment by regulating and controlling the microorganisms in the seawater sample, shorten the degradation detection time, and deduce the maximum degradation rate of the detected material in the real seawater, so the invention has the advantages ofThe method is beneficial to efficiently and accurately evaluating the degradation performance of the high polymer material in a real seawater environment in a laboratory.

Description

Method for detecting aerobic biodegradation performance of high polymer material in marine environment
Technical Field
The invention relates to the evaluation of biodegradability of a high polymer material, in particular to the detection of aerobic biodegradability of the high polymer material in a marine environment.
Background
Microorganisms are the determining factor affecting the rate of biodegradation of polymeric materials such as polyesters in the marine environment. When specific microorganisms required by biodegradation of high polymer materials such as polyester exist in seawater, the high polymer materials are biodegraded, the degradation rate is increased along with the increase of the number of the microorganisms, and when the required specific microorganisms do not exist, the high polymer materials such as polyester can only be subjected to slow non-enzymatic hydrolysis in water, and the final biodegradation process cannot be completed.
The microorganism species and quantity distribution in real seawater environment vary with sea area and depth, except that the microorganism density in the near sea area, especially in the inner gulf and estuary area is slightly higher, and can be separated to 10 per ml2～103Individual bacterial colonies, sometimes more than 106Besides, in deep sea water, the types and the amounts of microorganisms are very small, and one bacterial colony is probably not separated from each milliliter of deep sea water.
In view of the influences of a plurality of factors such as the number and the types of microorganisms and the environmental temperature, the actual degradation rates of the same material in the natural seawater in different areas are different, and even in the same area, different seasons and different time periods, the actual degradation rates of the same material in the natural seawater are greatly different.
In the existing detection standards (such as ASTM standards and national standards) for the degradation performance of plastics, the adopted water body is from natural seawater, or microorganisms of specific types are added into brine configured in a laboratory, the number and types of microorganisms in the water body are not regulated, and because a plurality of variables existing in the degradation environment cannot be considered, the degradation performance and the degradation rate of the same material detected in the natural seawater have great difference, so the degradation performance under the seawater environment cannot be truly reflected.
Disclosure of Invention
In view of the above problems in the prior art, the present invention is directed to provide an improved method for detecting the aerobic biodegradation performance of a polymer material in a marine environment, which can simulate a real marine environment and simultaneously increase the degradation rate and shorten the degradation time of the polymer material by controlling the types and the amounts of microorganisms in a seawater sample.
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) regulating and controlling the number of microorganisms in the seawater sample to be 103-107CPU/mL；
(iii) Placing a sample to be detected in the seawater sample obtained by regulation and control in the step (ii), and performing a degradation test in an aerobic environment for a preset time;
(iv) and (3) periodically measuring the accumulated release amount of carbon dioxide generated by degradation, and evaluating the biodegradation performance of the high polymer material according to the change of the carbon dioxide gas release amount along with the degradation time.
In some embodiments of the invention, step (ii) regulates the number of microorganisms in the seawater sample by enrichment, culture or dilution to 103-107CPU/mL。
In some embodiments of the present invention, the seawater sample obtained by the step (ii) regulation contains microorganisms that specifically degrade the high molecular material to be detected. The microorganism for specifically degrading the high molecular material to be detected comprises at least one of the following microorganism species: bacillus (Bacillus sp), Enterobacter (Enterobacter sp.), Rhodococcus (Rhodococcus sp), gracillus (gracillus sp), Alpha-proteobacterium (Alpha-proteobacteria), Beta-proteobacterium (Beta-proteobacteria), Gamma-proteobacterium (Gamma-proteobacteria), sphingobacterium (sphingobacterium), flavobacterium (flavobacterium) in some embodiments of the invention, the detection method of the invention further comprises the steps of:
absorbing carbon dioxide gas generated by degradation, and reacting to generate carbonate ions;
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 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, the content of carbonate ions in absorbed hydrazine can be accurately and sensitively detected by anion chromatography even in the case of a small amount of carbon dioxide released at the initial stage of degradation due to the low detection limit of ion chromatography.
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 mineralization rate of the polymer material may be further converted according to the carbon dioxide gas release amount, and the biodegradation performance may be evaluated based on the mineralization rate, where the conversion formula of the mineralization rate is as follows:
Figure BDA0002493414590000031
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 considers a plurality of influence factors of the high polymer material in the seawater degradation, can better simulate the real seawater degradation environment on one hand by regulating and controlling microorganisms in a seawater sample, and on the other hand, improves the degradation rate by regulating and controlling the quantity of the microorganisms, shortens the degradation time, and further deduces the maximum degradation rate which can be reached by the detection material in the real seawater, so that the method is favorable for a laboratory to efficiently and accurately evaluate the degradation performance of the high polymer material in the real seawater environment.
In addition, when the accumulated release amount of carbon dioxide is calculated based on the content of carbonate ions, the content of the carbonate ions is measured by an ion chromatograph, so that the detection method provided by the invention has high sensitivity and high detection limit, and the detection automation is convenient to realize.
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Fig. 1A shows the cumulative release of carbon dioxide over a degradation period of 90 days for test samples according to examples 1 to 6 of the present invention.
FIG. 1B shows the calculated mineralization rate as a function of time for the cumulative release of carbon dioxide over the 90 day degradation period for the test samples of examples 1 to 6 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, a seawater sample may be extracted from natural seawater, and then the number of microorganisms in the extracted seawater sample is adjusted to 10 by adjusting it to enrichment, culture or dilution3-107CPU/mL. The components and contents of the microorganisms and the required inorganic nutrients in the seawater sample can be determined according to the existing standard (for example, ASTM D6691-2009), and the inorganic nutrients can be adjusted according to the number of the microorganisms. 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 degradation tests can be set so as to improve detection efficiency.
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 vessel, and a gas buffer tank and an air flow may be provided in the gas flow path as required in order to appropriately control the gas flow rateAnd (6) metering. 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 material2The gas can be collected by absorbing hydrazine with at least one alkali solution with a certain concentration, and in order to improve the absorption efficiency, if necessary, CO can be added2The 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):
Figure BDA0002493414590000052
wherein (CO)2)TCumulative CO production for the sample2Content of (g), (CO)2)BCumulative production for blank groupRaw CO2Content (mg) and ThCO2Theoretical generation of CO for a sample2Total content (mg) (see equation 2).
Figure BDA0002493414590000051
Wherein M isTOTTo test the total dry solids (mg) of the material, CTOTThe total organic carbon content (%) of the test material was determined.
In the detection method of the present invention, the carbon dioxide generated by the degradation of the polymer material can be detected by other detection means known in the art, such as a respirator, an infrared spectrometer, a gas chromatograph, an acid-base titrator, and the like.
Example 1
Collecting natural seawater from around Tianjin harbor in China, displaying the microorganism content therein to be 945CPU/mL by flat plate counting, putting 3L of the natural seawater sample into a 5L degradation reaction tank, and adding inorganic nutrients required by microorganisms into the natural seawater: NH concentration of 0.5g/L4Cl and KH 0.1g/L2(PO4) And then 1.5g of film-shaped sample PBSG50 to be tested is put into the natural seawater for degradation experiment, and carbon dioxide generated by degradation is introduced into hydrazine absorbed by 0.1mol/L NaOH aqueous solution to generate carbonate ions. The degradation test lasts for three months, samples are taken from the hydrazine absorption solution every 15 days, and the content of carbonate ions in the extracted hydrazine absorption solution is determined by adopting 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 and the mineralization rate (i.e., the degradation rate) at each sampling time were estimated from the carbonate ion concentration. The measurement results are shown in table 1 below, fig. 1A and fig. 1B.
Figure BDA0002493414590000061
Example 2
Collecting natural seawater from around Tianjin harbor in China, counting by flat plate to show that the microorganism content is 945CPU/mL, and enriching the microorganisms by filtration to increase the microorganism concentration to 6.34x104CPU/mL, 3L of the natural seawater sample is put into a 5L degradation reaction tank, and inorganic nutrients required by microorganisms are added into the tank: NH concentration of 0.5g/L4Cl and KH 0.1g/L2(PO4) And then putting 1.5g of film-shaped sample PBSG50 to be tested into the natural seawater sample for a degradation experiment, introducing carbon dioxide generated by degradation into carbon dioxide absorption hydrazine containing 0.1mol/L NaOH aqueous solution, continuing the degradation experiment for three months, sampling from the absorption hydrazine solution every 15 days, and determining the content of carbonate ions in the extracted absorption hydrazine solution by adopting 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 and the mineralization rate (i.e., the degradation rate) at each sampling time were estimated from the carbonate ion concentration. The measurement results are shown in table 1 below, fig. 1A and fig. 1B.
Example 3
Collecting natural seawater from around Tianjin harbor in China, transporting to laboratory, displaying microorganism content therein as 945CPU/mL by plate counting, and enriching microorganism by centrifugation to increase microorganism concentration to 8.14x106CPU/mL, 3L of the natural seawater sample is put into a 5L degradation reaction tank, and inorganic nutrients required by microorganisms are added into the tank: NH concentration of 0.5g/L4Cl and KH 0.1g/L2(PO4) Then 1.5g of film-shaped sample PBSG50 to be tested is put into the natural seawater for degradation experiment, carbon dioxide generated by degradation is led into absorption hydrazine filled with 0.1mol/L NaOH aqueous solution, the degradation lasts for three months, and every other monthThe hydrazine absorption solution was sampled for 15 days, and the content of carbonate ions in the extracted hydrazine absorption solution was measured 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 and the mineralization rate (i.e., the degradation rate) at each sampling time were estimated from the carbonate ion concentration. The measurement results are shown in table 1 below, fig. 1A and fig. 1B.
Example 4
The microorganism in the seawater is enriched by using a microorganism in-situ enrichment device near Tianjin harbor in China to obtain the microorganism with the content of 7.12x106Taking 3L of water with CPU/mL from the water body, putting the water body into a 5L degradation reaction tank, and adding inorganic nutrients required by microorganisms into the water body: NH concentration of 0.5g/L4Cl and KH 0.1g/L2(PO4) And then putting 1.5g of film-shaped sample PBSG50 to be tested for a degradation experiment, introducing carbon dioxide generated by degradation into carbon dioxide absorption hydrazine containing 0.1mol/L NaOH aqueous solution, degrading for three months, sampling from the absorption hydrazine solution every 15 days, and determining the content of carbonate ions in the extracted absorption hydrazine 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 and the mineralization rate (i.e., the degradation rate) at each sampling time were estimated from the carbonate ion concentration. The measurement results are shown in table 1 below, fig. 1A and fig. 1B.
Example 5
Collecting natural seawater from around Tianjin harbor in China, displaying microorganism content of 945CPU/mL by plate counting, performing enrichment culture on microorganism, separating microorganism, adding into 5L, and loweringDecomposing 3L natural seawater in the reaction tank, and adding into the natural seawater to increase the microorganism content in the seawater to 8.51x106CPU/mL, and adding inorganic nutrients required by microorganisms into the seawater: 0.5g/L NH at a degree of 0.5g/L4Cl and KH 0.1g/L2(PO4) And then putting 1.5g of film-shaped sample PBSG50 to be tested into the natural seawater for a degradation experiment, introducing carbon dioxide generated by degradation into carbon dioxide absorption hydrazine containing 0.1mol/L NaOH aqueous solution, continuing degradation for three months, sampling from the absorption hydrazine solution every 15 days, and determining the content of carbonate ions in the extracted absorption hydrazine 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 and the mineralization rate (i.e., the degradation rate) at each sampling time were estimated from the carbonate ion concentration. The measurement results are shown in table 1 below, fig. 1A and fig. 1B.
Example 6
Collecting natural seawater from around Tianjin harbor in China, displaying microorganism content of 945CPU/mL by plate counting, performing enrichment culture on microorganism, separating microorganism, adding into 3L natural seawater in 5L degradation reaction tank to increase microorganism content in seawater to 3.56x109CPU/mL, and adding inorganic nutrients to the seawater: NH concentration of 0.5g/L4Cl and KH 0.1g/L2(PO4) And then putting 1.5g of film-shaped sample PBSG50 to be tested into the natural seawater for a degradation experiment, introducing carbon dioxide generated by degradation into carbon dioxide absorption hydrazine containing 0.1mol/L NaOH aqueous solution, degrading for three months, sampling from the absorption hydrazine solution every 15 days, and determining the content of carbonate ions in the extracted absorption hydrazine 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 H2SO4Flow rate of 0.5mL/min, column temperature of 30 ℃, and quantitative ring 100 μ L, was sampled with a 858 autosampler and analyzed by a conductivity detector. Three replicates of each sample were averaged. The cumulative amount of carbon dioxide produced and the mineralization rate (i.e., the degradation rate) at each sampling time were estimated from the carbonate ion concentration. The measurement results are shown in table 1 below, fig. 1A and fig. 1B.
TABLE 1 cumulative release of carbon dioxide generated in three months of degradation and corresponding degradation rates estimated based on carbonate ion content in examples 1 to 6
Figure BDA0002493414590000081
As can be seen from the test results shown in table 1 above and fig. 1A and 1B, in the detection method of the above embodiment, because the number of microorganisms in the seawater sample is effectively controlled, the degradation rate of the polymer material is improved while the real seawater degradation environment is simulated as much as possible, the degradation detection time is shortened, and the laboratory can efficiently and accurately evaluate the degradation performance of the polymer material in the real seawater 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.

Claims (10)

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) regulating and controlling microorganisms in the seawater sample, wherein the quantity of the microorganisms is regulated and controlled to be 103-107CPU/mL；
(iii) Placing a sample to be detected in the seawater sample regulated and controlled in the step (ii), and performing a degradation test in an aerobic environment for a preset time;
(iv) and (3) periodically measuring the accumulated release amount of carbon dioxide generated by degradation, and evaluating the biodegradation performance of the high polymer material according to the change of the carbon dioxide gas release amount along with the degradation time.
2. The method of claim 1, wherein step (ii) regulates the microbial population in the seawater sample by enrichment, culture, or dilution.
3. The method of claim 1, wherein the conditioned seawater sample of step (ii) contains microorganisms that specifically degrade the polymeric material to be detected.
4. The method according to claim 3, wherein the microorganism specifically degrading the polymeric material to be detected comprises at least one of the following microbial genera: bacillus, Enterobacter, Rhodococcus, Gracillus, alpha-proteus, beta-proteus, gamma-proteus, Sphingobacterium, Flavobacterium.
5. The method of any of claims 1 to 4, further comprising:
absorbing carbon dioxide gas generated by degradation, and reacting to generate carbonate ions;
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.
6. The method of claim 5, wherein absorbing the carbon dioxide gas with at least one alkali solution produces carbonate ions.
7. The method of claim 5, wherein the carbonate ion content is detected using anion chromatography.
8. The method of claim 6, wherein the alkali solution is capable of forming water soluble carbonate ions with carbon dioxide.
9. The method of claim 5, wherein at least two alkali solutions are used to absorb hydrazine to absorb carbon dioxide gas and generate carbonate ions, and the absorbed hydrazine is connected in series with each other.
10. 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:
Figure FDA0002493414580000021
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).
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