Method for separating and purifying diatom in deep-sea sediment

Abstract

The invention relates to the technical field of biology, in particular to a method for separating and purifying diatom in deep-sea sediment. The invention discloses a method for separating and purifying diatom in deep-sea sediment, which comprises the following steps: adding a hydrogen peroxide solution to remove organic matters in a sample, adopting ultrasonic-assisted double-aperture (100 [mu] m and 10 [mu] m) screening to effectively remove ineffective components outside a target diameter range in advance, and then removing carbonate by using diluted hydrochloric acid to obtain the diatom in the deep-sea sediment. And centrifuging, discarding supernatant, and drying at low temperature to obtain purified diatom. According to the method, physical screening and the ultrasonic vibration effect are fused, the screening precision and efficiency are improved, the burden of subsequent separation treatment is relieved through preposed screening operation, use of other corrosive chemical reagents is reduced, the heavy liquid flotation step is abandoned, the experimental period is shortened, the extraction abundance and form integrity of diatom are remarkably improved, and the method is suitable for industrial production. And a more reliable and environment-friendly analysis basis is provided for the fields of deep sea diatom ecology research, paleoenvironment reconstruction and the like.

Description

Technical Field

[0001] The invention relates to the field of biotechnology, and in particular to a method for separating and purifying diatoms in deep-sea sediments. Background Art

[0002] Diatoms are a type of single-celled plants with chromatophores. They exist in groups in almost all aquatic environments and are widely found in rivers, lakes, oceans and other water environments. They are characterized by wide distribution, many species, large numbers and rapid reproduction. The individual sizes of diatoms in different seas vary greatly. According to statistics, 99.6% of diatom sediments in the deep seas of the Pacific, Indian and Atlantic Oceans have a diameter of 20-90μm. Diatoms in deep-sea sediments refer to diatoms whose siliceous skeletons are deposited on the seabed after death, forming a large number of diatom fossils, not living diatoms.

[0003] Diatoms are sensitive to environmental changes. Their species composition, quantity changes and geographical distribution depend on various physical and chemical conditions, so they contain rich ecological and environmental information. The distribution and composition of diatom species are very sensitive to indicators such as water salinity, depth, temperature, and nutritional changes. Different combinations of diatom species can indicate different sedimentary environments (lakes, deltas, continental shelves, deep seas, etc.). Diatoms are considered to be one of the effective indicators for judging changes in sedimentary environments in rivers, lakes, and oceans. The cell walls of diatoms are highly resistant to dissolution and can be preserved in sediments for a long time. Their morphology, size, and pattern can be used as a basis for species identification. In the early 20th century, researchers used them as indicator organisms and widely used them in paleoceanography research. Diatom oxygen isotopes are an important paleoclimate research method developed in recent years, and the separation and purification of diatoms is an important part of this research.

[0004] Diatom oxygen isotope analysis requires extremely high purity of diatom samples. Therefore, sediment samples containing diatoms obtained in the field (coarse sand, fine sand, silt, mud, soil, etc.) must first be extracted in the laboratory to remove impurities such as organic matter, clay, quartz sand, etc., in order to obtain diatoms with high purity and few impurities. The laboratory treatment method to obtain a large number of intact and pure diatom fossils is an important step in conducting environmental change research. Most reports at home and abroad mainly use purification methods for diatom isotopes. The purification process includes the removal of organic matter, carbonates, calcium, heavy liquid flotation and centrifugal purification. Hydrochloric acid and heavy liquid are mainly used as the main reagents to remove calcium and organic matter in sediment samples, and diatoms with a specific gravity of 2.4 are flotated by heavy liquid. However, for marine sediments with high silica content, the current processing methods still result in a certain amount of silica and quartz sand in the processed samples, which is not as good as expected. It also often leads to the loss of some diatom species, causing a certain amount of artificial deviation in the diatom combination finally obtained by counting, affecting the accuracy and objectivity of environmental reconstruction. Summary of the invention

[0005] In view of this, the present invention proposes a method for separating and purifying diatoms from deep-sea sediments, which significantly optimizes the efficiency of removing impurities such as silica, clay minerals and organic matter, and avoids the complicated process of harmful solution preparation and subsequent recovery and treatment by eliminating the heavy liquid flotation step. The present invention ensures higher purity pretreatment results for diatom samples, and also significantly improves the abundance and quality of diatom extraction, providing a more reliable and environmentally friendly analytical basis for deep-sea diatom ecology research and paleoenvironment reconstruction.

[0006] The technical solution of the present invention is achieved in this way:

[0007] The present invention, in a first aspect, provides a method for separating and purifying diatoms in deep-sea sediments, comprising the following steps:

[0008] S1. Take 1g of dry sample of deep-sea sediment and put it into a reaction container. Add 9-12mL of hydrogen peroxide solution to fully react, so that the particles in the sample are fully dispersed, and then add pure water to wash repeatedly until neutral;

[0009] S2, the solution obtained in step S1 is subjected to ultrasonic filtration using mesh sieves with a pore size of 100 μm and a pore size of 10 μm, samples with a particle size range of 10-100 μm are collected and transferred to a new container, dilute hydrochloric acid is added, fully reacted for 24 hours, and then purified water is added to repeatedly wash until neutral;

[0010] S3. Transfer the sample into a round-bottom test tube, centrifuge it, discard the supernatant and keep the solution containing solids at the bottom, and dry it at low temperature.

[0011] In some preferred embodiments, in step S2, during ultrasonic filtration, the sample solution first passes through a mesh sieve with a pore size of 100 μm and then passes through a mesh sieve with a pore size of 10 μm; after ultrasonic filtration, the sample retained above the mesh sieve with a pore size of 10 μm is a sample with a particle size in the range of 10-100 μm.

[0012] In some preferred embodiments, in step S2, the reaction conditions of the ultrasonic filtration are: using an ultrasonic oscillator with a frequency of 40 kHz and a power of 120 W for 5 minutes of oscillation filtration.

[0013] In some preferred embodiments, in step S1, the specific steps of adding the hydrogen peroxide solution include: first adding a small amount of hydrogen peroxide solution dropwise, observing the reaction, and if overflow occurs, transferring the solution to a larger reaction container to avoid excessively violent reaction; then adding the remaining hydrogen peroxide solution in 3-5 portions until no more bubbles are generated in the sample, indicating that all organic matter has been removed.

[0014] In some preferred embodiments, in step S1, the concentration of the hydrogen peroxide solution is 30 wt % and the reaction time is 2 h.

[0015] In some preferred embodiments, in step S2, the concentration of the dilute hydrochloric acid is 10 wt %, and the volume is 10-50 mL, and the volume varies according to the composition of the sample.

[0016] In some preferred embodiments, in step S3, the centrifugation speed and time are 4000 rpm and 3 min.

[0017] In some preferred embodiments, in step S3, the reaction conditions of low-temperature drying are 40° C. and 1-3 h.

[0018] The present invention, in a second aspect, provides application of the method for separating and purifying diatoms in deep-sea sediments in pre-treatment of diatom sediment samples, wherein the samples are collected from the deep sea, including the deep sea waters of the Pacific Ocean, the Indian Ocean and the Atlantic Ocean.

[0019] The present invention, in a third aspect, provides the application of the method for separating and purifying diatoms in deep-sea sediments in extracting pure deep-sea diatom fossils.

[0020] The beneficial effects of the present invention include at least the following:

[0021] The present invention proposes a deep-sea sediment sample pretreatment process optimized by ultrasonic-assisted dual-aperture (100μm and 10μm) screening technology, which significantly improves the abundance and extraction efficiency of diatoms while taking into account environmental friendliness. The present invention combines physical screening with ultrasonic vibration effects, and effectively reduces the burden of subsequent processing steps by pre-removing invalid components outside the target diameter range. This step realizes the preliminary classification of coarse and fine particles, effectively eliminating the interference of large-sized impurities and fine particles, and at the same time introduces ultrasonic vibration to act on the surface of the screen, which improves the penetration ability of fine particles and sticky materials (such as silt, clay particles, etc.), and also significantly enhances the screening accuracy, effectively reduces the risk of screen clogging, and ensures the continuity and stability of the screening process.

[0022] The present invention significantly reduces the amount of chemical reagents (such as hydrochloric acid) used, and completely abandons the heavy liquid flotation step, greatly reducing the emission of harmful substances and the complexity of subsequent treatment. At the same time, by optimizing the drying and centrifugation processes, the number of operations is reduced to a single time, effectively shortening the experimental cycle, reducing energy consumption and costs.

[0023] The present invention ensures that the diatom samples reach a high purity level in the pre-treatment stage, effectively delaying the potential damage to the effective components of diatoms by subsequent acid treatment. In addition, experimental results show that the present invention also significantly improves the extraction abundance and morphological integrity of diatoms, providing a more reliable and environmentally friendly analytical basis for deep-sea diatom ecology research and paleoenvironment reconstruction. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] In order to more clearly illustrate the technical solutions in the embodiments of the present invention, the following briefly introduces the drawings required for use in the description of the embodiments. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without creative work.

[0025] Figure 1 The microscopic examination results of the sample before sieving on the mesh screen in Example 1 of the present invention are as follows;

[0026] Figure 2 The microscopic examination results of the diatom sample obtained after the treatment in Example 1 of the present invention are as follows;

[0027] Figure 3 These are the microscopic examination results of the samples obtained after step 3 in comparative example 2 (as shown in FIG. A) and example 1 (as shown in FIG. B) of the present invention. DETAILED DESCRIPTION

[0028] In order to make the purpose, technical scheme and advantages of the present application clearer, the present application is further described in detail below in conjunction with the examples. It should be understood that the specific embodiments described herein are only used to explain the present application and are not intended to limit the present application. The reagents not described separately in detail in the present application are all conventional reagents and can be obtained from commercial channels; the methods not described in detail are all conventional experimental methods and can be obtained from the prior art.

[0029] Unless otherwise defined, all technical and scientific terms used in the present invention have the same meaning as those commonly understood by those skilled in the art of the present invention. The terms used in the specification of the present invention are only for the purpose of describing specific embodiments and are not intended to limit the present invention.

[0030] The terms "including" and "having" and any variations thereof of the present invention are intended to cover non-exclusive inclusions. For example, a process, method, device, product or equipment comprising a series of steps is not limited to the listed steps or modules, but may optionally include steps not listed, or may optionally include other steps inherent to these processes, methods, products or equipment.

[0031] In the present invention, the term "multiple" refers to two or more than two. "And / or" describes the association relationship of the associated objects, indicating that three relationships may exist. For example, S and / or B can represent: S exists alone, S and B exist at the same time, and B exists alone. The character " / " generally indicates that the associated objects are in an "or" relationship.

[0032] The dry sample of deep-sea sediment in the embodiment of the present invention comes from the Philippine Basin (coordinates 130.403194E-139.937621E, 14.278944N-19.387962N, water depth 3519-5317m), and the collection time is September 25, 2023. The fresh samples collected are mud lumps or silt sediments with high water content (water content 50-95%). First, 10g of fresh samples need to be weighed, moved into a beaker and placed in an oven to dry and remove moisture (80°C drying for 6 hours). After drying, the dried sample is ground and crushed in an agate mortar according to the degree of consolidation, and a crushed sample with a silt particle size is obtained, which is moved into a beaker and waits for laboratory pretreatment.

[0033] Example 1

[0034] 1. Take 1g of dry sample of deep-sea sediment and put it into a 100mL beaker. Prepare 10mL of 30wt% hydrogen peroxide solution. First, add a small amount of hydrogen peroxide solution and observe the reaction. If there is overflow, transfer it to a larger reaction container to avoid excessive reaction. Then add the remaining hydrogen peroxide solution in 3 times and react for 2h to allow the sample to react fully and the sediment particles to be fully dispersed until no bubbles are generated in the sample, indicating that all organic matter has been removed.

[0035] 2. Add pure water to wash the sample until it is neutral (pH = 7). The sample microscopic examination results are as follows: Figure 1 As shown, the sample contains a lot of dark clay, silt and other impurities;

[0036] 3. The sample solution was passed through sieves with pore sizes of 100 μm and 10 μm respectively, and filtered by ultrasonic oscillator (frequency 40 KHZ, power 120 W) for 5 min, retaining samples with particle sizes ranging from 10 to 100 μm;

[0037] 4. Move the solids left on the 10 μm sieve after filtration into a beaker, add 10 mL of 10 wt% dilute hydrochloric acid (the volume varies according to the actual conditions such as the lithology and organic matter content of the sample), stir, and fully react for 24 hours;

[0038] 5. Pour pure water into the beaker several times to clean until the solution is neutral (pH = 7);

[0039] 6. Transfer the solution in the beaker into a round-bottom test tube (10 mL) and centrifuge for 3 minutes (4000 rpm);

[0040] 7. Pour off the clear liquid at the top of the test tube and keep the solution containing solids at the bottom;

[0041] 8. Move the test tube into the dryer and dry it at 40℃ for 1-3 hours until the solution at the bottom of the test tube disappears;

[0042] 9. Add acetone to the dried test tube and place it in a test tube rack for examination under a microscope.

[0043] Microscopic examination results Figure 2 As shown, the results show that the impurities in the samples in the field of view are removed more thoroughly, and the diatoms are intact in morphology without obvious damage.

[0044] Comparative Example 1

[0045] 1. Take 1g of dry sample and put it into a 100mL beaker, add 20mL of 30wt% hydrogen peroxide to make the sample fully react and the sediment particles disperse;

[0046] 2. Add purified water several times to rinse until neutral (pH = 7);

[0047] 3. Add 50 mL of 10 wt% dilute hydrochloric acid (the volume varies depending on the lithology, organic matter content, etc. of the sample), stir, and react fully for 24 hours;

[0048] 4. Pour pure water into the beaker several times to clean until the solution is neutral (pH = 7);

[0049] 5. Pour the sample into a 50 mL centrifuge tube and dry it;

[0050] 6. Use heavy liquid with a specific gravity of 2.4 (ZnI2 solution, volume mass 2.2g / ml) for flotation;

[0051] 7. Collect the flotation solution into a 10 mL centrifuge tube;

[0052] 8. Filter with a 10 μm mesh under ultrasonic action;

[0053] 9. Put the filtered flotation solution into a centrifuge and centrifuge for 3 minutes (speed 3000rpm), pour out the upper clear liquid, and keep the solid solution at the bottom;

[0054] 10. Move the test tube into the dryer and dry it at low temperature;

[0055] 11. Add acetone to the dried test tube and place it in a test tube rack for examination under a microscope.

[0056] The diatoms were separated and extracted from 10 different deep-sea sediment dry samples by the methods of Example 1 and Comparative Example 1 (the same parameters or operation methods as those of Example 1 were used for all the parts not specified in this comparative example). The diatom extracts obtained were identified and counted under a microscope.

[0057] Diatom abundance = number of diatom shells in the sample (pieces) / dry weight of sample (g)

[0058] The results are as follows:

[0059]

[0060]

[0061] It can be seen from the test results that the method for separating and purifying diatoms provided by the present invention significantly improves the extraction abundance of diatoms in deep-sea sediments.

[0062] Comparative Example 2

[0063] The step 3 in Example 1 was changed to: ultrasonic filtration using only a single 10 μm mesh sieve, and the other experimental steps remained unchanged.

[0064] The diatom microscopic examination results obtained after step 3 in Comparative Example 2 and Example 1 are as follows: Figure 3 As shown in the figure, by comparing the two methods, the sample treated by ultrasonic filtration with a single-aperture 10 μm mesh only has more impurities, mainly coarse quartz particles, debris, carbon chips, wood chips and silt particles.

[0065] Comparative Example 3

[0066] Modify steps 3-5 in Example 1 to:

[0067] 3. Transfer the solid into a beaker, add 30 mL of 10 wt% dilute hydrochloric acid (the volume varies depending on the lithology, organic matter content, etc. of the sample), and stir to fully react;

[0068] 4. Pour pure water into the beaker several times to clean until the solution is neutral (pH = 7);

[0069] 5. The sample solution was passed through a sieve with a pore size of 100 μm and a sieve with a pore size of 10 μm, and filtered with an ultrasonic oscillator (frequency 40 KHZ, power 120 W) for 5 minutes, retaining samples with a particle size range of 10-100 μm;

[0070] The rest of the experimental steps remain unchanged.

[0071] Since impurities containing carbonates, calcium and other components are not filtered out, 30 mL of hydrochloric acid is required (taking the sample at the Philippine Sea sampling point 130.403194E, 14.278944N as an example), and the chemical reaction time of the acid reagent is also increased by about 1 hour. The method of first performing ultrasonic filtration and then adding hydrochloric acid can filter out invalid impurities containing carbonates, calcium and other components through a mesh screen in advance, which requires 10 ml of hydrochloric acid and greatly reduces the chemical reaction time of the acid reagent.

[0072] Example 2

[0073] 10 samples were selected and separated and purified by the methods in Example 1 and Comparative Example 1, and the identification data on the microscope slides were counted (3 slides were counted for each sample), and the acquisition rate of diatoms, whether the morphology was complete (whether there was damage), and C (organic matter) content were counted.

[0074] The calculation formula for the acquisition rate of diatoms is:

[0075] R=NM / NS×100%

[0076] Where: R represents the acquisition rate (or recovery rate) of diatoms, 100%; NM represents the number of diatoms actually detected from the sample; NS represents the theoretical maximum value of the standard number of diatoms in the sample.

[0077] The C content of diatom shells was determined by quantitative analysis of the C element using an element analyzer.

[0078] The test results are as follows:

[0079]

[0080] It can be seen from the test results that the diatom separation and purification method provided by the present invention significantly improves the acquisition rate and morphological integrity of diatoms, and at the same time, the removal rate of organic impurities is also significantly improved.

[0081] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included in the protection scope of the present invention.

Claims

1. A method for separating and purifying diatoms from deep-sea sediments, characterized in that: The steps include: S1. Take 1g of dry sample of deep-sea sediment and put it into a reaction container. Add 9-12mL of hydrogen peroxide solution to fully react, so that the particles in the sample are fully dispersed, and then add pure water to wash repeatedly until neutral; S2, the solution obtained in step S1 is subjected to ultrasonic filtration using mesh sieves with a pore size of 100 μm and a pore size of 10 μm, samples with a particle size range of 10-100 μm are collected and transferred to a new container, dilute hydrochloric acid is added, fully reacted for 24 hours, and then purified water is added to repeatedly wash until neutral; S3. Transfer the sample into a round-bottom test tube, centrifuge it, discard the supernatant and keep the solution containing solids at the bottom, and dry it at low temperature.

2. The method according to claim 1, characterized in that In the step S2, during ultrasonic filtration, the sample solution first passes through a mesh sieve with a pore size of 100 μm and then passes through a mesh sieve with a pore size of 10 μm; after ultrasonic filtration, the sample retained above the mesh sieve with a pore size of 10 μm is a sample with a particle size in the range of 10-100 μm.

3. The method according to claim 1, characterized in that In step S2, the reaction conditions of the ultrasonic filtration are: using an ultrasonic oscillator with a frequency of 40 kHz and a power of 120 W for 5 minutes of oscillation filtration.

4. The method according to claim 1, characterized in that: In step S1, the specific steps of adding the hydrogen peroxide solution include: firstly adding a small amount of hydrogen peroxide solution dropwise, observing the reaction, and if there is overflow, transferring to a larger reaction container to avoid excessive reaction; then adding the remaining hydrogen peroxide solution in 3-5 times until no bubbles are generated in the sample, indicating that all organic matter has been removed.

5. The method according to claim 1, characterized in that In the step S1, the concentration of the hydrogen peroxide solution is 30 wt % and the reaction time is 2 h.

6. The method according to claim 1, characterized in that In step S2, the concentration of the dilute hydrochloric acid is 10wt% and the volume is 10-50mL.

7. The method according to claim 1, characterized in that In step S3, the centrifugal speed and time are 4000 rpm and 3 min.

8. The method according to claim 1, characterized in that In step S3, the reaction conditions of low-temperature drying are 40° C. and 1-3 h.

9. Application of the method according to any one of claims 1 to 8 in the pretreatment of diatom sediment samples, characterized in that: The samples were collected from the deep sea, including the deep waters of the Pacific Ocean, the Indian Ocean and the Atlantic Ocean.

10. Use of the method according to any one of claims 1 to 8 in extracting pure diatom fossils from the deep sea.

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