CN112978738A - Method for extracting pure diatom shells from marine unicellular diatoms - Google Patents
Method for extracting pure diatom shells from marine unicellular diatoms Download PDFInfo
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- 238000000034 method Methods 0.000 title claims abstract description 48
- 241000206761 Bacillariophyta Species 0.000 title claims abstract description 20
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- CEAZRRDELHUEMR-URQXQFDESA-N Gentamicin Chemical compound O1[C@H](C(C)NC)CC[C@@H](N)[C@H]1O[C@H]1[C@H](O)[C@@H](O[C@@H]2[C@@H]([C@@H](NC)[C@@](C)(O)CO2)O)[C@H](N)C[C@@H]1N CEAZRRDELHUEMR-URQXQFDESA-N 0.000 description 1
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- 230000018109 developmental process Effects 0.000 description 1
- LRCFXGAMWKDGLA-UHFFFAOYSA-N dioxosilane;hydrate Chemical compound O.O=[Si]=O LRCFXGAMWKDGLA-UHFFFAOYSA-N 0.000 description 1
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- C—CHEMISTRY; METALLURGY
- C01—INORGANIC CHEMISTRY
- C01B—NON-METALLIC ELEMENTS; COMPOUNDS THEREOF; METALLOIDS OR COMPOUNDS THEREOF NOT COVERED BY SUBCLASS C01C
- C01B33/00—Silicon; Compounds thereof
- C01B33/113—Silicon oxides; Hydrates thereof
- C01B33/12—Silica; Hydrates thereof, e.g. lepidoic silicic acid
- C01B33/18—Preparation of finely divided silica neither in sol nor in gel form; After-treatment thereof
-
- C—CHEMISTRY; METALLURGY
- C01—INORGANIC CHEMISTRY
- C01P—INDEXING SCHEME RELATING TO STRUCTURAL AND PHYSICAL ASPECTS OF SOLID INORGANIC COMPOUNDS
- C01P2002/00—Crystal-structural characteristics
- C01P2002/80—Crystal-structural characteristics defined by measured data other than those specified in group C01P2002/70
- C01P2002/82—Crystal-structural characteristics defined by measured data other than those specified in group C01P2002/70 by IR- or Raman-data
-
- C—CHEMISTRY; METALLURGY
- C01—INORGANIC CHEMISTRY
- C01P—INDEXING SCHEME RELATING TO STRUCTURAL AND PHYSICAL ASPECTS OF SOLID INORGANIC COMPOUNDS
- C01P2004/00—Particle morphology
- C01P2004/01—Particle morphology depicted by an image
- C01P2004/03—Particle morphology depicted by an image obtained by SEM
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- Preparation Of Compounds By Using Micro-Organisms (AREA)
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Abstract
The invention discloses a method for extracting pure diatom shells from marine unicellular diatoms, which comprises the following steps: (1) preparing algae mud; (2) treating the algae mud by an acid-thermal method; (3) and (5) washing and drying. Compared with the prior art, the invention has the following advantages: the operation process is simple and easy to implement; the integrity is good, the structure of the diatom shell treated by the mixed acid solution of sulfuric acid and nitric acid is complete, and the pores are clear; the cleanliness is high, organic matters and harmful solvent residues are not left on the surfaces of the diatom shells, and the purity is high; the invention provides a simple, convenient and efficient method for extracting and separating high-purity diatom shells from diatom shell-containing unicellular organisms such as marine diatoms, and is beneficial to further research of industrial large-scale extraction and separation processes of the diatom shells.
Description
Technical Field
The invention relates to extraction of frustules, in particular to a method for extracting complete and pure frustules from unicellular diatoms.
Background
Diatoms are a class of photoautotrophic, unicellular eukaryotic algae widely distributed throughout the global marine and freshwater ecosystems. It is one of the most important primary productivities in marine ecosystems, producing 45-50 million tons of organic carbon each year, accounting for 40% of the marine primary productivities and 20% of the global primary productivities. SiliconThe algae has strong adaptability to the environment, grows rapidly, and some species can be cultured in a large scale, and can accumulate a large amount of biomass in a short time. The diatom shell (diatom frustule), the cell wall of diatoms, is a strong shell that takes up the silicates from the environment and is deposited by the process of biomineralization, consisting mainly of amorphous hydrated Silica (SiO)2‧xH2O) is formed.
The diatom shell is used as a novel material with a unique structure, the structural fineness of the diatom shell exceeds that of a plurality of existing advanced materials, and the diatom shell is widely applied and researched in the aspects of chemical industry, environment, building materials and the like due to the excellent characteristics of a highly-ordered three-dimensional porous structure, large specific surface area, high adsorbability, mechanical stability, heat resistance and the like. In recent years, the potential of frustules as biomedical materials has also been developed. For example, Aw et al (DOI: 10.1177/0885328212441846) explored the feasibility of diatomaceous earth as a novel drug carrier in 2011: the administration capacity of the diatomite to hydrophobic (indomethacin) and hydrophilic (gentamicin) drugs is respectively evaluated, and research results show that the diatomite has good administration effect, the drug loading rate is 14-22 wt%, and the sustained administration can reach more than 2 weeks; researches in 2013 of Hongbo Zhang et al (DOI: 10.1016/j. biomaterials.2013.08.035) show that the diatomite can maintain and control the release of prednisone (prednisone), change the absorption mode of the prednisone and obviously improve the medication safety of the prednisone. A research result of 2016 (DOI: 10.1021/acsami.6b12317) shows that the frustules have good imbibing performance, and the surface of the frustules carries negative charges due to the existence of a large amount of silanol groups, so that the frustules can be used as an excellent hemostatic material to replace a synthetic silicon dioxide nano material. Therefore, there is a need to improve the existing technology to obtain a large amount of complete and clean frustules, thereby allowing the development of the application potential of the frustules in other fields.
According to the research of documents in the prior art, Chinese patent CN201210352321.9 discloses a method for separating and extracting diatom shells from diatoms, dilute hydrochloric acid is adopted to remove carbonate in diatom cells in the extraction process, deionized water is added for multiple times to wash and remove impurities, and organic matters in diatom bodies are removed through high-temperature calcination, so that the extraction of diatom shells is realized. The main disadvantages of this patent are: the operation process is complicated, the diatom shells have low purity and are adhered to each other; multiple centrifugation and calcination lead to the crushing of the shell and the reduction of the number of silanol groups, which affects the downstream application.
Chinese patent CN201780038312.3 discloses a frustule extracted from benthic pinnate diatoms harvested by industrial biofilm treatment, which has the main disadvantages of: the method is more suitable for the pinnate diatom, and for the central diatom, hydrogen peroxide is not enough to remove all organic matters, and continuous high-temperature calcination can damage the surface groups of the silica shells, so that the application of the diatom shells in the micro-nano field is influenced.
The Master thesis of Hainan university, namely the research on the culture of marine diatom, the extraction of siliceous shells and the adsorption performance thereof and the research on the extraction of siliceous shells of Alhainanensis and the adsorption performance thereof, disclose a method for extracting siliceous shells (combination of acid washing and roasting), and the method has the following defects: the operation process is complicated (gradient ethanol dehydration, acid washing, water washing and roasting), and the diatom shell is collapsed due to long-time high-temperature calcination, the structure is damaged, and the content of silanol on the surface of the diatom shell is reduced.
Therefore, how to simply and conveniently extract the frustules and keep the complete shape of the siliceous shells is the key for extracting the frustules, and in order to simplify the treatment process and further improve the treatment efficiency and quality of the frustules, the prior art needs to be improved to obtain high-quality frustules, so that the high-purity frustules can be conveniently used in different fields.
Disclosure of Invention
In order to solve various defects in the existing extraction process of the frustules, the invention provides a simple and convenient method for extracting the frustules.
The present invention differs from the methods disclosed in the prior art described above in that: the method adopts the mixed acid solution of concentrated sulfuric acid and concentrated nitric acid to remove organic matters of the frustules, realizes high-purity separation and extraction of different frustules, has simple operation process, does not need calcination, has high efficiency, and ensures cleanness and completeness of the frustules.
The invention provides a method for extracting pure diatom shells from marine unicellular diatoms, which comprises the following steps:
(1) preparing algae mud;
(2) treating the algae mud by an acid-thermal method;
(3) washing and drying to obtain the frustules.
The specific method for preparing the algae mud in the step (1) is to precipitate algae liquid of marine unicellular diatoms in a centrifugal or filtering mode to obtain the algae mud.
The specific method for treating the algae mud by the acid thermal method in the step (2) comprises the following steps: in order to fully remove organic matters on the surface of diatom and ensure the cleanliness of diatom shells, the mixed solution of strong acid (concentrated sulfuric acid and concentrated nitric acid) is added into the diatom ooze for acid-heat treatment.
The mass fractions of the concentrated sulfuric acid and the concentrated nitric acid are respectively 98% and 65%.
The mixed solution of concentrated sulfuric acid and concentrated nitric acid is prepared from the following components in percentage by volume: sulfuric acid: nitric acid =1:1 (V/V).
The acid-thermal method comprises the following steps: adding concentrated sulfuric acid into oceanic unicellular diatom, fully and uniformly mixing, heating in a water bath at 60 ℃ for 15-60 min, and standing at room temperature for 24-48 h by utilizing most organic matters on the surfaces of diatom carbide shells, which are strong in oxidizing property, dehydrating property and corrosiveness of the concentrated sulfuric acid; adding equal volume of concentrated nitric acid, heating at 60 deg.C to clarify, removing residual organic matter and pigment on the surface of frustules by strong oxidizing property of concentrated nitric acid, improving cleanness of frustules, and standing at room temperature for 12-24 hr.
The specific method for washing and drying in the step (3) is as follows: in order to avoid the damage of centrifugal force to the diatom shells, the diatom shells subjected to acid treatment are naturally filtered through bolting silk, and are washed for 3-10 times by distilled water, so that the residual acid in the diatom shells is removed; washing with anhydrous ethanol for 3-10 times, removing residual water and organic matter and pigment, and drying at 60 deg.C to obtain pure frustules.
The marine unicellular diatom is a marine unicellular organism containing a siliceous shell, and specifically comprises the following components: haichia willebrand (S. willebrand) (S. willebrand)Thalassiosira weissflogii) Cryocyclodinium sp (Anocyclodinium sp.) (Cyclotella cryptica) Pseudo-micro Alhainan algae (A. sp.) (B.)Thalassiosirasp., navicula (I), Zhou Hao (I) ((II))Navicula avium) Zhouyuan algae (Zhouyuan)Naviculasp., oval algae (A), (B), (C), (D), (ECocconeiopsis orthoneoides) Twill algae (A) and (B)Pleurosigma indicum) Round sifting algae (Yuanqu)Coscinodiscussp.) is used.
Further preferred of the present invention are:
collecting 1L of algae solution by centrifugation or filtration, adding 25 mL of concentrated sulfuric acid into diatom ooze, fully and uniformly mixing, heating in 60 ℃ water bath for 45 min, and standing at room temperature for 24 h; continuously adding the concentrated nitric acid with the same volume, heating to be clear at 60 ℃, and standing for 24 hours at room temperature. Collecting the treated algae cells by bolting silk filtration, washing with distilled water for 5 times, washing with absolute ethyl alcohol for 5 times, and drying at 60 ℃ to obtain pure diatom shells.
The diatom shells separated and extracted by the method are complete in structure, free of damage, transparent in shell and clear in pore space, and the microscopic appearance of the original diatom shells is kept through observation of a scanning electron microscope; analyzing the diatom shells by adopting an energy dispersive X-ray spectrometer (EDXS), and finding that organic matters on the diatom shells are basically and completely removed, wherein the main components are Si and O; the infrared spectrum result shows that the diatom shells present the characteristic peak of the silicon dioxide; the element analysis result shows that the C element of the diatom shell is less than 5 percent, basically no organic matter remains, and the diatom shell can be regarded as purer SiO2。
Compared with the prior art, the invention has the following advantages:
1. the operation process is simple. The extraction material used in the invention is unicellular seaweed which can be directly cultured, collected and extracted, and the operation process is simple.
2. The integrity is good. The diatom shells subjected to acid heat treatment are collected by using a natural filtration method, the shell structure is complete and transparent, the pores are clear, and high-temperature roasting is not needed, so that damage to the shell structure and properties caused by repeated centrifugation and high temperature is avoided.
3. The cleanliness is good. After the strong mixed acid solution is used for extraction, no organic matter residue exists on the surfaces of the diatom shells, and the purity is high.
4. The invention is suitable for extracting the frustules of different species, and provides a new biological material source for the research in the field of nanotechnology.
5. Provides reference for industrial large-scale acquisition of pure frustules.
Drawings
FIG. 1 shows Cyclotella miniata (C.sub.Cyclotella cryptica) Haematococcus welshii (Haematococcus weizii) (II)Thalassiosiraweissflogii) And oval algae (Cocconeiopsis orthoneoides) Scanning electron microscope images of the prepared frustules.
Wherein a-d in FIG. 1 represent: a. whole frustules of Cyclotella minor; b. local amplification of the edge of the diatom shell of the small spirulina; c. haichia willebrand (S. willebrand) (S. willebrand)T. weissflogii) The overall morphology of the diatom shell of (a); d. oval algae (A), (B), (C)Cocconeiopsis orthoneoides) Overall appearance of diatom shell.
FIG. 2 shows Cyclotella miniata (C.sub.Cyclotella cryptica) And Haematococcus welshii: (Thalassiosira weissflogii) Energy spectrum of (2). Wherein a is the result of energy spectrum analysis of Cyclotella tenella, b is Haematococcus welviensis: (T. weissflogii) The result of the energy spectrum analysis.
FIG. 3 shows Cyclotella miniata (C.sub.Cyclotella cryptica) Haematococcus welshii (Haematococcus weizii) (II)Thalassiosira weissflogii) And oval algae (Cocconeiopsis orthoneoides) An infrared spectrum of (1).
FIG. 4 shows Cyclotella miniata (C.sub.Cyclotella cryptica) Haematococcus welshii (Haematococcus weizii) (II)Thalassiosiraweissflogii) And oval algae (Cocconeiopsis orthoneoides) Elemental analysis histogram.
Detailed Description
In order to explain the method for separating and extracting intact diatom shells from diatoms according to the present invention in detail, the following description is further provided in conjunction with the accompanying drawings.
Several Marine diatoms used in the experiments were inoculated into f/2 liquid medium in a certain ratio (Guillard RL (1975) Culture of seaweed for Feeding Marine Animals, in Culture of Marine Animals, eds Smith WL, Chanley MH (Plenum, New York),pp 29-60.), air-cultured at 23 deg.C (ventilation amount of 150 ml/min), and illumination intensity of 40 μmol m-2s-1The light-dark period is 12 h:12 h.
Example 1: purified chlorella shell
Culturing the above cultured Cyclotella cryptica (C. encyclopedia)Cyclotella cryptica) Subpackaging the algae liquid into 100 mL centrifuge tubes, balancing, and centrifuging in a centrifuge with centrifugal force of 2000 g for 5 min. After the centrifugation was completed, the supernatant was decanted, and the diatom oozes in each tube were combined and washed 3 times with distilled water to remove inorganic salts and some impurities. Adding 25 mL concentrated sulfuric acid into the algae mud as a treatment solution, mixing the algae cells and the concentrated sulfuric acid, and heating in a 60 ℃ water bath for 45 min. Placing the heated mixed solution at room temperature for 24 h; adding the concentrated nitric acid with the same volume into the mixed solution again, heating to be clear at 60 ℃, and standing for 24 hours at room temperature. The treated diatom shells are naturally filtered and collected through the bolting silk, damage to the diatom shells caused by centrifugal force is avoided, then the diatom shells are washed for 5 times by distilled water, washed for 5 times by absolute ethyl alcohol to remove residual water and impurities, placed in a 60 ℃ drying oven for drying, and the white powder on the bolting silk is collected, namely the diatom shells.
Adhering the diatom shells of the small ring algae on a double-sided adhesive tape, spraying gold on the double-sided adhesive tape, observing under a scanning electron microscope, and preliminarily evaluating the feasibility of the scheme. Respectively analyzing the main components and the group components of the Cyclotella by using an energy dispersive X-ray spectrometer (EDXS), an element analyzer and an infrared spectrum (FI-IR).
FIG. 1 is a scanning electron micrograph of Cyclotella minitans. As can be seen from fig. 1a-1b, the frustules of the chlorella obtained by the method provided by the embodiment have a complete structure, organic matters are removed, the chlorella is transparent, and hierarchical porous structures with different scales can be seen.
FIG. 2 shows the result of energy spectrum analysis of Cyclotella, from which it can be seen that the shells of Cyclotella purified by this method contain Si and O as main components and only a very small amount of C element.
FIG. 3 is the infrared spectrum of Cyclotella, from which it can be seen that the Cyclotella capsulata shell shows typical dioxygenThe peak of silicon oxide (Si-O-Si, Si-OH) was 1440 cm other than that of silicon oxide-1And 1640 cm-1There is also a small fraction of organic material peaks.
FIG. 4 shows the results of quantitative analysis of the main elemental composition of frustules using an elemental analyzer, and it was found that the C content of the frustules was < 5%, which was considered to be pure SiO2A material.
Example 2: purified Haematococcus williamsii shell
Culturing the above cultured Haematococcus Weissn: (Thalassiosira weissflogii) And subpackaging the algae liquid into 100 mL centrifuge tubes, balancing, and centrifuging in a centrifuge with the centrifugal force of 2000 g for 5 minutes. After the centrifugation was completed, the supernatant was poured off, and the algal slurry in each tube was combined and washed 3 times with distilled water. The method of extracting pure frustules was the same as in example 1.
The diatom shell of the Haichia willebrand is stuck on a double-sided adhesive tape, and then is subjected to simple gold spraying, and the result shows that the Haichia willebrand shell has a complete structure, a transparent pore diameter and no organic matter residue as shown in a scanning electron microscope picture of the Haichia willebrand (figure 1 c).
FIG. 2 is the result of energy spectrum analysis of the Haichia williamsii, and it can be seen from FIG. 2b that the main elements on the surface of the Haichia williamsii shell are Si and O, and only a very small amount of C element exists, indicating that most organic matters are removed.
FIG. 3 shows Haematococcus williamsii (Haematococcus williamsii)Thalassiosira weissflogii) The infrared spectrogram of the shell can draw conclusions or results that: the Haematococcus williamsii shell shows characteristic peaks (Si-O-Si, Si-OH) in silicon dioxide, and shows that the frustules are substantially pure SiO2；
FIG. 4 shows the result of quantitative analysis of the residual organic matter in diatom ootheca by an elemental analyzer, which shows that the content of C element remained in Haematococcus williamsii is only 2.9%, and it can be regarded as pure SiO2。
Example 3: purifying oval algae hulls
Mixing the cultured Coccomys ovatus (A), (B) and (C)Cocconeiopsis orthoneoides) Filtering with 2500 mesh bolting silk, collecting algae cell, washing with distilled water for 3 times to removeInorganic salts and some impurities. The extraction method of the ootheca diatoms shells is the same as that of example 1. The main components of the frustules are quantitatively analyzed by an element analyzer through the shape analysis of a Scanning Electron Microscope (SEM) and the analysis of an energy dispersive X-ray spectrometer (EDXS) of the extracted sample.
Fig. 1 is a scanning electron microscope image of the ootheca, and the result shows that the ootheca has a complete structure, retains the original appearance and microstructure of the shell, and has no organic matter residue.
FIG. 3 is an infrared spectrum of the oval algae, showing that the oval algae shell exhibits characteristic peaks (Si-O-Si, Si-OH) of silica with substantially no organic matter remaining.
FIG. 4 shows the results of the elemental analysis of the oval algae, from which it can be seen that the content of C in the shells of oval algae after the treatment of the present invention is only about 1.8%, demonstrating that the shells of diatoms after the treatment of the present invention can be regarded as substantially pure SiO2。
The above disclosure is only a preferred embodiment of the present invention, and the method of purifying algal shells is equally applicable to other marine unicellular organisms containing siliceous shells, such as Haematococcus williamsii (Haematococcus willebrand)Thalassiosira weissflogii) Cryocyclodinium sp (Anocyclodinium sp.) (Cyclotella cryptica) Pseudo-micro Alhainan algae (A. sp.) (B.)Thalassiosirasp., navicula (I), Zhou Hao (I) ((II))Navicula avium) Zhouyuan algae (Zhouyuan)Naviculasp., oval algae (A), (B), (C), (D), (ECocconeiopsis orthoneoides) Twill algae (A) and (B)Pleurosigma indicum) Round sifting algae (Yuanqu)Coscinodiscussp.) is used.
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| CN114229856A (en) * | 2022-01-19 | 2022-03-25 | 中南大学 | Method for extracting diatom shells |
| CN115786328A (en) * | 2022-11-29 | 2023-03-14 | 青岛大学 | Rapid nucleic acid extraction method based on diatom frustule |
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| CN114229856A (en) * | 2022-01-19 | 2022-03-25 | 中南大学 | Method for extracting diatom shells |
| CN115786328A (en) * | 2022-11-29 | 2023-03-14 | 青岛大学 | Rapid nucleic acid extraction method based on diatom frustule |
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