CN112960781A

CN112960781A - Organic pollutant degradation method based on biological nanometer heterozygous system

Info

Publication number: CN112960781A
Application number: CN202110302117.5A
Authority: CN
Inventors: 雍阳春; 杨雪瑾; 王兴强
Original assignee: Jiangsu University
Current assignee: Jiangsu University
Priority date: 2021-03-22
Filing date: 2021-03-22
Publication date: 2021-06-15
Anticipated expiration: 2041-03-22
Also published as: CN112960781B

Abstract

The invention provides an organic pollutant degradation method based on a biological nano hybrid system, which comprises the following steps: inoculating and activating Shewanella to culture to obtain a bacterial liquid; centrifuging the bacterial liquid, adding the centrifuged bacterial sludge into an anaerobic reaction buffer solution, and adding a water-soluble ferric salt solution and a water-soluble sulfur source salt solution to obtain a shewanella-nano ferrous sulfide heterozygous system; preparing a reaction buffer solution containing organic pollutants; centrifuging and washing the obtained shewanella-nano ferrous sulfide hybrid system, and adding the obtained shewanella-nano ferrous sulfide hybrid system into a reaction buffer solution of the organic pollutants again to obtain an organic pollutant degradation system; and (4) sampling at fixed time, and measuring the concentration of the organic pollutants in the sample at different times after high-speed centrifugation. The invention utilizes the advantage complementation of microbial reduction power and high-efficiency catalytic performance of the nano material, and obviously improves the treatment efficiency of organic compounds while improving the treatment efficiency of the nano material per unit mass.

Description

Organic pollutant degradation method based on biological nanometer heterozygous system

Technical Field

The invention belongs to the technical field of biochemistry, and particularly relates to an organic pollutant degradation method based on a biological nanometer heterozygous system.

Background

In recent years, with the increase of industrialization degree, the discharge amount of industrial wastewater containing a large amount of organic pollutants such as organic dyes, various aromatic compounds, halogenated hydrocarbons and the like is greatly increased, and the pollutants generally have teratogenicity, carcinogenicity, mutagenicity and high toxicity, and have good bioaccumulation and persistence, thereby causing serious threats to the ecological environment and human health. Therefore, the establishment of an efficient and durable organic pollutant degradation method is significant.

The traditional treatment method of the organic wastewater mainly comprises physical methods such as a membrane separation method, an extraction method and an adsorption method, chemical methods such as an electrochemical method, a catalytic reduction method and an advanced oxidation method, and biological treatment methods such as aerobic, anaerobic and biological adsorption. Although the physical method is simple and low in cost, the pollutants cannot be removed efficiently, and the method is often used for pretreatment of wastewater; the chemical method has fast reaction, good pollutant removing effect but serious consumption, complex synthesis conditions and easy secondary pollution of water. In comparison, the microorganisms can utilize the energy of the original substrate in the wastewater to reduce and detoxify the pollutants, so that the organic pollutants in the water body are reduced, meanwhile, the nutrient substances in the water body can be reduced through biological metabolism, the energy consumption is reduced from the other side, and the wastewater is more environment-friendly and does not produce a large amount of byproducts. However, biological methods also have problems, such as the difficulty of degradation of organic contaminants and the limitation of the efficiency of biological methods due to biotoxicity. In the newly emerging field of nano material catalysts, nano ferrous sulfide is widely applied to wastewater treatment due to high catalytic efficiency and strong reducibility.

At present, noble metal materials such as nano palladium, gold and the like are mostly used for treating organic pollutants in a microorganism-nano material hybrid system, or pollutants are degraded through reactions such as photocatalysis and the like after a composite material is synthesized, and the problems of high material cost, large reaction energy consumption, complex reaction conditions and the like exist.

Disclosure of Invention

Aiming at the technical problems, the invention provides an organic pollutant degradation method based on a biological nanometer hybrid system, which combines the characteristics of electron generation by metabolism of an electroactive microorganism Shewanella and transmembrane electron transfer to realize continuous regeneration of a nanometer iron sulfide material in the biological preparation and degradation processes of nanometer ferrous sulfide, utilizes the complementation of the advantages of microbial reduction force and efficient catalytic performance of a nanometer material, and obviously improves the treatment efficiency of organic compounds while improving the treatment efficiency of the nanometer material in unit mass.

The technical scheme of the invention is as follows: a method for degrading organic pollutants based on a biological nano hybrid system comprises the following steps:

step (1), Shewanella is inoculated, activated and cultured to obtain a bacterial liquid;

step (2) centrifuging the bacterial liquid obtained in the step (1), adding the centrifuged bacterial sludge into an anaerobic reaction buffer solution, adding a water-soluble ferric salt solution and a water-soluble sulfur source salt solution, and culturing in a shaking table to obtain a shewanella-nano ferrous sulfide hybrid system;

adding nutrient substances and different organic pollutants into the reaction buffer solution to obtain the reaction buffer solution containing the organic pollutants;

step (4) centrifuging and washing the Shewanella-nano ferrous sulfide hybrid system obtained in the step (2), adding the Shewanella-nano ferrous sulfide hybrid system into the reaction buffer solution containing the organic pollutants obtained in the step (3) again to obtain an organic pollutant degradation system, and culturing the system in a shaking table;

and (5) sampling in the organic pollutant degradation system obtained in the step (4) at regular time, and measuring the concentration of the organic pollutants in the samples at different times after high-speed centrifugation.

In the above embodiment, the concentration OD of the bacterial liquid obtained in the step (1)₆₀₀The value is 0.5-4.

In the above scheme, the final concentration OD of Shewanella in the anaerobic reaction buffer solution in the step (2)₆₀₀The value is 0.05-5.

In the scheme, in the step (2), the water-soluble ferric salt is ferric chloride, ferric nitrate, ferric sulfate or ferric citrate, and the water-soluble sulfur source salt is sodium thiosulfate, sodium sulfite, sodium low sulfate or sodium sulfate.

In the above scheme, the final concentration of the water-soluble ferric salt solution and the water-soluble sulfur source salt solution in the reaction buffer solution in the step (2) is more than 50 μ M.

In the above scheme, the culture conditions in the step (2) are as follows: the time is more than 5h, the rotating speed is 50-300rpm, and the temperature is 4-37 ℃.

In the above scheme, the nutrient added in step (3) includes lactate, formate, acetate, pyruvate or a plurality of mixed nutrients.

In the above scheme, the final concentration of Shewanella after the re-suspension of the hybrid system in the step (4) is OD₆₀₀＝0.05-5。

In the above scheme, the temperature of the shaking culture in the step (4) is 4-37 ℃, and the rotation speed is 50-300 rpm.

In the scheme, the experimental environments of the steps (2) to (5) are controlled in an anaerobic environment.

Compared with the prior art, the invention has the beneficial effects that:

according to the method, a Shewanella-nanometer ferrous sulfide hybrid system is constructed, intracellular electrons generated by metabolism of the Shewanella are transferred to an extracellular nanometer iron sulfide catalyst through a transmembrane electron transfer mechanism, ferric iron to ferrous iron generated by pollutant reduction and degradation are reduced, continuous regeneration of extracellular ferrous sulfide is realized, the loss of nanometer materials is reduced by combining the advantages of biological reducing power and strong catalytic performance of the nanometer ferrous sulfide, and efficient and lasting degradation of organic pollutants is realized.

Drawings

FIG. 1 is a schematic diagram of Shewanella-nano ferrous sulfide hybrid system for degrading organic pollutants;

FIG. 2 is a bivalent iron change process in the construction process of a Shewanella-nanometer ferrous sulfide hybrid system;

FIG. 3 is a SEM representation of Shewanella and Shewanella-nano ferrous sulfide hybrid system;

FIG. 4 is an EDS (EDS characterization chart) of the biosynthetic nano ferrous sulfide;

FIG. 5 is a graph showing the effect of Shewanella-nano ferrous sulfide hybrid system on degrading methyl violet, wherein the Shewanella-nano ferrous sulfide hybrid system comprises Shewanella, biosynthetic nano ferrous sulfide and Shewanella-nano ferrous sulfide hybrid system;

FIG. 6(a) is a diagram showing the effect of Shewanella-nanometer ferrous sulfide hybrid system in degrading nitrophenol, wherein the Shewanella-nanometer ferrous sulfide hybrid system comprises Shewanella, biosynthetic nanometer ferrous sulfide and Shewanella-nanometer ferrous sulfide hybrid systems synthesized under different conditions; (b) is the change of ferrous iron in the process of degrading nitrophenol in a Shewanella-nanometer ferrous sulfide heterozygous system and a biosynthetic nanometer ferrous sulfide control group.

Detailed Description

The present invention is specifically described below with reference to examples, which are intended to better understand the technical spirit of the present invention, but the scope of the present invention is not limited to the following embodiments.

A method for degrading organic pollutants based on a biological nano hybrid system comprises the following steps:

step (2) centrifuging the bacterial liquid obtained in the step (1), adding bacterial sludge into an anaerobic reaction buffer solution, adding a water-soluble ferric salt solution and a water-soluble sulfur source salt solution, and culturing in a shaking table to obtain a Shewanella-nano ferrous sulfide hybrid system;

adding nutrient substances and different organic pollutants into the reaction buffer solution to obtain the reaction buffer solution containing pollutants with a certain concentration;

step (4) centrifuging and washing the Shewanella-nano ferrous sulfide hybrid system obtained in the step (2), adding the Shewanella-nano ferrous sulfide hybrid system into the reaction buffer solution containing the organic pollutants obtained in the step (3) again to obtain a new organic pollutant degradation system, and placing the new organic pollutant degradation system in a shaking table;

step (5) sampling in the organic pollutant degradation system in the step (4) at regular time, and measuring the concentration of pollutants in the samples at different times after high-speed centrifugation;

preferably, the culture conditions of the Shewanella in the step (1) are that the temperature is 4-37 ℃, the shaking revolution is 50-300rpm, and the bacterial liquid is adjusted to OD₆₀₀The culture was stopped at 0.5-4.

Preferably, the Shewanella is Shewanella oneidensis MR-1 purchased from ATCC American type culture Collection under the strain number ATCC700550 in the step (1).

Preferably, the reaction buffer in step (2) is prepared by mixing the anaerobically treated LB liquid medium and M9 medium at a volume ratio of 20:80-1:99, and adding sodium lactate to a final concentration of 0.1-50 mM.

Preferably, the centrifugation in step (2) is carried out at 3000-7000rpm for 4-10 min.

Preferably, the final concentration of Shewanella in the reaction buffer of step (2) is OD₆₀₀The value is 0.05-5.

Preferably, the final concentration of the water-soluble ferric salt solution and the water-soluble sulfur source salt solution in the buffer solution in the step (2) is more than 50 μ M; the water-soluble ferric salt is ferric chloride, ferric nitrate, ferric sulfate or ferric citrate, and the water-soluble sulfur source salt is sodium thiosulfate, sodium sulfite, sodium sulfate or sodium sulfate.

Preferably, the culture conditions in step (2) are: the time is more than 5h, the rotating speed is 50-300rpm, and the temperature is 4-37 ℃.

Preferably, the nutrients added in step (3) include lactate, formate, acetate, pyruvate or various mixed nutrients.

Preferably, the washing in step (3) is performed by washing with M9 medium.

Preferably, the final concentration of Shewanella in the degradation system in the step (4) is maintained as OD₆₀₀＝0.05-5，

Preferably, in the step (4), the temperature of the shaking table culture is 4-37 ℃, and the shaking rotation speed is 50-300 rpm.

Preferably, the timed sampling in step (5) is within 0-35h, the high-speed centrifugation is at 10000-12000rpm, and the centrifugation time is 3-5 min.

Electrons generated in cells in the Shewanella metabolic process are transferred to the nano iron sulfide catalyst on the surface of the cells through a transmembrane electron transfer mechanism, and ferric iron generated by pollutant degradation is reduced to ferrous iron, so that the high-efficiency and durable degradation of organic pollutants is realized by combining the advantages of biological reduction and nano material catalysis. In the shewanella @ nano ferrous sulfide hybrid system, the cost of iron sulfide is low, the shewanella @ nano ferrous sulfide hybrid system has strong catalytic performance without reaction conditions such as photocatalysis, and the like, and meanwhile, the shewanella has higher electron transfer efficiency when being used as an electroactive microorganism. By the synergistic effect of the Shewanella and the nano ferrous sulfide, the biological reduction is combined with the catalytic advantage of the nano material, and the high-efficiency degradation of the organic pollutants can be directly realized.

Example 1:

(1) preparing LB culture medium comprising tryptone 10g/L, yeast extract 5g/L, and sodium chloride 5g/L, pH ═ 7; preparing M9 culture medium containing Na₂HPO₄·12H₂O 17.8g/L、KH₂PO₄ 3g/L、NaCl 0.5g/L、NH₄Cl 10.5g/L, 10mM sodium lactate, 0.1mM CaCl₂And 1mM MgSO₄(ii) a Preparing 0.1M ferric chloride solution and 0.1M sodium thiosulfate mother liquor;

(2) and then, Shewanella (purchased from ATCC American type culture Collection, the strain number ATCC700550) is inoculated into an LB culture medium for culture, and the volume ratio of the inoculum size of the Shewanella inoculated into the LB culture medium to the LB culture medium is 0.4: 100, respectively; then culturing at 30 ℃ and shaking at the rotation speed of 200rpm for 12h to obtain bacterial liquid, wherein the surface of the bacterial liquid is basically smooth and has no obvious particles, as shown in figure 3 a;

(2) preparing a reaction buffer solution of a shewanella-nano ferrous sulfide nano system: the LB liquid medium and the M9 medium were mixed well at a volume ratio of 20:80 to obtain a mixed solution. Carrying out anaerobic treatment: heating to boil, blowing nitrogen, autoclaving, adding sterile sodium lactate as nutrient substance to make its final concentration reach 18 mM;

(3) centrifuging overnight-cultured Shewanella bacteria liquid at 5000rpm for 5min, adding the precipitate bacteria mud into 300mL buffer solution, and controlling the final concentration at OD₆₀₀0.1; adding an iron chloride solution and a sodium thiosulfate solution into a reaction buffer solution inoculated with the bacterial sludge, wherein the final concentration of the iron chloride and the sodium thiosulfate is 100 mu M; culturing in 30 deg.C shaking table at shaking rotation speed of 200rpm for 13h to obtain Shewanella-nanometer ferrous sulfide heterozygous system, wherein the color of the system gradually turns black, the concentration of ferrous iron gradually increases, and obvious nanometer ferrous sulfide particles are outside cells after synthesis, as shown in FIG. 2 and FIG. 3 b.

(4) Methyl violet dyes were added to the reaction buffer solutions obtained in step (2) in an anaerobic workstation using 1mL syringes, respectively, to a final concentration of 7.5 mg/L.

(5) Taking the Shewanella-nano ferrous sulfide heterozygote system obtained in the step (3) out of an anaerobic workstation, centrifuging at 6000rpm for 8min, washing by using fresh M9, and suspending into 60mL of reaction buffer solution containing organic pollutants in the step (4), wherein the final concentration of Shewanella is kept to be OD₆₀₀After being set at 0.1, the mixture was finally placed in a 30 ℃ shaker.

(6) And (3) regularly and uniformly mixing the degradation system in an anaerobic workstation within 0-35h, taking out the degradation system by using a 1mL injector, centrifuging the sample at 12000rpm for 5 minutes, and finally measuring the concentration of the pollutants in the supernatant by using a spectrophotometer.

The constructed Shewanella-nano ferrous sulfide heterozygote system is resuspended in wastewater containing methyl violet under an anaerobic condition, the initial concentration of p-nitrophenol is controlled to be 7.5mg/L, the sampling time is 0h, 0.25h, 1h, 2h, 14h, 26h and 34h, the obtained sample is centrifuged at 12000rpm for 5min, the methyl violet concentration is measured by a spectrophotometer at 580nm, and a degradation curve is drawn, as shown in figure 5.

Example 2:

this example 2 differs from example 1 in that the degradation of methyl violet by Shewanella under the same conditions was measured singly without adding ferric chloride and sodium thiosulfate, and a degradation curve was plotted as a control, as shown in FIG. 5.

Example 3:

the difference between this example 3 and example 1 is that after the shewanella-nano ferrous sulfide hybrid system is prepared, high-pressure moist heat sterilization at 120 ℃ for 15-20min is performed, centrifugation is performed at 6000rpm for 8min, and then biological nano ferrous sulfide is obtained after fresh M9 washing, resuspension and ultrasound, the degradation condition of single biological nano ferrous sulfide to methyl violet is determined under the same conditions, and a degradation curve is drawn as a control, as shown in fig. 5.

Example 4:

taking out the Shewanella-nano ferrous sulfide hybrid system synthesized in the embodiment 1 in an anaerobic workstation, centrifuging at 8000rpm for 5min, discarding the supernatant, and centrifuging and washing 3 times by using anoxic water; centrifuging at 8000rpm for 5min, discarding the supernatant, washing with 75%, 100% ethanol and acetone respectively, drying the final centrifuged precipitate in an anaerobic workstation, grinding the black solid into powder with agate mortar after the precipitate is dried, and taking a proper amount of powder sample for EDS elemental analysis characterization, as shown in FIG. 4.

Example 5:

the difference between the example 5 and the example 1 is that the constructed Shewanella-nanometer ferrous sulfide hybrid system is resuspended in the wastewater containing p-nitrophenol under anaerobic condition, the initial concentration of the p-nitrophenol is controlled to be 70 μ M, the sampling time is 0h, 2h, 5h, 10h, 24h and 35h, the obtained sample is centrifuged at 12000rpm for 5min, the supernatant is taken, the concentration of the nitrophenol in the water sample is measured by a spectrophotometer at 400nm, and a degradation curve is drawn (as shown in FIG. 6 a).

Example 6:

this example 6 is different from example 1 in that paranitrophenol was added to the reaction buffer without adding ferric chloride and sodium thiosulfate, and the degradation of paranitrophenol by Shewanella under the same conditions was measured singly and a degradation curve was plotted as a control, as shown in FIG. 6 a.

Example 7:

the difference between this example 7 and example 3 is that the biological nano ferrous sulfide is washed and then resuspended in the reaction buffer solution added with p-nitrophenol, the degradation of p-nitrophenol by a single biological nano ferrous sulfide is measured under the same conditions, and a degradation curve is drawn as a control, as shown in fig. 6 a.

Example 8:

this example differs from example 1 in that Shewanella-nano ferrous sulfide hybrid system was synthesized with 50. mu.M aqueous ferric chloride solution and sodium thiosulfate solution, and 5000. mu.M aqueous ferric chloride solution and sodium thiosulfate solution, respectively, washed, centrifuged, and resuspended in reaction buffer containing p-nitrophenol to control Shewanella OD₆₀₀0.1, the degradation of p-nitrophenol was determined as shown in FIG. 6aShown in the figure.

Example 9:

the difference between this example 9 and example 5 is that the sampling time was 0h and 35h, the obtained sample was centrifuged at 12000rpm for 5min to take a precipitate, the divalent iron change was measured by o-phenanthroline chromogenic method after digestion with hydrochloric acid, and a histogram was plotted as a control, as shown in fig. 6 b.

FIG. 1 is a schematic diagram of organic pollutant degradation by a Shewanella-nanometer ferrous sulfide hybrid system, and the Shewanella consumes a carbon source such as sodium lactate and the like in a metabolic process, and synthesizes nanometer ferrous sulfide extracellularly by taking an iron source and a sulfur source of ferric trichloride and sodium thiosulfate as electron receptors to form the Shewanella-nanometer ferrous sulfide hybrid system. As shown in fig. 1, electrons generated in the process of cell metabolism are transmitted to the outside of cells through cytochromes and the like, and are continuously transmitted to the nano ferrous sulfide outside the cells, trivalent iron generated in the process of catalyzing and degrading pollutants by the ferrous sulfide is converted into divalent iron, so that the nano ferrous sulfide is continuously regenerated, the loss of the nano ferrous sulfide is greatly reduced while organic compounds are degraded by utilizing the efficient catalytic performance of the nano ferrous sulfide, and thus, the advantages of biological reducing force and strong catalytic performance of the nano ferrous sulfide are combined in the redox cycle process, so that the efficient and continuous degradation of the organic pollutants is realized.

Fig. 2 is a diagram showing the change of the content of the ferrous iron in the process of constructing the shewanella-nano ferrous sulfide hybrid system, wherein the content of the ferrous iron is gradually increased along with the change of time, and the system color is gradually blackened in the synthesis process through observation.

FIG. 3(a) is a SEM electron microscope picture of Shewanella, FIG. 3(b) is a SEM electron microscope picture of a Shewanella-nano ferrous sulfide hybrid system synthesized by taking ferric chloride and sodium thiosulfate as an iron source and a sulfur source, from which Shewanella in an oval shape and nano ferrous sulfide outside Shewanella in the hybrid system can be seen.

Fig. 4 is an EDS spectrum of biological nano ferrous sulfide, which shows the existence of iron and sulfur elements, and proves that the extracellular nanoparticles are iron sulfide.

FIG. 5 is a graph showing the effect of Shewanella-nano ferrous sulfide hybrid system on degrading methyl violet, wherein the Shewanella-nano ferrous sulfide hybrid system comprises Shewanella, biosynthetic nano ferrous sulfide and Shewanella-nano ferrous sulfide hybrid system; the removal efficiency of the Shewanella-nano ferrous sulfide hybrid system to methyl violet is 4.15 times of that of wild Shewanella and 3.58 times of that of biological nano ferrous sulfide respectively obtained from the figure 5.

FIG. 6(a) is a diagram showing the effect of Shewanella-nano ferrous sulfide hybrid system on degrading p-nitrophenol, wherein Shewanella, bio-synthesized nano ferrous sulfide, and Shewanella-nano ferrous sulfide hybrid systems synthesized under different conditions are included; from the figure, FeCl at 100. mu.M can be obtained₃And Na₂S₂O₃The removal efficiency of the synthesized Shewanella-nano ferrous sulfide heterozygous system on p-nitrophenol is 2.6 times that of wild Shewanella and 8.82 times that of biological nano ferrous sulfide respectively; FeCl at 50. mu.M₃And Na₂S₂O₃And 5000. mu.M FeCl₃And Na₂S₂O₃The degradation efficiency of the synthesized Shewanella-nano ferrous sulfide hybrid system is slightly reduced, but the efficiency is still improved by 2-4.8 times compared with that of wild Shewanella and biological nano ferrous sulfide.

FIG. 6 (b) shows the change of divalent iron in the degradation process of p-nitrophenol in the Shewanella-nano ferrous sulfide hybrid system and the biosynthetic nano ferrous sulfide control group; from the figure, it can be obtained that the ferrous content of the single nanometer ferrous sulfide system is gradually reduced along with the gradual reduction of the ferrous content in the degradation process, while the ferrous content of the Shewanella-nanometer ferrous sulfide system is hardly reduced after the single degradation of 70 mu M p-nitrophenol.

It is therefore intended that the following appended claims be interpreted as including all such alterations and permutations as fall within the true spirit and scope of the invention.

Claims

1. A method for degrading organic pollutants based on a biological nano hybrid system is characterized by comprising the following steps:

2. The method for degrading organic pollutants based on biological nano hybrid system according to claim 1, wherein the concentration OD of the bacterial liquid obtained in the step (1)₆₀₀The value is 0.5-4.

3. The method for degrading organic pollutants based on biological nano hybrid system according to claim 1, wherein the final concentration OD of Shewanella in the anaerobic reaction buffer in the step (2)₆₀₀The value is 0.05-5.

4. The method for degrading organic pollutants based on biological nano hybrid system according to claim 1, wherein the water-soluble ferric salt in the step (2) is ferric chloride, ferric nitrate, ferric sulfate or ferric citrate, and the water-soluble sulfur source salt is sodium thiosulfate, sodium sulfite, sodium sulfate or sodium sulfate.

5. The method for degrading organic pollutants based on biological nano hybrid system according to claim 1, wherein the final concentration of the water-soluble ferric salt solution and the water-soluble sulfur source salt solution in the reaction buffer solution in the step (2) is more than 50 μ M.

6. The method for degrading organic pollutants based on biological nano hybrid system according to claim 1, wherein the culturing conditions in the step (2) are as follows: the time is more than 5h, the rotating speed is 50-300rpm, and the temperature is 4-37 ℃.

7. The method for degrading organic pollutants based on biological nano hybrid system according to claim 1, wherein the nutrients added in the step (3) comprise one or more mixed nutrients of lactate, formate, acetate and pyruvate.

8. The method for degrading organic pollutants by using biological nano hybrid system as claimed in claim 1, wherein the final concentration of Shewanella after the re-suspension of the hybrid system in the step (4) is OD₆₀₀＝0.05-5。

9. The method for degrading organic pollutants in bionano hybrid system according to claim 1, wherein the temperature of shaking table in the step (4) is 4-37 ℃.

10. The method for degrading organic pollutants in a bionano hybrid system as claimed in claim 1, wherein the experimental environment of the steps (2) to (5) is controlled under anaerobic environment.