Method for synergistically strengthening denitrification of anaerobic ammonia oxidation system based on tea polyphenol and ferric iron

Abstract

The invention relates to the technical field of wastewater treatment, and discloses a method for synergistically strengthening denitrification of an anaerobic ammonia oxidation system based on tea polyphenol and ferric iron, and the method comprises the following steps: in an anaerobic ammonia oxidation reactor, inoculating anaerobic ammonia oxidation sludge, adding wastewater, adding tea polyphenol-Fe < 3 + >, and treating the wastewater. According to the invention, the tea polyphenol can promote the anaerobic ammonium oxidation bacteria to absorb ferric iron and enhance the biological utilization of iron element in the anaerobic ammonium oxidation system. Iron elements absorbed by the anaerobic ammonium oxidation bacteria can be used for synthesizing key functional enzymes, the metabolic activity of anaerobic ammonium oxidation and dissimilatory nitrate reduction into ammonium of the anaerobic ammonium oxidation bacteria is enhanced, meanwhile, coupling of two nitrogen conversion ways is promoted, the total nitrogen removal rate is synergistically increased, and the denitrification efficiency of an anaerobic ammonium oxidation system is enhanced.

Description

Technical Field

[0001] The invention relates to the technical field of wastewater treatment, and in particular to a method for denitrification of an anaerobic ammonia oxidation system based on the synergistic enhancement of tea polyphenols and trivalent iron. Background Art

[0002] The anaerobic ammonium oxidation (Anammox) process is a sustainable wastewater biological treatment technology with the advantages of low aeration energy consumption, low sludge production, and no need for external carbon source. It has good application potential in the treatment of high ammonia nitrogen wastewater such as landfill leachate, sludge digestate, and coking wastewater.

[0003] The principle of anaerobic ammonium oxidation process is that anaerobic ammonium oxidizing bacteria convert ammonia nitrogen (NH4 + −N) and nitrite (NO2 − −N) is converted into nitrogen gas, while a small amount of nitric nitrogen (NO3 − −N).

[0004] However, in actual engineering applications, anaerobic ammonium oxidizing bacteria grow slowly and are sensitive to environmental conditions, resulting in problems such as limited system denitrification efficiency. In addition, the anaerobic ammonium oxidation process produces 11% nitrate nitrogen, further limiting the total nitrogen removal efficiency (NRE) of the system. Although some anaerobic ammonium oxidizing bacteria (such as Candidatus Brocadia and Candidatus Jettenia can reduce nitrate nitrogen to nitrite nitrogen and ammonia nitrogen through the dissimilatory nitrate reduction to ammonium (DNRA) pathway, using small molecular organic matter as electron donors. However, its DNRA activity is relatively low, making it difficult to effectively consume the nitrate nitrogen produced during anaerobic ammonium oxidation.

[0005] Iron, as an important trace element for microbial growth and metabolism, participates in the formation of Anammox functional enzymes such as hydrazine synthase (HZS) and hydrazine dehydrogenase (HDH), as well as DNRA functional enzymes such as periplasmic dissimilatory nitrate reductase (NAP), cytoplasmic NADH-dependent nitrite reductase (NIR), and periplasmic dissimilatory nitrite reductase (NRF), which helps to enhance the Anammox and DNRA metabolic activity of anaerobic ammonia-oxidizing bacteria.

[0006] However, in the anaerobic ammonium oxidation system (pH 7.0~8.5, redox potential -100~400mV), iron mainly exists in the form of trivalent iron, while anaerobic ammonium oxidizing bacteria can only directly absorb dissolved divalent iron, which limits the application of iron in enhancing the anaerobic ammonium oxidation process.

[0007] Tea polyphenols (TP) are important cytokines for the uptake of iron by microorganisms in natural waters, and can stably chelate dissolved trivalent iron (Fe 3+). Introducing tea polyphenols into the anaerobic ammonium oxidation system is expected to promote the absorption of trivalent iron by anaerobic ammonium oxidizing bacteria, thereby enhancing their Anammox and DNRA metabolic activities, and can synergistically improve the total nitrogen removal rate and enhance the denitrification efficiency of the anaerobic ammonium oxidation system through the coupling of the Anammox and DNRA nitrogen conversion pathways. Summary of the invention

[0008] The purpose of the present invention is to provide a method for synergistically enhancing the denitrification of an anaerobic ammonium oxidation system based on tea polyphenols and trivalent iron in order to address the problem that the denitrification activity of anaerobic ammonium oxidizing bacteria is low during actual application, resulting in limited total nitrogen removal rate of the anaerobic ammonium oxidizing system.

[0009] In order to achieve the above object, the first aspect of the present invention provides a method for denitrification of an anaerobic ammonium oxidation system based on the synergistic enhancement of tea polyphenols and trivalent iron, the method comprising:

[0010] In the anaerobic ammonium oxidation reactor, anaerobic ammonium oxidation sludge is inoculated, wastewater is added, and then tea polyphenols-Fe are added. 3+ , to treat the wastewater.

[0011] The method provided by the present invention also has at least the following beneficial effects: (1) The present invention promotes the absorption of trivalent iron by anaerobic ammonium oxidizing bacteria by adding tea polyphenols, which can effectively enhance the biological utilization of iron and increase the intracellular iron content of anaerobic ammonium oxidizing bacteria.

[0012] (2) The present invention uses tea polyphenols-Fe 3+ Promoting the synthesis of Anammox and DNRA functional enzymes is beneficial to enhancing the Anammox and DNRA metabolism of anaerobic ammonium oxidizing bacteria and strengthening the denitrification efficiency of the anaerobic ammonium oxidizing system.

[0013] (3) The tea polyphenols used in the present invention are low in price and dosage, and have good cost-effectiveness.

[0014] (4) The method of the present invention is simple to operate, environmentally friendly, economical and efficient, and is suitable for improving the denitrification efficiency of anaerobic ammonia oxidation processes in environments with low iron concentrations, such as municipal wastewater and natural surface water.

[0015] (5) Introducing tea polyphenols-Fe into the anaerobic ammonium oxidation system 3+ It is beneficial to promote the absorption of trivalent iron by anaerobic ammonium oxidizing bacteria and enhance the biological utilization of iron in the system, thereby promoting the metabolism of Anammox and DNRA by anaerobic ammonium oxidizing bacteria and enhancing the denitrification efficiency of the anaerobic ammonium oxidizing system, which has guiding significance for the engineering application of anaerobic ammonium oxidation. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1is a graph showing changes in intracellular iron content and NRE in the anaerobic ammonium oxidation reactors of Examples 1 to 7 and Comparative Example 1;

[0017] Figure 2 is a graph showing the change of nitrogen concentration and NRE in the anaerobic ammonium oxidation reactor of Comparative Example 2 with the operation time;

[0018] Figure 3 is a graph showing the change of nitrogen concentration and NRE of the inlet and outlet water in the anaerobic ammonium oxidation reactor of Comparative Example 3 with the operation time;

[0019] Figure 4 is a graph showing changes in nitrogen concentration and NRE in the anaerobic ammonium oxidation reactor of Example 8 over the operating time;

[0020] Figure 5 It is a composition diagram of anaerobic ammonium-oxidizing bacteria in the sequencing batch reactor (SBR) of Example 8 of the present invention and Comparative Examples 2 and 3 in the inoculated anaerobic ammonium-oxidizing sludge and stage III;

[0021] Figure 6 In stage III, the relative abundance diagram of nitrogen conversion functional genes in the SBR reactors of the above-mentioned Example 8 and Comparative Examples 2 and 3;

[0022] Figure 7 This is a graph showing the contribution of nitrogen conversion functional gene species in the SBR reactors of Example 8 and Comparative Examples 2 and 3 in Phase III. DETAILED DESCRIPTION

[0023] The endpoints and any values ​​of the ranges disclosed in this article are not limited to the precise ranges or values, and these ranges or values ​​should be understood to include values ​​close to these ranges or values. For numerical ranges, the endpoint values ​​of each range, the endpoint values ​​of each range and the individual point values, and the individual point values ​​can be combined with each other to obtain one or more new numerical ranges, which should be considered as specifically disclosed in this article.

[0024] In the present invention, "NER" means the total nitrogen removal rate of the anaerobic ammonium oxidation system; "Anammox" means the anaerobic ammonium oxidation pathway; "DNRA" means the dissimilatory nitrate reduction pathway; " Candidatus Brocadia, Candidatus Jettenia, Candidatus "Kuenenia" indicates three different genera of anaerobic ammonium-oxidizing bacteria; "HZS" indicates hydrazine synthase; "HDH" indicates hydrazine dehydrogenase; "NAP" indicates periplasmic dissimilatory nitrate reductase; "NADH" indicates reduced coenzyme I; "NIR" indicates cytosolic NADH-dependent nitrite reductase; and "NRF" indicates "periplasmic dissimilatory nitrite reductase".

[0025] As mentioned above, the first aspect of the present invention provides a method for denitrification of an anaerobic ammonium oxidation system based on the synergistic enhancement of tea polyphenols and trivalent iron, the method comprising:

[0026] In the anaerobic ammonium oxidation reactor, anaerobic ammonium oxidation sludge is inoculated, wastewater is added, and then tea polyphenol-Fe3+ is added to treat the wastewater.

[0027] Preferably, the addition concentration of the anaerobic ammonium oxidation sludge is 2000 mg SS / L.

[0028] Further preferably, the inoculated anaerobic ammonium oxidation sludge contains anaerobic ammonium oxidizing bacteria Candidatus Brocadia and anaerobic ammonium oxidizing bacteria Candidatus Jettenia.

[0029] Preferably, the preparation method of the tea polyphenol-Fe3+ is to complex tea polyphenol and FeCl3 at a molar concentration of 1:1.

[0030] More preferably, the continuous addition concentration of the tea polyphenol-Fe3+ is 12.5~150 μmol / L.

[0031] Preferably, in the short-term startup phase, the continuous addition concentration of the tea polyphenol-Fe3+ is 25 μmol / L; in the long-term stable operation phase, the continuous addition concentration of the tea polyphenol-Fe3+ is 12.5 μmol / L; wherein, the short-term startup phase is preferably 1 to 30 days, and the long-term stable operation phase is preferably greater than 30 days.

[0032] Preferably, the temperature in the anaerobic ammonium oxidation reactor is controlled at 30-35°C, the pH is 7.8±0.5, and the dissolved oxygen is <0.1 mg / L.

[0033] In the present invention, the technical principle of synergistically strengthening the denitrification of the anaerobic ammonium oxidation system based on tea polyphenols and trivalent iron is as follows: tea polyphenols can promote the absorption of trivalent iron by anaerobic ammonium oxidizing bacteria and enhance the biological utilization of iron in the anaerobic ammonium oxidation system. The iron absorbed by anaerobic ammonium oxidizing bacteria can be used to synthesize functional enzymes of the Anammox process such as HZS and HDH, as well as functional enzymes of the DNRA process such as NAP, NIR, and NRF, to enhance the Anammox and DNRA metabolic activities of anaerobic ammonium oxidizing bacteria, and at the same time promote the coupling of the two nitrogen conversion pathways, synergistically improve the total nitrogen removal rate, and enhance the denitrification efficiency of the anaerobic ammonium oxidation system.

[0034] The present invention will be described in detail below by way of examples. In the following examples, unless otherwise specified, the raw materials are all commercially available.

[0035] Example 1

[0036] Take 2000 mg SS / L anaerobic ammonium oxidation sludge and place it in a 100 mL serum bottle, add 100 mL synthetic wastewater to form an anaerobic ammonium oxidation reactor, and then continue to add tea polyphenol-Fe at a concentration of 25 μmol / L. 3+ , treat wastewater;

[0037] The anaerobic ammonium oxidation sludge is uniformly pretreated for about 45 days before inoculation, during which no iron and tea polyphenols are added to the influent, and other components are added normally to eliminate the interference of iron in the inoculated sludge as much as possible;

[0038] The synthetic wastewater is artificially prepared in the laboratory, and in the synthetic wastewater, the ammonia nitrogen concentration is 50 mg / L, the nitrite nitrogen concentration is 60 mg / L, the pH is 7.5±0.1, and the dissolved oxygen is <0.1 mg / L;

[0039] The anaerobic ammonium oxidation reactor was operated in SBR mode for 7 days, with 2 cycles per day, a single cycle duration of 12 hours, and the temperature was controlled at 32-34°C and the rotation speed was 180 rpm by a constant temperature shaker;

[0040] The anaerobic ammonium oxidation reactor replaces the synthetic wastewater once every half a day, and the water replacement ratio is 100%;

[0041] The tea polyphenols-Fe 3+ The concentration molar ratio of tea polyphenols to Fe is 1:1 3+ Complexed.

[0042] Example 2

[0043] The method of Example 1 was followed, except that the tea polyphenol-Fe 3+ The continuous addition concentration is 12.5 μmol / L, and the remaining steps and parameters are the same as in Example 1.

[0044] Example 3

[0045] The method of Example 1 was followed, except that the tea polyphenol-Fe 3+ The continuous addition concentration is 37.5 μmol / L, and the remaining steps and parameters are the same as in Example 1.

[0046] Example 4

[0047] The method of Example 1 was followed, except that the tea polyphenol-Fe 3+ The continuous addition concentration is 50 μmol / L, and the remaining steps and parameters are the same as in Example 1.

[0048] Example 5

[0049] The method of Example 1 was followed, except that the tea polyphenol-Fe3+ The continuous addition concentration is 75 μmol / L, and the remaining steps and parameters are the same as in Example 1.

[0050] Example 6

[0051] The method of Example 1 was followed, except that the tea polyphenol-Fe 3+ The continuous addition concentration is 100 μmol / L, and the remaining steps and parameters are the same as in Example 1.

[0052] Example 7

[0053] The method of Example 1 was followed, except that the tea polyphenol-Fe 3+ The continuous addition concentration is 150 μmol / L, and the remaining steps and parameters are the same as in Example 1.

[0054] Comparative Example 1

[0055] The method of Example 1 was followed, except that tea polyphenol-Fe was not added. 3+ The remaining steps and parameters are the same as in Example 1.

[0056] Example 8

[0057] Take 2000mg SS / L anaerobic ammonium oxidation sludge and place it in a 1L SBR reactor with an effective volume, add 1L synthetic wastewater, and continuously add tea polyphenols-Fe in stage I to stage III. 3+ , for wastewater treatment;

[0058] The anaerobic ammonium oxidation sludge is pretreated for about 45 days before inoculation, during which no iron and tea polyphenols are added to the influent, and other components are added normally to eliminate the interference of iron in the inoculated sludge as much as possible;

[0059] The wastewater has an ammonia nitrogen concentration of 100 mg / L, a nitrite nitrogen concentration of 120 mg / L, a pH of 7.5±0.1, and dissolved oxygen <0.1 mg / L;

[0060] The SBR reactor was operated for 75 days, with 3 cycles per day, a single cycle lasting 7 hours, the temperature was controlled at 32-34°C by water bath heating, and the stirring speed was 30 rpm;

[0061] The SBR reactor replaces the wastewater every 8 hours, with a water replacement ratio of 50%;

[0062] The stage I is from the 1st day to the 30th day, and in the stage I, the tea polyphenol-Fe 3+ The addition concentration was 25 μmol / L;

[0063] The stage II is from the 31st day to the 45th day, and in the stage II, the tea polyphenol-Fe 3+ The addition concentration was 25 μmol / L;

[0064] The stage III is from the 46th day to the 75th day, and in the stage III, the tea polyphenol-Fe 3+ The addition concentration was 12.5 μmol / L;

[0065] The tea polyphenols-Fe 3+ The concentration molar ratio of tea polyphenols to Fe is 1:1 3+ Complexed.

[0066] Comparative Example 2

[0067] The method of Example 8 was followed, except that tea polyphenol-Fe was not added. 3+ The remaining steps and parameters are the same as in Example 8.

[0068] Comparative Example 3

[0069] The method of Example 8 was followed, except that FeCl3 was used instead of the tea polyphenol-Fe 3+ , and FeCl3 with a concentration of 25 μmol / L was continuously added in stage I to stage III, and the remaining steps and parameters were the same as those in Example 8.

[0070] Test Example 1

[0071] After the experiment, the intracellular iron content and NRE changes in the anaerobic ammonium oxidation reactors of Examples 1 to 7 and Comparative Example 1 were detected. The results are as follows Figure 1 shown.

[0072] Depend on Figure 1 It can be seen that the NRE and intracellular iron content in the anaerobic ammonium oxidation reactor increased with the increase of tea polyphenol-Fe 3+ However, when tea polyphenols-Fe 3+ When the concentration was higher than 25 μmol / L, the NRE and intracellular iron content in the anaerobic ammonium oxidation reactor tended to be stable, which indicated that the tea polyphenol-Fe 3+ The appropriate addition concentration is 25 μmol / L (i.e., the tea polyphenol-Fe in Example 1 of the present invention). 3+ of dosing concentration).

[0073] Moreover, compared with Comparative Example 1 (without adding tea polyphenols-Fe 3+ ), the NRE in the anaerobic ammonium oxidation reactors of Examples 1 to 7 of the present invention increased by 3.2 to 6.7 times, and the intracellular iron content increased by 91% to 188%, which indicates that tea polyphenols-Fe3+ The introduction of promoted the absorption of trivalent iron by anaerobic ammonium oxidizing bacteria, and then anaerobic ammonium oxidizing bacteria used iron to enhance the metabolism of Anammox, DNRA, etc., significantly enhancing the denitrification efficiency in the anaerobic ammonium oxidation reactor.

[0074] Test Example 2

[0075] During the operation of the reactor, the denitrification efficiency in the SBR reactor of the above-mentioned Example 8 and Comparative Examples 2 and 3 was detected. The results are as follows: Figure 2~Figure 4 shown.

[0076] Depend on Figure 2~Figure 4 It can be seen that in stage I, adding 25μmol / L tea polyphenols-Fe 3 + After that, in Example 8 of the present invention, NRE first increased rapidly and then stabilized at 87.7%; however, in stage II, long-term addition of 25 μmol / L tea polyphenols-Fe 3+ It is not conducive to the stable operation of the anaerobic ammonium oxidation system, causing its NRE to drop to 68.4%; in stage III, tea polyphenols-Fe 3+ After the addition concentration was reduced to 12.5 μmol / L, the denitrification efficiency of Example 8 of the present invention recovered rapidly, and NRE finally stabilized to 88.8%, indicating that in the long-term stable operation stage, tea polyphenols-Fe 3+ The appropriate dosage concentration is 12.5 μmol / L.

[0077] Secondly, the denitrification performance in the reactors of Comparative Example 2 and Comparative Example 3 was maintained at a low level, with the NRE in the reactor of Comparative Example 2 being 12.2% and the NRE in the reactor of Comparative Example 3 being 13.5%, indicating that the addition of Fe alone 3+ The denitrification efficiency of the anaerobic ammonium oxidation system cannot be effectively improved. However, in stage III, the NRE in the reactor of Example 8 of the present invention can be stabilized at 88.8%, and at the same time, ∆NO3 − −N / ∆NH4 + −N ratio is 0.23, which is significantly lower than that of comparative example 2 (∆NO3 − −N / ∆NH4 + −N ratio is 0.27) and comparative example 3 (∆NO3 − −N / ∆NH4 + −N ratio is 0.27), indicating that tea polyphenols-Fe 3+ The addition of anammox promoted the metabolism of Anammox and DNRA at the same time, thereby enhancing the denitrification efficiency of the anaerobic ammonium oxidation system.

[0078] Test Example 2

[0079] During the operation of the reactor, the composition of anaerobic ammonium-oxidizing bacteria in the inoculated anaerobic ammonium-oxidizing sludge and the SBR reactors of Example 8 and Comparative Examples 2 to 3 in Stage III was detected, and the results were as follows: Figure 5 shown.

[0080] Depend on Figure 5 It can be seen that the inoculated anaerobic ammonium oxidation sludge contains Candidatus Brocadia, Candidatus Jettenia, Candidatus Kuenenia three anaerobic ammonium oxidizing bacteria genera, and Candidatus Brocadia was the main component (accounting for 80%). Compared with the inoculated sludge, the absolute abundance of anaerobic ammonia-oxidizing bacteria (2×10 9 →6×10 7 , 3×10 7 copies / g VSS) and relative abundance (16%→4%, 2%) decreased significantly; while the absolute abundance of anaerobic ammonia-oxidizing bacteria in the reactor of Example 8 of the present invention (9×10 8 copies / g VSS) and relative abundance (12%) remained basically unchanged, but Candidatus Jettenia was enriched, and its proportion in anaerobic ammonia-oxidizing bacteria increased significantly from 17% to 39%, indicating that tea polyphenols-Fe 3+ Helps maintain the proliferation of anaerobic ammonium oxidizing bacteria in the anaerobic ammonium oxidizing system, especially Candidatus Proliferation of Jettenia.

[0081] Test Example 2

[0082] During the operation of the reactor, in stage III, the relative abundance of nitrogen conversion functional genes and the species contribution of nitrogen conversion functional genes in the SBR reactors of Example 8 and Comparative Examples 2 to 3 were detected, and the results were as follows: Figure 6 and Figure 7 shown.

[0083] Depend on Figure 6 It can be seen that compared with Comparative Examples 2 and 3, the hz , hhd Genes and genes related to DNRA metabolism napAB , nirBD , nbq The relative abundance of the genes in Example 8 of the present invention increased significantly, indicating that tea polyphenols-Fe 3+ Strengthened the Anammox and DNRA metabolism of the anaerobic ammonium oxidation system.

[0084] Furthermore, anaerobic ammonium-oxidizing bacteria Candidatus Brocadia and Anammox bacteria Candidatus Jettenia is a system that performs Anammox ( hz and hhd gene) and DNRA ( nbq Gene) metabolism. Figure 7 It can be seen that the anaerobic ammonia oxidizing bacteria in the reactor of comparative example 2 contribute 95% hz Gene, 77% hhd Gene, 5% nbq Gene, anaerobic ammonia oxidizing bacteria in the comparative reactor 3 contributed 88% hz Gene, 57% hhd Gene, 3% nbq Gene, anaerobic ammonia oxidizing bacteria in the reactor of Example 8 of the present invention contribute 99% hz Gene, 99% hhd Gene, 17% nbq Compared with Comparative Examples 2 and 3, the contribution of anaerobic ammonia oxidizing bacteria to the functional genes of the Anammox and DNRA pathways in Example 8 of the present invention increased significantly, indicating that tea polyphenols-Fe 3+ It mainly promotes the metabolism of Anammox and DNRA of anaerobic ammonia-oxidizing bacteria.

[0085] In summary, Figure 1 to Figure 7 It can be seen that tea polyphenols-Fe 3+ It can promote the absorption of trivalent iron by anaerobic ammonium oxidizing bacteria, enhance the metabolism of Anammox and DNRA of anaerobic ammonium oxidizing bacteria, and significantly enhance the denitrification efficiency of the anaerobic ammonium oxidizing system. 3+ The appropriate dosage concentration is 25 μmol / L in the short-term startup phase and 12.5 μmol / L in the long-term stable operation phase.

[0086] The preferred embodiments of the present invention are described in detail above, but the present invention is not limited thereto. Within the technical concept of the present invention, the technical solution of the present invention can be subjected to a variety of simple modifications, including the combination of various technical features in any other suitable manner, and these simple modifications and combinations should also be regarded as the contents disclosed by the present invention and belong to the protection scope of the present invention.

Claims

1. A method for denitrification of anaerobic ammonium oxidation system based on tea polyphenols and trivalent iron, characterized in that: The method includes: In the anaerobic ammonium oxidation reactor, anaerobic ammonium oxidation sludge is inoculated, wastewater is added, and then tea polyphenols-Fe are added. 3+ , to treat the wastewater.

2. The method according to claim 1, characterized in that The addition concentration of the anaerobic ammonium oxidation sludge is 2000 mgSS / L.

3. The method according to claim 1, characterized in that The inoculated anaerobic ammonium oxidation sludge contains anaerobic ammonium oxidizing bacteria Candidatus Brocadia and Anammox bacteria Candidatus Jettenia.

4. The method according to claim 1, characterized in that: The tea polyphenols-Fe 3+ The preparation method is to complex tea polyphenols and FeCl3 with a molar concentration of 1:

1.

5. The method according to claim 1, characterized in that The tea polyphenols-Fe 3+ The continuous addition concentration is 12.5~150μmol / L.

6. The method according to claim 1, characterized in that In the short-term startup phase, the tea polyphenols-Fe 3+ The continuous addition concentration is 25 μmol / L; in the long-term stable operation stage, the tea polyphenol-Fe 3+ The continuous addition concentration is 12.5 μmol / L; wherein the short-term startup phase is preferably 1 to 30 days, and the long-term stable operation phase is preferably >30 days.

7. The method according to claim 1, characterized in that The temperature in the anaerobic ammonium oxidation reactor was controlled at 30~35℃, the pH at 7.8±0.5, and the dissolved oxygen <0.1mg / L.

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