CN108707598B - Method for enhancing denitrification of denitrifying bacteria by taking filamentous fungi as carrier - Google Patents

Method for enhancing denitrification of denitrifying bacteria by taking filamentous fungi as carrier Download PDF

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CN108707598B
CN108707598B CN201810533296.1A CN201810533296A CN108707598B CN 108707598 B CN108707598 B CN 108707598B CN 201810533296 A CN201810533296 A CN 201810533296A CN 108707598 B CN108707598 B CN 108707598B
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denitrifying bacteria
talaromyces flavus
mycelium
suspension
denitrifying
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CN108707598A (en
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缪恒锋
周梦娟
阮文权
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Jiangnan University
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    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12NMICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N11/00Carrier-bound or immobilised enzymes; Carrier-bound or immobilised microbial cells; Preparation thereof
    • C12N11/16Enzymes or microbial cells immobilised on or in a biological cell
    • CCHEMISTRY; METALLURGY
    • C02TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
    • C02FTREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
    • C02F3/00Biological treatment of water, waste water, or sewage
    • C02F3/34Biological treatment of water, waste water, or sewage characterised by the microorganisms used
    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12NMICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/14Fungi; Culture media therefor
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    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12NMICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/20Bacteria; Culture media therefor
    • CCHEMISTRY; METALLURGY
    • C02TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
    • C02FTREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
    • C02F2101/00Nature of the contaminant
    • C02F2101/10Inorganic compounds
    • C02F2101/16Nitrogen compounds, e.g. ammonia

Abstract

The invention discloses a method for strengthening denitrification of denitrifying bacteria by taking filamentous fungi as a carrier, belonging to the technical field of biological sewage treatment. The method comprises the following steps: inoculating a proper amount of sludge-water mixed liquor to the denitrification enrichment culture solution for culture to obtain a bacterial suspension of denitrifying bacteria; inoculating Talaromyces flavus S1 slant culture medium into PDB culture medium, and performing shake culture to obtain mature mycelium pellet; inoculating the denitrifying bacteria suspension into a denitrifying culture solution containing mature mycelium pellets to enhance the denitrification effect of denitrifying bacteria, and repeatedly collecting the mycelium pellets. The Talaromyces flavus S1 mycelium pellet used in the method has better denitrification effect than the existing Trichoderma virrid mycelium pellet immobilized denitrifying bacteria, not only has the advantages of simple operation, good sedimentation performance, high repeated utilization rate and the like, but also enhances the denitrification effect of the denitrifying bacteria, provides basis for the practical application of the denitrifying bacteria in the future, and develops the practical application prospect of Talaromyces flavus S1.

Description

Method for enhancing denitrification of denitrifying bacteria by taking filamentous fungi as carrier
Technical Field
The invention relates to a method for strengthening denitrification of denitrifying bacteria by taking filamentous fungi as a carrier, belonging to the technical field of biological sewage treatment.
Background
In recent years, the unreasonable use of nitrogenous fertilizers, messy discharge of sewage, and untreated leakage of landfill leachate by humans has resulted in a large amount of nitrate in the water body. And an excess of nitrate, which threatens human health, such as acute methemoglobinemia and blue infant syndrome. Therefore, in recent years, efforts have been made to remove nitrates from groundwater. Compared with physical and chemical methods, the method has the limitations of high cost, poor effect, difficult implementation and the like, and the biological method is used for removing nitrate pollution in water and is concerned by people.
Nitrate removal in water is mainly reduced by denitrification, and microorganisms involved in denitrification are diverse. Microorganisms capable of undergoing denitrification are generally referred to collectively as denitrifying microorganisms, such as Proteobacteria, bacteriodes, Firmicutes, Gracilobacter, and the like. Under the anoxic or anaerobic condition, denitrifying bacteria convert nitrate in water into N with self carbon source and external carbon source as electron donor, nitrate and nitrite as electron acceptor, nitrate reductase, nitrite reductase, etc2Thereby completing the removal of nitrate in the water body and achieving the purposes of the removal effect of total nitrogen and the removal of organic matters. Therefore, denitrifying bacteria play an important role in removing nitrate in water and are highly favored.
In the actual application process, the effect of directly adding denitrifying bacteria into the water body cannot achieve an ideal effect, and the strengthening technology is used in the actual application of the water body in order to avoid the loss of microorganisms in the water body. The reinforced microorganism technology is characterized in that a target microorganism is fixed in a carrier by means of physics, chemistry and the like, the concentration of the microorganism is increased on the basis of ensuring the original activity of the microorganism, the microorganism is protected, and the impact resistance is improved. Meanwhile, the product is recycled and utilized to be enhanced, so that the product classification is facilitated, and the waste phenomenon is reduced. Therefore, a proper strengthening mode is found, the denitrifying bacteria are fixed, and the method has great application prospect in water denitrification. Generally, the method is divided into physical adsorption, chemical adsorption and biological adsorption according to the strengthened fixing type, and common physical and chemical adsorption materials comprise polyurethane foam, polyvinyl alcohol, sodium alginate and the like, but the activity of denitrifying microorganisms is influenced to a certain extent, and the denitrification effect of a water body is influenced.
At present, the nitrogen removal is carried out by using Trichoderma viride mycelium pellets in a reported mycelium pellet culture mode, wherein paracoccus (a denitrifying bacterium) and Trichoderma viride are co-cultured to obtain mixed mycelium pellets to remove river sewage pollutants. The method has high removal effect, but has the defects of long culture time, low success rate and the like of the mixed mycelium pellet.
Disclosure of Invention
Based on the problems, the invention provides a more efficient denitrifying bacteria immobilization method aiming at the practical application of denitrifying bacteria, and the method adopts mycelium pellets of a filamentous fungus Talaromyces flavus S1 (cyanobacteria) as an immobilization carrier of the denitrifying bacteria to further treat the sewage, thereby effectively improving the sewage treatment effect. Compared with the existing method for denitrifying by adopting Trichoderma virrid mycelium pellets as immobilized carriers, the invention adopts the mode of firstly culturing the mycelium pellets and then inoculating denitrifying bacteria, thereby greatly improving the success rate of mycelium pellet preparation and effectively shortening the time of immobilizing the denitrifying bacteria by the mycelium pellets; meanwhile, the invention can aim at most denitrifying bacteria, and avoids the interaction between certain denitrifying bacteria and the strains capable of preparing mycelium pellets. In a word, the method is more efficient, and provides a basis for the Talaromyces flavus S1 mycelium pellet to be applied to the water body treatment of denitrifying bacteria.
The mycelium pellet is prepared by the filamentous fungus Talaromyces flavus S1, so that the mycelium pellet becomes an excellent carrier for strengthening microorganisms, and a denitrifying bacteria immobilization method taking the Talaromyces flavus S1 mycelium pellet as the carrier, which is more efficient than the Trichoderma virid mycelium pellet, is established.
The first purpose of the invention is to provide immobilized denitrifying bacteria which are obtained by taking Talaromyces flavus S1 mycelium pellets as an immobilized carrier.
In one embodiment, the preparation of the immobilized denitrifying bacteria comprises:
(1) obtaining denitrifying bacteria suspension and Talaromyces flavus S1 mycelial pellets;
(2) and simultaneously inoculating the denitrifying bacteria suspension and mature Talaromyces flavus S1 mycelial pellets into the wastewater to be treated to obtain the immobilized denitrifying bacteria taking Talaromyces flavus S1 bacteria as carriers.
The second purpose of the invention is to provide a method for enhancing the denitrification effect of denitrifying bacteria, which takes Talaromyces flavus S1 mycelium pellets as immobilized carriers of the denitrifying bacteria, and then utilizes the immobilized denitrifying bacteria to carry out denitrification treatment.
In one embodiment, the method comprises:
(1) obtaining denitrifying bacteria suspension and Talaromyces flavus S1 mycelial pellets;
(2) the denitrifying bacteria suspension and the mature Talaromyces flavus S1 mycelium pellet are simultaneously inoculated into the wastewater to be treated to obtain the denitrifying bacteria mycelium pellet taking the Talaromyces flavus S1 as a carrier, and the denitrifying bacteria mycelium pellet is cultured for a period of time.
In one embodiment, the method of preparing the bacterial suspension of denitrifying bacteria comprises:
(A) culturing denitrifying bacteria: taking a proper amount of sludge-water mixed liquor of an anoxic tank of a sewage treatment plant, inoculating the sludge-water mixed liquor into a denitrification culture solution, carrying out anaerobic shaking culture, carrying out continuous culture for three times, and centrifuging to obtain denitrifying bacteria thallus;
(B) preparing a denitrifying bacteria bacterial suspension: taking a proper amount of thalli to be resuspended in sterilized water to obtain a bacterial suspension of denitrifying bacteria;
in one embodiment, the slurry-water mixture in step (a) is washed with a phosphate buffer (pH 7), and sieved with a 200-mesh sieve to remove impurities.
In one embodiment, in the step (A), the activated sludge and the distilled water are diluted in a volume ratio of 1:4, and after standing for 1 hour, 10ml of the middle layer sludge-water mixture is taken, 90ml of the denitrification culture solution is added, and the mixture is placed in a conical flask.
In one embodiment, the denitrification culture solution in step (A) comprises the following main components: KNO3 2g/L,C6H12O6 1.298g/L,MgSO4·7H20.2g/L of O, 2ml/L of trace elements, 50ml/L of phosphate buffer (pH 7.5) and the balance of water.
In one embodiment, the phosphate buffer of step (a) is composed of Na2HPO40.908g/L and NaH2PO40.25 g/L.
In one embodiment, the anaerobic condition of step (a) is argon gas for 10 minutes to remove excess oxygen.
In one embodiment, the denitrifying bacteria thallus in step (A) is obtained by centrifugation at 4000r/min for 15 minutes.
In one embodiment, the bacterial suspension of the denitrifying bacteria of step (A) is 1g/100 ml.
In one embodiment, the culture conditions of step (a) are: the rotating speed is 130r/min, the temperature is 28 +/-2 ℃, and the culture period time is 5 days.
In one embodiment, the preparation of the Talaromyces flavus S1 mycelial pellets comprises:
(a) preparation of a bacterial suspension of Talaromyces flavus S1: inoculating a slant culture medium of Talaromyces flavus S1 into sterilized water filled with glass beads, placing the sterilized water in a shaking table for shaking, and carrying out blood cell counting to obtain a spore bacteria suspension of Talaromyces flavus S1;
(b) culturing Talaromyces flavus S1 mycelial pellets: taking a proper amount of spore bacterium suspension, inoculating the spore bacterium suspension into a PDB culture medium, and performing shake culture for 3-5 days to obtain mature mycelium pellets;
in one embodiment, the filamentous fungus Talaromyces flavus S1 in step (a) is obtained from environmental and civil engineering institute of university in south Jiangnan from solid waste control and resource laboratory screening, and the specific characteristics are referred to in research on the balling property and dehydration property improvement of the filamentous fungus Talaromyces flavus S1 in sludge.
In one embodiment, the shake culture conditions in step (a) are: the temperature is 28 +/-2 ℃, the rotating speed is 130r/min, and the culture time is 5-6 days.
In one embodiment, the blood cell count in step (a) is 7.8 × 107~9.3×107And (2) per liter.
In one embodiment, the spore suspension in step (b) is inoculated in an amount of 10% by volume.
In one embodiment, the PDB medium in step (b) comprises the following main components: peeling and cooking potato at a ratio of 200g/L, 20g/L C6H12O6Mixing, and autoclaving at 121 deg.C for 20 min.
In one embodiment, the denitrifying bacteria suspension in step (2) is added at a volume ratio of 0.5-2% to 5g of Talaromyces flavus S1 mycelial pellets in an amount of 3-8g/100mL of wastewater.
In one embodiment, the suspension of denitrifying bacteria in step (2) is added at 1% volume ratio, together with 5g of Talaromyces flavus S1 mycelial pellets, to 100ml of simulated wastewater.
In one embodiment, the culture conditions in step (2) are: the temperature is 28 +/-2 ℃, the rotating speed is 100-.
In one embodiment, the shake culture conditions in step (2) are: the temperature is 28 +/-2 ℃, the rotating speed is 130r/min, and the culture time is 1.8 days.
The invention has the beneficial effects that:
the invention prepares the mycelium pellet of Talaromyces flavus S1, and the mycelium pellet is used as a carrier to immobilize denitrifying bacteria. The denitrifying bacteria are adsorbed on the surface and inside of the mycelium pellet by utilizing the unique spatial structure and biocompatibility of the mycelium pellet, so that the denitrifying bacteria mixed mycelium pellet taking the mycelium pellet as the center is formed. The biomass of denitrifying bacteria is improved, and the impact resistance is also improved. When the denitrifying bacteria inhibitor is applied to sewage, the denitrifying capability of denitrifying bacteria is obviously enhanced.
The invention uses Talaromyces flavus S1 as a carrier to carry out immobilization of denitrifying bacteria, thereby strengthening the denitrification effect of the immobilized denitrifying bacteria. The method has the advantages of simple operation, good settling property, high reusability and the like, can effectively immobilize the nitrate nitrogen removal capability of denitrifying bacteria, and enhances the removal effect of total nitrogen in the water body, thereby improving the deterioration condition of water quality. And the immobilized denitrifying bacteria have high recovery rate and can reduce the pollution to the environment. The invention not only provides a new application prospect for the application of Talaromycesflavus S1 bacteria, but also provides a more efficient immobilization method for removing nitrate in water.
Drawings
FIG. 1 is the nitrate removal of example 1;
FIG. 2 is the total nitrogen removal of example 1;
FIG. 3 is a graph showing nitrate removal during recycling of mycelial pellets in example 1;
FIG. 4 is a graph of total nitrogen removal during recycling of the mycelial pellets of example 1;
FIG. 5 shows the nitrate removal of actual wastewater;
FIG. 6 shows the nitrate removal of actual wastewater;
FIG. 7 shows the nitrate removal situation of mycelium pellets in actual wastewater treatment recycling;
FIG. 8 shows the total nitrogen removal of mycelium pellets for actual wastewater treatment recycling.
Detailed Description
In order to more clearly illustrate the present invention, the present invention is further illustrated below with reference to experimental cases. It is to be understood by persons skilled in the art that the following detailed description is illustrative and not restrictive, and is not to be taken as limiting the scope of the invention.
Example 1:
(1) enrichment culture of denitrifying bacteria: taking a plurality of sludge in an anoxic pond of a sewage treatment plant. It was washed with phosphate buffer (pH 7) and passed through a 200 mesh screen to remove excess impurities. Activated sludge and distilled water were diluted at a volume ratio of 1: 4. After standing for 1 hour, 10ml of the middle layer slurry mixture was taken and placed in a conical flask, 90ml of the denitrification culture solution (tables 1 and 2) was added, and argon gas was introduced to remove oxygen gas and ensure anaerobic conditions. And when the nitrate concentration of the culture solution is reduced to a certain degree, taking the mixed solution to carry out centrifugation at 4000r/min for 15 minutes to obtain the denitrifying bacteria. The denitrifying bacteria with high relative abundance can be obtained by repeating the culture for three times by the same method.
(2) Preparing bacterial suspension of denitrifying bacteria: resuspending 1g of the denitrifying bacteria deposit in (1) with 100ml of sterile water to obtain a bacterial suspension of the denitrifying bacteria, and storing in a refrigerator at 4 ℃.
(3) Inoculating a slant culture medium of Talaromyces flavus S1 into sterilized water filled with glass beads, placing the sterilized water in a shaking table for shaking, and carrying out blood cell counting to obtain spore bacteria suspension of Talaromyces flavus S1; inoculating the mycelium into a PDB culture medium, and performing shake culture for 5-6 days to obtain mature mycelium pellets;
(4) inoculating a slant culture medium of Trichoderma viride into sterilized water filled with glass beads, placing the slant culture medium into a shaking table for shaking, and carrying out blood cell counting to obtain a spore bacteria suspension of Trichoderma viride; inoculating the mycelium into a PDB culture medium, and performing shake culture for 5-6 days to obtain mature mycelium pellets;
(5) inoculating denitrifying bacteria suspension and mature Talaromyces flavus S1 mycelial pellets (or Trichoderma virid mycelial pellets) into 100ml of simulated wastewater (Table 3) with the nitrate nitrogen concentration of 277mg/L, the COD of 650mg/L, the pH of 7.5 and the temperature of 25 ℃ simultaneously to obtain denitrifying bacteria mycelial pellets taking Talaromyces flavus S1 bacteria (or Trichoderma virid bacteria) as carriers;
(6) experiments were divided into a blank group 1 (culture medium only added group), a blank group 2 (culture medium and mycelium pellet only added group), an unfixed denitrifying bacteria group (culture medium and denitrifying bacteria added group), a Trichoderma virrid mycelium pellet fixed group (denitrifying bacteria, Trichoderma virrid mycelium pellet and culture medium added group) and a Talaromyces flavus S1 mycelium pellet (denitrifying bacteria, Talaromyces flavus S1 mycelium pellet and culture medium added group), and the removal rates of nitrate nitrogen and total nitrogen were measured every 3 hours, as shown in FIGS. 2 and 3, respectively.
(7) Collecting mycelium pellets in the trichoderma mycelium pellet fixed group (added with denitrifying bacteria, trichoderma mycelium pellets and a culture solution group) and the Talaromyces flavus S1 mycelium pellets (added with denitrifying bacteria, Talaromyces flavus S1 mycelium pellets and a culture solution group) in the step 6), washing the mycelium pellets for multiple times by using a phosphate buffer solution to remove the denitrifying bacteria in the mycelium pellets, adding 1% of denitrifying bacteria by volume ratio, re-inoculating the denitrifying bacteria into a denitrifying culture solution, and measuring final nitrate nitrogen and total nitrogen concentration of the denitrifying bacteria in a cycle of 1.8 days to explore the recycling effect of the mycelium pellets;
as shown in FIG. 1, for nitrate removal in different experimental groups, mature Trichoderma virrid mycelium pellets and 5g Talaromyces flavus S1 mycelium pellets were added to 100ml of denitrification culture solution (tables 1 and 2), and 1ml of denitrification bacterial suspension was added to analyze nitrate adsorption capacity of the mycelium pellets. The results show that: the self-adsorption nitrate capacity of the mycelium pellet is only 24.34mg/L, the removal rate is 8.83 percent, the nitrate removal amount of only inoculated denitrifying bacteria is 168.52mg/L, the removal rate is 61.20mg/L, and the removal rate of the denitrifying bacteria after immobilization is increased, but the difference exists. The trichodermavid mycelium pellet of the prior document is adopted for immobilization, only 206.93mg/L of nitrate is removed, and the removal rate is 75.15%. The removal rate of immobilized denitrifying bacteria using the Talaromyces flavus S1 mycelium pellets is enhanced to 223.40mg/L and 81.14%, and compared with the existing mycelium pellets, the removal rate is increased by 5.99%.
As shown in fig. 2: the total nitrogen removal condition of different experimental groups. The total nitrogen removal of the mycelium pellet is only 36.35mg/L, and the total nitrogen removal rate is only 12.50%. The total nitrogen removal effect of the immobilized denitrifying bacteria is improved. The total nitrogen removal of the Talaromyces flavus S1 mycelial sphere immobilized denitrifying bacteria prepared by the experiment can reach 225.64mg/L, the removal rate is 78.41%, and the total nitrogen removal rate is 41.62mg/L higher than that of the Trichodermavirid mycelial sphere immobilized total nitrogen removal amount 184.02mg/L adopted in the existing literature, and the removal rate is 14.46% higher.
As shown in fig. 3: the nitrate nitrogen removal capacity in the recycling of Trichoderma viride mycelium pellets and Talaromyces flavus S1 mycelium pellets. The results show that: after 5 times of cyclic utilization, the two mycelium pellets still keep higher immobilization effect. After 5 times of utilization, the nitrate removal rate of the Trichoderma virrid mycelium pellet is converted from 75.15% to 74.20%, while the nitrate removal rate of the Talaromyces flavus S1 mycelium pellet prepared by the invention is converted from 81.14% to 76.68% after 5 times of utilization. Starting from the nitrate removal rate: the recycling performance of Talaromyces flavus S1 mycelium pellet is better than that of Trichodermavirid mycelium pellet reported in the literature.
As shown in fig. 4: the total nitrogen removal capacity of the Trichoderma viride mycelium pellet and Talaromyces flavus S1 mycelium pellet in recycling is provided. The results show that: after 5 times of recycling, the total nitrogen removal rate of the two mycelium pellets is still kept at a higher level. The total nitrogen removal rate of the trichoderma virid mycelium pellet was converted from 63.95% to 62.81%, and the total nitrogen removal effect was not impaired. After 5 times of recycling, the total nitrogen removal rate of the Talaromyces flavus S1 mycelium pellet is reduced from 78.41% to 68.46%, and the total nitrogen removal rate is still higher than that reported in the existing literature.
TABLE 1 Denitrification broth principal Components
TABLE 2 Trace element composition in Denitrification broth
Table 3 simulation of wastewater Components in examples
Example 2:
(1) enrichment culture of denitrifying bacteria: taking a plurality of sludge in an anoxic pond of a sewage treatment plant. It was washed with phosphate buffer (pH 7) and passed through a 200 mesh screen to remove excess impurities. Activated sludge and distilled water were diluted at a volume ratio of 1: 4. Standing for 1 hour, taking 10ml of the mud-water mixture in the middle layer, placing the mixture in a conical flask, adding 90ml of denitrification culture solution, and introducing argon to remove oxygen so as to ensure anaerobic conditions. And when the nitrate concentration of the culture solution is reduced to a certain degree, taking the mixed solution to carry out centrifugation at 4000r/min for 15 minutes to obtain the denitrifying bacteria. The denitrifying bacteria with high relative abundance can be obtained by repeating the culture for three times by the same method.
(2) Preparing bacterial suspension of denitrifying bacteria: resuspending 1g of the denitrifying bacteria deposit in (1) with 100ml of sterile water to obtain a bacterial suspension of the denitrifying bacteria, and storing in a refrigerator at 4 ℃.
(3) Inoculating a slant culture medium of Talaromyces flavus S1 into sterilized water filled with glass beads, placing the sterilized water in a shaking table for shaking, and carrying out blood cell counting to obtain spore bacteria suspension of Talaromyces flavus S1; inoculating the mycelium into a PDB culture medium, and performing shake culture for 5-6 days to obtain mature mycelium pellets;
(4) inoculating a slant culture medium of Trichoderma viride into sterilized water filled with glass beads, placing the slant culture medium into a shaking table for shaking, and carrying out blood cell counting to obtain a spore bacteria suspension of Trichoderma viride; inoculating the mycelium into a PDB culture medium, and performing shake culture for 5-6 days to obtain mature mycelium pellets;
(5) the denitrifying bacteria suspension, mature Talaromyces flavus S1 mycelial pellets and Trichoderma virid mycelial pellets are simultaneously inoculated into 100ml of actual wastewater (Table 4) with the nitrate nitrogen concentration of 138mg/L, the COD of 693mg/L, the pH of 7 and the temperature of 10 ℃ to obtain the denitrifying bacteria mycelial pellets taking the Talaromyces flavus S1 as a carrier;
(6) the experiment was divided into a blank group (only actual wastewater group), an unfixed denitrifying bacteria group (added actual wastewater and denitrifying bacteria group), a Trichoderma virrid mycelium pellet fixed group (added denitrifying bacteria, Trichoderma virrid mycelium pellet and actual wastewater group) and a Talaromyces flavus S1 mycelium pellet (added denitrifying bacteria, Talaromyces flavus S1 mycelium pellet and actual wastewater group) fixed group, and the removal rates of nitrate nitrogen and total nitrogen were measured every 3 hours, as shown in FIGS. 5 and 6, respectively.
(7) Collecting mycelium pellets in the trichoderma mycelium pellet fixed group (added with denitrifying bacteria, trichoderma mycelium pellets and actual wastewater group) and the Talaromyces flavus S1 mycelium pellets (added with denitrifying bacteria, Talaromyces flavus S1 mycelium pellets and actual wastewater group) in the step 6), washing for multiple times by using a phosphate buffer solution to remove the denitrifying bacteria in the mycelium pellets, adding 1% of denitrifying bacteria by volume ratio, re-inoculating the denitrifying bacteria into a denitrifying culture solution, and measuring final nitrate nitrogen and total nitrogen concentration of the bacteria in a cycle of 1.8 days to explore the recycling effect of the mycelium pellets;
FIG. 5 shows the nitrate removal of mycelium pellets in actual wastewater. The results show that: only 1% of denitrifying bacteria are used for treating actual wastewater, the treatment effect can be only reduced to 51.00mg/L, 46.56mg/L and the removal rate is 47.72%. After the immobilization, the nitrate removal rate is improved. The nitrate removal rate of denitrifying bacteria is increased to 50.48% by using the Trichoderma virrid mycelium pellet immobilized group, while the nitrate removal rate of denitrifying bacteria is reduced to 20.13mg/L and 77.43mg/L by using Talaromyces flavus S1 mycelium pellet immobilized denitrifying bacteria, and the nitrate removal rate is 79.37%, which is higher than that of the prior document by 28.89%.
FIG. 6 shows the total nitrogen removal from the mycelium pellets in actual wastewater. The results show that: the immobilized denitrifying bacteria facilitate the removal of total nitrogen. Denitrifying bacteria were added directly to treat the actual wastewater with a total nitrogen removal of 47.25%. And the total nitrogen of the immobilized denitrifying bacteria carried out by the Trichodermavirid mycelium pellet reported in the literature is reduced to 53.51mg/L, the total nitrogen is removed by 49.25mg/L, and the removal rate is 47.92%. The total nitrogen removal rate is greatly improved by adopting the Talaromyces flavus S1 mycelium pellets for immobilization, the total nitrogen in the sewage is reduced from 102.76mg/L to 23.33mg/L after being treated for 42 hours, the removal rate reaches 77.29%, and the effect is improved by 29.37% compared with the Trichodermavirid mycelium pellet immobilization in the prior document.
FIG. 7 shows the nitrate removal during recycling of two mycelium pellets. The results show that: after 5 times of cyclic use, the nitrate removal capacity of the immobilized denitrifying bacteria is changed slightly, the immobilized denitrifying bacteria still have higher immobilization capacity, and the nitrate nitrogen removal rate (76.1-79.37%) of the Talaromyces flavus S1 mycelium pellet is higher than the immobilization effect (48.12-50.48%) of the Trichoderma virrid mycelium pellet.
FIG. 8 shows the total nitrogen removal during recycling of two mycelium pellets. The results show that: after the mycelium pellet is repeatedly used for 5 times, the total nitrogen removal rate of the mycelium pellet is not obviously changed. The total nitrogen removal rate of the immobilized Trichodermavirid mycelium pellet is 45.15-47.92%. The total nitrogen removal rate (76.23-77.29%) of the Talaromyces flavus S1 mycelium pellet which is utilized for 5 times is obviously higher than that of the mycelium pellet reported in the current literature.
TABLE 4 actual main Components of wastewater

Claims (9)

1. An immobilized denitrifying bacterium, wherein the immobilized denitrifying bacterium is a microorganism that is capable of producing a large amount of waterTalaromyces flavus S1The mycelium pellet is obtained by taking the mycelium pellet as an immobilized carrier; the preparation of the immobilized denitrifying bacteria comprises the following steps:
(1) obtaining a suspension of denitrifying bacteria andTalaromyces flavus S1a mycelium pellet;
(2) mixing the suspension of denitrifying bacteria with the matureTalaromyces flavus S1And simultaneously inoculating the mycelium pellets into the wastewater to be treated to obtain the immobilized denitrifying bacteria taking Talaromyces flavus S1 bacteria as carriers.
2. A method for strengthening denitrification effect of denitrifying bacteria is characterized in that the method is toTalaromyces flavus S1The mycelium pellet is used as an immobilized carrier of denitrifying bacteria, and the immobilized denitrifying bacteria are used for denitrification treatment.
3. The method of claim 2, wherein the method comprises:
(1) obtaining a suspension of denitrifying bacteria andTalaromyces flavus S1a mycelium pellet;
(2) mixing the suspension of denitrifying bacteria with the matureTalaromyces flavus S1The mycelium pellets are simultaneously inoculated into the wastewater to be treated to obtainTalaromyces flavus S1Culturing mycelium pellet of denitrifying bacteria with bacteria as carrier for a period of time.
4. The method according to claim 3, wherein the denitrifying bacteria suspension in step (2) is added in a volume ratio of 0.5-2%,Talaromyces flavus S1the addition amount of the mycelium pellets is 3-8g/100mL of wastewater.
5. The method according to claim 3, wherein the culture conditions in step (2) are: the temperature is 28 +/-2 ℃, the rotating speed is 100-.
6. The method of claim 3, wherein the denitrifying bacteria suspension is prepared by a method comprising:
(A) culturing denitrifying bacteria: taking a proper amount of sludge-water mixed liquor of an anoxic tank of a sewage treatment plant, inoculating the sludge-water mixed liquor into a denitrification culture solution, carrying out anaerobic shaking culture, continuously culturing for many times, and centrifuging to obtain denitrifying bacteria thallus;
(B) preparing a denitrifying bacteria bacterial suspension: and taking a proper amount of thalli to be resuspended in sterilized water to obtain a bacterial suspension of denitrifying bacteria.
7. The method of claim 3, wherein the step of applying the coating comprises applying a coating to the substrateTalaromyces flavus S1The preparation method of the mycelium pellet comprises the following steps:
(a) preparation ofTalaromyces flavus S1The bacterial suspension of (a): getTalaromyces flavus S1Inoculating the slant culture medium into sterilized water containing glass beads, shaking in a shaking table, and counting blood cells to obtainTalaromyces flavus S1The spore bacterium suspension of (a);
(b) culturingTalaromyces flavus S1Mycelium pellets: and taking a proper amount of spore bacterium suspension, inoculating the spore bacterium suspension into a PDB culture medium, and performing shake culture for 3-5 days to obtain mature mycelium pellets.
8. The method according to claim 6, wherein the denitrification culture solution in the step (A) mainly comprises the following components: KNO3 2 g/L,C6H12O6 1.298 g/L,MgSO4·7H20.2g/L of O, 2ml/L of trace elements, 50ml/L of phosphate buffer (pH = 7.5), and the balance of water.
9. The method of claim 7, wherein the spore suspension in step (b) is inoculated in an amount of 10% by volume.
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