CN110452846B - Paracoccus denitrificans and biological denitrification method by combining paracoccus denitrificans with mineralization bed - Google Patents
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Abstract
The invention provides paracoccus denitrificans and a method for biological denitrification by combining the paracoccus denitrificans with a mineralization bed, and provides an autotrophic denitrification paracoccus denitrificans TNS-1 strain, mineralized garbage is selected as a main component of a packed bed, so that the resource utilization of waste is realized, the total nitrogen is removed by adopting a biological enhancement effect, the method is environment-friendly, has no secondary pollution, is low in treatment cost, and is a wastewater denitrification method with low energy consumption, environmental protection and low carbon.
Description
Technical Field
The invention relates to paracoccus denitrificans and further relates to a method for biological denitrification by combining paracoccus denitrificans with a mineralization bed, belonging to the field of sewage treatment.
Background
The mineralized refuse means that after being degraded in a refuse landfill for several years, easily degradable substances are completely or nearly completely degraded, the easily degradable substances basically reach a stable state, the surface sedimentation amount is very small, leachate and gas which are naturally generated are little or not generated, and the mass fraction of the biodegradable substances is reduced to below 3%. In the aspect of appearance composition, except large-particle inorganic substances and non-degradable organic substances such as plastics, glass, stones, animal bones and the like, the rest are black dispersed granular soil-like substances with uniform texture. The mineralized refuse has good porous structure and huge surface area, is attached with abundant microbial population, and is a good biological filler.
Since the last 90 s, numerous scholars have started intensive research on mineralized refuse, and the results show that mineralized refuse has good treatment effect on ammonia nitrogen, but the problem of overhigh nitrate nitrogen content in effluent is not solved, so that the total nitrogen of the effluent is high. One reason for this is that in the conventional heterotrophic denitrification process, organic substances are required to be additionally provided as a carbon source and an electron donor, and when the carbon source in the system is insufficient and the carbon-nitrogen ratio is too low, the denitrification process of nitrate nitrogen is hindered.
Paracoccus denitrificans is a facultative inorganic autotrophic gram-negative bacterium which can perform heterotrophic growth by utilizing organic matters such as various saccharides and amino acids and can also perform heterotrophic growth in H2、O2、CO2And carrying out autotrophic growth in the presence of partial inorganic salt. When no molecular oxygen exists in the system, the paracoccus denitrificans can utilize nitrate or nitrite as a final electron acceptor to carry out a denitrification process.
Disclosure of Invention
In order to solve the problem that low-carbon high-nitrogen wastewater is difficult to be subjected to conventional denitrification and reasonably utilize mineralized waste, the invention provides an autotrophic denitrifying paracoccus denitrificans TNS-1, provides an expanded culture method thereof, and combines the method with a mineralized waste packed bed to realize the removal of the total nitrogen in the high-nitrogen wastewater.
The invention adopts the following technical scheme:
the invention provides paracoccus denitrificans, which is characterized in that: paracoccus denitrificans (Paracoccus, Denitrificans) TNS-1 with the preservation number of CGMCC NO. 17156.
The invention also provides a method for biological denitrification by combining paracoccus denitrificans and a mineralization bed, which is characterized by comprising the following steps:
the method comprises the following steps: strain culture
Primary culture medium: 5.0g/L peptone, 1.0g/L potassium nitrate, pH 7.0, high pressure steam sterilizing at 121 deg.C for 20min,
secondary culture medium: na (Na)2S2O3·5H2O 5.0，KNO3 1.0g/L，K2HPO4 2.0g/L，NaHCO3 1.0g/L，MgCl2·6H2O 0.5g/L，FeSO4·7H2O0.01 g/L, adjusted to a predetermined pH,
taking activated paracoccus denitrificans (Paracoccus) TNS-1 strain with the preservation number of CGMCC NO:17156, aseptically inoculating the activated paracoccus denitrificans (Paracoccus) TNS-1 strain into a primary culture medium according to the inoculation amount of 1%, carrying out shaking culture at 30 ℃ and 120r/min for 24-48h, taking the primary strain, inoculating the primary strain into a secondary culture medium according to the inoculation amount of 1-2%, carrying out expanding culture, and carrying out standing culture for 5-7d to obtain a secondary expanded culture solution;
step two: packed bed preparation
Pretreating mineralized garbage: the mineralized refuse is screened to remove large particle substances such as glass, metal, plastic, stone and the like to obtain a soil-like material with the particle size of less than 30mm,
the denitrification packed bed comprises the following components in percentage by mass: 60 percent of mineralized refuse, 20 percent of sulfur particles and 20 percent of particle activated carbon, the components are evenly mixed according to the proportion and are filled into a filling tank,
step three: strain adsorption
Filling the expanded bacteria liquid into a filler tank until the top layer of the denitrification filler bed is just submerged, keeping the submerged state for 3-5 days until the strains are naturally adsorbed on the surface of the filler, wherein the filler tank after the strains are adsorbed is called a denitrification filler tank,
step four: oxygen removal
Before entering the denitrification filler tank, the high-nitrogen wastewater passes through a filter material tank filled with sponge iron to remove molecular oxygen, so that the ORP of the effluent is kept below-100 mv,
step five: denitrification operation
In the denitrification filling tank, equal amount of wastewater is respectively fed in and out every day.
The method for biological denitrification by combining paracoccus denitrificans with the mineralization bed further has the following characteristics: wherein the denitrification operation temperature of the fifth step is 10-50 ℃.
The method for biological denitrification by combining paracoccus denitrificans and a mineralization bed further has the following characteristics: wherein the denitrification operation temperature in the fifth step is 30 ℃.
The method for biological denitrification by combining paracoccus denitrificans and a mineralization bed further has the following characteristics: in the first step, the predetermined pH is 3.0 to 11.0.
The method for biological denitrification by combining paracoccus denitrificans with the mineralization bed further has the following characteristics: in the first step, the predetermined pH is 6.0 to 8.0.
Advantageous effects of the invention
The invention provides an autotrophic denitrification denitrogenation paracoccus TNS-1 strain, mineralized refuse is selected as a main component of a packed bed, resource utilization of waste is realized, total nitrogen is removed by adopting a biological strengthening effect, and the method is environment-friendly, free of secondary pollution and low in treatment cost, and is a low-energy-consumption, environment-friendly and low-carbon wastewater denitrification method.
Drawings
FIG. 1(a) is a graph of the effect of different temperatures on growth;
FIG. 1(b) is the effect of different pH on growth;
FIG. 2(a) is a graph showing the effect of different temperatures on denitrification;
FIG. 2(b) is a graph of the effect of different pH on denitrification;
FIG. 3 is the results of a pilot plant total nitrogen removal;
figure 4 is the results of pilot plant total nitrogen removal.
Paracoccus denitrificans (paracoccus. Denitrificans) TNS-1 is preserved in the China general microbiological culture Collection center (address: Beijing, West Lu No. 1, North Chen, Xilu, No. 3, the institute of microbiology, China academy of sciences, postal code: 100101) in 2019, 1 month and 10 days, and the preservation number is CGMCC NO: 17156.
Detailed Description
The following describes embodiments of the present invention with reference to the drawings.
Example 1
The culture method of paracoccus denitrificans TNS-1 provided by the invention is determined by the following steps:
1. strain activation
Seed medium (g/L): peptone 5.0, potassium nitrate 1.0, pH 7.0. Sterilizing with high pressure steam at 121 deg.C for 20 min.
Denitrogenation medium (g/L): na (Na)2S2O3·5H2O 5.0，KNO3 1.0，K2HPO4 2.0，NaHCO3 1.0，MgCl2·6H2O 0.5，FeSO4·7H2O0.01, and PH 8.0-8.5. Sterilizing with high pressure steam at 121 deg.C for 20 min.
Inoculating a loop of preserved Paracoccus denitrificans (Paracoccus, Denitrificans) TNS-1 slant strain into a sterilized seed culture medium under aseptic condition, and performing shake culture at 30 ℃ and 120r/min for 24-48h to obtain the strain.
2. Growth Condition test
1) Effect of different temperatures on growth
Inoculating the activated strain into a seed culture medium, wherein the inoculation amount is 1%, and the shake culture is carried out at 30 ℃ and 120 r/min. Respectively selecting 5 culture temperatures of 10 ℃, 20 ℃, 30 ℃, 40 ℃ and 50 ℃, measuring the absorbance change of the bacterial liquid, and inspecting the influence of different temperatures on the growth of the TNS-1 strain. As a result, as shown in FIG. 1(a), a suitable growth temperature for Paracoccus denitrificus (Paracoccus. Denitrificans) TNS-1 was 30-40 ℃.
2) Effect of different pH on growth
Inoculating the activated strain into a seed culture medium, wherein the inoculation amount is 1%, and the shake culture is carried out at 30 ℃ and 120 r/min. Respectively setting pH values of 3.0, 4.0, 5.0, 6.0, 7.0, 8.0, 9.0, 10.0 and 11.0, and totally 9 pH conditions, measuring absorbance change of the bacterial liquid, and examining the influence of different pH values of the culture liquid on the growth of the strain TNS-1. As a result, as shown in FIG. 1(b), Paracoccus denitrificus (Paracoccus) TNS-1 was suitably grown in a neutral environment, and the optimum pH range was 6.0 to 8.0.
3. Test of Denitrification Condition
1) Influence of different temperatures on nitrogen removal
Inoculating the seeds into denitrification culture medium according to the inoculation amount of 1%, respectively selecting culture temperatures of 10 ℃, 20 ℃, 30 ℃, 40 ℃ and 50 ℃ for 5 times, and standing for culture. And measuring the change of total nitrogen, and inspecting the influence of different temperatures on the denitrification performance of the TNS-1 strain. As a result, as shown in FIG. 2(a), the optimum temperature for denitrification of Paracoccus denitrificans (Paracoccus. Denitrificans) TNS-1 was 30 ℃.
2) Effect of different initial pH on Denitrification
Inoculating the seeds into a denitrification culture medium according to the inoculation amount of 1 percent, respectively setting the initial pH of the culture medium to be 3.0, 4.0, 5.0, 6.0, 7.0, 8.0, 9.0, 10.0 and 11.0, and keeping the culture medium still at 30 ℃ under the conditions of 9 pH. And measuring the total nitrogen change, and inspecting the influence of different initial pH values on the denitrification performance of the TNS-1 strain. As a result, as shown in FIG. 2(b), paracoccus denitrificans (Paracoccus. Denitrificans) TNS-1 showed the best denitrification effect at an initial pH of 8.0.
Example 2
Denitrification bench scale
1. Culture medium
Primary medium (g/L): peptone 5.0, potassium nitrate 1.0, pH 7.0. Sterilizing with high pressure steam at 121 deg.C for 20 min.
Secondary medium (g/L): na (Na)2S2O3·5H2O 5.0，KNO3 1.0，K2HPO4 2.0，NaHCO3 1.0，MgCl2·6H2O 0.5，FeSO4·7H2O 0.01，pH 8.0。
2. Strain culture
Taking activated paracoccus denitrificans (Paracoccus, Denitrificans) TNS-1 strain, aseptically inoculating into a primary culture medium according to 1% of inoculation amount, and carrying out shake culture at 30 ℃ and 120r/min for 24-48 h. Taking the first-stage strain, inoculating to the second-stage culture medium according to 1-2% of inoculation amount, performing amplification culture, and performing static culture for 5-7 days.
3. Strain adsorption
And pouring the expanded bacteria liquid into a filler tank until the top layer of the filler is just submerged, and keeping the submerged state for 3-5 days until the strains are naturally adsorbed on the surface of the filler.
4. Packed bed preparation
Pretreating mineralized garbage: the mineralized refuse comes from domestic refuse landfill in Shanghai old harbor and is more than 7 years old in heap. The excavated mineralized refuse is screened to remove large particle substances such as glass, metal, plastic, stone and the like, and the soil-like material with the particle size of less than 30mm is obtained.
The denitrification packed bed comprises the following components: 60% of mineralized refuse, 20% of sulfur particles and 20% of particle activated carbon. Mixing the above materials at a certain proportion, and packaging into a can.
5. Oxygen removal
Before denitrification, a high-nitrogen wastewater sample passes through a filter material tank filled with sponge iron particles to remove molecular oxygen, so that the ORP of effluent is kept below-100 mv.
6. Denitrification bench scale
High nitrogen wastewater quality: CODCr344 mg/L; 29mg/L ammonia nitrogen; TN 1466 mg/L.
2 glass jars with 5L small openings are taken, and a water drain valve is arranged close to the bottom of the jar. The No. 1 can is filled with the components of the denitrification packed bed, and is added with the paracoccus denitrificans TNS-1 secondary expanded culture solution, and the liquid level is stabilized until the surface of the filler is submerged. The tank No. 2 is only filled with mineralized refuse materials, no bacteria liquid is added, the high-nitrogen wastewater after equivalent deoxidation is added, the liquid level is enabled to submerge the surface of the filler after stabilization, and the tank No. 2 is used as a reference.
The tanks No. 1 and No. 2 respectively feed in and discharge the same amount of waste water every day. The hydraulic retention time was about 5 days.
As a result, as shown in FIG. 3, the total nitrogen removal rate of the effluent of the denitrification tank No. 1 after stable operation was about 80%. No. 2 control tank has no obvious removal of total nitrogen and no denitrification effect.
Example 3
Denitrification pilot scale test
1. Culture medium
Primary medium (g/L): peptone 5.0, potassium nitrate 1.0, pH 7.0. Sterilizing with high pressure steam at 121 deg.C for 20 min.
Secondary medium (g/L): na (Na)2S2O3·5H2O 5.0，KNO3 1.0，K2HPO4 2.0，NaHCO3 1.0，MgCl2·6H2O 0.5，FeSO4·7H2O 0.01，pH 8.0。
2. Strain culture
Taking activated paracoccus denitrificans (Paracoccus, Denitrificans) TNS-1 strain, aseptically inoculating into a primary culture medium according to 1% of inoculation amount, and carrying out shake culture at 30 ℃ and 120r/min for 24-48 h. Taking the first-stage strain, inoculating to the second-stage culture medium according to 1-2% of inoculation amount, performing propagation culture, and performing static culture for 5-7 d.
3. Strain adsorption
And pouring the expanded bacteria liquid into a filler tank until the top layer of the filler is just submerged, and keeping the submerged state for 3-5 days until the strains are naturally adsorbed on the surface of the filler.
4. Preparation of packed bed
Pretreating mineralized garbage: the mineralized refuse comes from domestic refuse landfill in Shanghai old harbor and is more than 7 years old in heap. The excavated mineralized refuse is screened to remove large particle substances such as glass, metal, plastic, stone and the like, and the soil-like material with the particle size of less than 30mm is obtained. In other embodiments, the heap age is not particularly limited as long as mineralized waste is formed.
The denitrification packed bed comprises the following components: 60% of mineralized refuse, 20% of sulfur particles and 20% of particle activated carbon. Mixing the above components at a certain proportion, and packaging into a can.
5. Oxygen removal
Before denitrification, a high-nitrogen wastewater sample passes through a filter material tank filled with sponge iron particles to remove molecular oxygen, so that the ORP of effluent is kept below-100 mv.
6. Denitrification pilot scale test
High nitrogen wastewater quality: CODCr344 mg/L; 29mg/L ammonia nitrogen; TN 1466 mg/L.
A50L polyethylene tank is taken, a water distributor is arranged at the tank opening, and water flows upwards and downwards. And filling the components of the denitrification packed bed in the tank, adding a paracoccus denitrificans TNS-1 secondary expanded culture solution, and stabilizing to ensure that the liquid surface is submerged on the surface of the packing.
The wastewater with equal amount of high nitrogen is fed in and discharged every day. The hydraulic retention time was about 5 days.
As shown in FIG. 4, the average removal rate of total nitrogen in the test water during denitrification was about 75% when the operation time was about 1 month. The denitrification effect is obvious.
The test is continuously operated for two months, and the results show that after the operation is stable, the total nitrogen removal rate is stable at 71-79%, and the average total nitrogen removal rate is 75%.
Claims (3)
1. A method for biological denitrification by combining paracoccus denitrificans with a mineralization bed is characterized by comprising the following steps:
the method comprises the following steps: strain culture
Primary culture medium: 5.0g/L peptone, 1.0g/L potassium nitrate, pH 7.0, high pressure steam sterilizing at 121 deg.C for 20min,
secondary culture medium: na (Na)2S2O3·5H2O 5.0，KNO3 1.0g/L，K2HPO4 2.0g/L，NaHCO3 1.0g/L，MgCl2·6H2O 0.5g/L，FeSO4·7H2O0.01 g/L, adjusted to a predetermined pH,
taking activated paracoccus denitrificans (Paracoccus) TNS-1 strain with the preservation number of CGMCC NO:17156, aseptically inoculating the activated paracoccus denitrificans (Paracoccus) TNS-1 strain into a primary culture medium according to the inoculation amount of 1%, carrying out shaking culture at 30 ℃ and 120r/min for 24-48h, taking the primary strain, inoculating the primary strain into a secondary culture medium according to the inoculation amount of 1-2%, carrying out expanding culture at the temperature of 30-40 ℃, and carrying out standing culture for 5-7d to obtain a secondary expanded culture solution;
step two: packed bed preparation
Pretreating mineralized garbage: the mineralized refuse is screened to remove large particle substances such as glass, metal, plastic, stone and the like to obtain a soil-like material with the particle size of less than 30mm,
the denitrification packed bed comprises the following components in percentage by mass: 60 percent of mineralized refuse, 20 percent of sulfur particles and 20 percent of particle activated carbon, the components are evenly mixed according to the proportion and are filled into a filling tank,
step three: strain adsorption
Filling the expanded bacteria liquid into a filler tank until the top layer of the denitrification filler bed is just submerged, keeping the submerged state for 3-5 days until the strains are naturally adsorbed on the surface of the filler, wherein the filler tank after the strains are adsorbed is called a denitrification filler tank,
step four: oxygen removal
Before entering the denitrification filler tank, the high-nitrogen wastewater passes through a filter material tank filled with sponge iron to remove molecular oxygen, so that the ORP of the effluent is kept below-100 mv,
step five: operation of denitrification
In a denitrification filling tank, equal amount of wastewater is respectively fed in and out every day, the hydraulic retention time is 5 days, and the denitrification operation temperature in the step is carried out at 30 ℃.
2. The method of claim 1, wherein the biological denitrification is performed by using paracoccus denitrificans in combination with a mineralization bed, the method comprising:
in the first step, the predetermined pH is 3.0 to 11.0.
3. The method of claim 2, wherein the biological denitrification is performed by using paracoccus denitrificans in combination with a mineralization bed, the method comprising:
in the first step, the predetermined pH is 6.0 to 8.0.
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