CN110669715A - Domestication and evaluation method for nitrobacteria flora in culture sewage - Google Patents

Domestication and evaluation method for nitrobacteria flora in culture sewage Download PDF

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CN110669715A
CN110669715A CN201910908774.7A CN201910908774A CN110669715A CN 110669715 A CN110669715 A CN 110669715A CN 201910908774 A CN201910908774 A CN 201910908774A CN 110669715 A CN110669715 A CN 110669715A
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梁晶晶
王熙涛
王金龙
张宗国
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QINGDAO SHANGDE BIOTECHNOLOGY Co Ltd
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Abstract

The invention relates to a method for domesticating and evaluating nitrobacteria flora in aquaculture sewage, which comprises the steps of carrying out enrichment culture on the aquaculture sewage by adopting an enrichment culture medium, carrying out passage domestication for 7-10 times, carrying out domestication for 7 days each generation, adding high-concentration ammonium salt and nitrite during the domestication, carrying out flora diversity analysis and evaluation by adopting PCR-DGGE in the passage process until the flora composition is stable, the highest ammonia oxidation rate is more than 30mg/L.d, and the highest nitrite oxidation rate is more than 150mg/L.d, thus obtaining the stable nitrobacteria flora. And finally, analyzing and evaluating the composition of the bacterial groups by adopting a high-throughput sequencing technology. The bacterial group is preserved by adopting a vacuum freeze drying method, and the obtained autotrophic and heterotrophic nitrobacteria are mixed and expanded during the expansion culture. The culture expanding liquid can be applied to a livestock and poultry breeding sewage treatment tank in a membrane hanging manner, so that the sewage treatment nitration process is effectively shortened, and the ammonia nitrogen in effluent is reduced.

Description

Domestication and evaluation method for nitrobacteria flora in culture sewage
Technical Field
The invention belongs to the technical field of sewage treatment, and particularly relates to a method for domesticating and evaluating nitrobacteria flora in aquaculture sewage.
Background
A large amount of sewage generated in the culture process is a great problem in the current culture pollution treatment. According to statistics in the first national pollution source census publication published in 2 months 2010, the livestock and poultry breeding manure is used as the main part of the agricultural pollution source, and the COD (chemical oxygen demand) and the ammonia nitrogen discharge amount of the livestock and poultry breeding manure are 1268.26 ten thousand t and 71.73 ten thousand t respectively, account for 96 percent and 38 percent of the total discharge amount of the agricultural pollution source, and account for 41.9 percent and 41.5 percent of the total COD and ammonia nitrogen discharge amount of the country. The removal of nitrogen and phosphorus in the livestock and poultry breeding sewage treatment is always the bottleneck of the breeding sewage treatment.
The key steps of sewage denitrification comprise nitrification and denitrification. Nitrification is the oxidation of ammonia to nitrates by microbial conversion. The process can be divided into two stages, namely, a nitrosation (ammonia oxidation) stage and a nitrification (nitrite oxidation) stage, which are respectively performed by two types of microorganisms, nitrite bacteria, namely, Ammonia Oxidizing Bacteria (AOB), oxidizing ammonia into nitrite, and nitrate bacteria, namely, Nitrite Oxidizing Bacteria (NOB), oxidizing nitrite into nitric acid. Since both AOB and NOB grew very slowly, the doubling time required 8h even for the fastest growing nitrosolonas europaea. The ammoxidation by AOB is the rate-limiting step in denitrification efficiency.
The nitrification process is not only completed by autotrophic bacteria, but heterotrophic bacteria can also participate in nitrification, i.e. heterotrophic nitrifiers and aerobic denitrifying bacteria exist. The autotrophic nitrifying bacteria are generally inhibited in a growth competitive disadvantage in an environment with high organic matter concentration and low dissolved oxygen, so that the removal effect of autotrophic nitrification is poor, but the heterotrophic nitrifying bacteria can grow in a large amount under the condition. Most researches are carried out by directionally enriching nitrobacteria and then separating and purifying to obtain a single nitrobacteria strain for expanded production, which is because AOB and NOB are slow in propagation and have multiple autotrophic strains, the actual efficiency of conventional industrial pure culture is very low, ammonia nitrogen is converted into nitrate in application, the synergistic effect of multiple nitrite bacteria and nitrate bacteria is needed, and the single strain for industrial pure culture cannot play a good effect. The development of a bacterial group (bacterial system) containing autotrophic nitrifying bacteria and heterotrophic nitrifying bacteria and the finding of a stable storage and effective use method thereof have great significance for biological denitrification of sewage.
Disclosure of Invention
The invention provides a method for domesticating and evaluating nitrobacteria flora in aquaculture sewage, aiming at the treatment of the aquaculture sewage. After enrichment culture is carried out on the culture sewage by adopting an enrichment culture medium, subculturing is carried out for 7-10 times at 30 ℃ and 180r/min, each generation of acclimation is carried out for 7 days, high-concentration ammonium salt and nitrite are added during the acclimation, and PCR-DGGE is adopted during the subculturing process to carry out analysis and evaluation on the diversity of the bacterial flora until the composition of the bacterial flora is stable, the highest ammonia oxidation rate is greater than 30mg/L.d, and the highest nitrite oxidation rate is greater than 150mg/L.d, so that the stable nitrobacteria bacterial flora is obtained. And finally, analyzing and evaluating the composition of the bacterial groups by adopting a high-throughput sequencing technology. The bacterial group is preserved by adopting a vacuum freeze drying method, and the obtained autotrophic and heterotrophic nitrobacteria are mixed and expanded during the expansion culture. The culture expanding liquid can be applied to a livestock and poultry breeding sewage treatment tank in a membrane hanging manner, so that the sewage treatment nitration process is effectively shortened, and the ammonia nitrogen in effluent is reduced.
In order to realize the purpose of the invention, the invention is realized by adopting the following technical scheme:
a method for domesticating and evaluating nitrobacteria flora in culture sewage comprises the steps of carrying out enrichment culture on the culture sewage by adopting an enrichment culture medium, carrying out passage domestication for 7-10 times, carrying out domestication for 7 days every generation, and adding high-concentration ammonium salt and nitrite during the domestication period until the flora composition is stable.
The directional enrichment medium comprises the following components: peptone 0.5g/L, yeast powder 1.0g/L, (NH)4)2SO40.05g/L、NaNO20.01g/L、MgSO4·7H2O 0.25g/L、KH2PO40.25g/L、NaCl 1.0g/L、CaCO33mg/L，pH 8.0±0.2，116℃20min。
Further, inoculating the enriched flora into an acclimatization culture medium according to the inoculum size of 10%, culturing at 30 ℃ for 7 days at 180r/min, transferring into a new acclimatization culture medium according to the inoculum size of 10%, carrying out passage acclimatization for 7-10 times, adding high-concentration ammonium salt and nitrite during 7 days of each generation of culture, and carrying out flora diversity analysis and evaluation by PCR-DGGE in the passage process until the flora composition is stable, the highest ammonia oxidation rate is more than 30mg/L.d, and the highest nitrite oxidation rate is more than 150 mg/L.d. And finally, analyzing and evaluating the composition of the bacterial groups by adopting a high-throughput sequencing technology.
The culture medium for training the nitrosation bacterial groups comprises the following components: peptone 0.5g/L, yeast powder 1.0g/L, (NH)4)2SO40.5g/L、NaNO21.0g/L、MgSO4·7H2O 0.25g/L、KH2PO40.25g/L、NaCl 1.0g/L、CaCO33mg/L。
Further, the bacterial groups need to be preserved by a vacuum freeze drying method, and the obtained autotrophic and heterotrophic nitrobacteria are mixed and expanded during the expansion culture. The culture expanding liquid can be applied to a livestock and poultry breeding sewage treatment tank in a membrane hanging manner, so that the sewage treatment nitration process is effectively shortened, and the ammonia nitrogen in effluent is reduced.
The invention has the advantages and technical effects that: the nitrifying bacteria flora is obtained through domestication and orientation of sludge or sewage, the stable nitrifying bacteria flora capable of efficiently treating nitrogen pollution of livestock and poultry breeding sewage is obtained, and the nitrifying bacteria flora is evaluated through a PCR-DGGE technology and a high-throughput sequencing technology, so that the diversity and the determined composition of the flora are determined.
Drawings
FIG. 1 is the nitrite change during the enrichment process in the examples.
FIG. 2 shows the ammonia nitrogen change during the enrichment process in the examples.
FIG. 3 is the nitrite degradation rate after directional acclimation in the examples.
FIG. 4 shows the degradation rate of ammoniacal nitrogen after the directional acclimation in the example.
FIG. 5 is the microbial diversity change during acclimatization passage of wastewater from Hui duck farm in Changle in example, wherein A is freshly taken wastewater; b is the enriched sewage; 1-10 is the passage frequency in the acclimatization process.
Detailed Description
The present invention is further described in detail with reference to the following specific examples in conjunction with the accompanying drawings.
Example 1 enrichment and acclimatization of the nitrifying bacterial flora
The enrichment medium used in the invention comprises the following components: peptone 0.5g/L, yeast powder 1.0g/L, (NH)4)2SO40.5g/L、NaNO20.1g/L、MgSO4·7H2O 0.25g/L、KH2PO40.25g/L、NaCl 1.0g/L、CaCO33mg/L，pH 8.0±0.2，116℃20min。
The domestication culture medium comprises the following components: peptone 0.5g/L, yeast powder 1.0g/L, (NH)4)2SO40.5g/L、NaNO21.0g/L、MgSO4·7H2O 0.25g/L、KH2PO40.25g/L、NaCl 1.0g/L、CaCO33mg/L，116℃20min。。
Simulating sewage: NaNO22g/L、MgSO4·7H2O50mg/L、CaCO33mg/L、KH2PO4150mg/L、FeSO4·7H2O 0.15mg/L、(NH4)2Mo7O24·4H2O50ug/L, NaCl 500mg/L (pH adjusted to 8.6).
The detection methods of ammonia nitrogen and nitrite nitrogen are a Nashin reagent method and a naphthyl ethylenediamine hydrochloride spectrophotometry respectively.
Screening the source of the sewage/sludge sample:
(1) the Qingdao root technology center levelness breeding laboratory breeding sewage;
(2) activated sludge in an aerobic tank of a Qingdao root-source fermentation sewage treatment system;
(3) 1 of the breeding wastewater of the pig farm in Qingdao, Jimo and Jiaozhou;
(4) weifang Changle Zhonghui duck farm cultivation sewage;
(5) the Qingdao root technology center red island culture laboratory culture sewage.
Respectively inoculating the collected sewage or sludge into a sterilized directional enrichment culture medium according to the inoculation amount of 10%, culturing at 30 ℃ for 180r/min, sampling at regular time, detecting the concentrations of ammonia nitrogen and nitrite nitrogen, culturing for 7 days, and taking 3 enrichment samples with the first removal rates of ammonia nitrogen and nitrite nitrogen as the directionally domesticated flora for reservation.
The results show that the first 3 ammonia nitrogen and nitrite nitrogen removal rates are as follows: no. 1 flat broiler breeding laboratory sewage, No. 2 root fermentation sewage treatment aerobic pond sludge and No. 4 Changle Zhonghui duck farm sewage (see the figure 1 and the figure 2).
Inoculating No. 1, No. 2 and No. 4 enrichment solutions into acclimation culture medium according to 10% inoculum size, respectively, culturing at 30 deg.C and 180r/min for 7 days per generation, inoculating into new acclimation culture medium according to 10% inoculum size, performing subculture for 7-10 times, and adding 1% 1000mg/L (NH) for each generation until 3 days4)2SO4Mother liquor and 1% 4000mg/LNaNO2And in the passage process, performing flora diversity analysis and evaluation through PCR-DGGE (polymerase chain reaction-denaturing gradient gel electrophoresis) until the flora diversity is not changed, and selecting a group with the highest ammonia oxidation rate of more than 30mg/L.d and the highest nitrite nitrogen oxidation rate of more than 150mg/L.d as a final nitrobacteria flora.
And (3) putting the bacterium group into the simulated sewage according to the addition amount of 1-5 ml/L, and tracking the change condition of ammonia nitrogen and nitrite. The measurement results of the ammonia nitrogen degradation rate and the nitrite degradation rate show that: no. 1 and No. 4, namely the highest ammonia oxidation rates of the bacterium groups obtained by domesticating the wastewater of the flat broiler chicken breeding laboratory and the wastewater of the Changle China coma duck farm are respectively 64.7mg/L.d and 45.8mg/L.d, and the highest nitrite nitrogen oxidation rates are respectively 164mg/L.d and 344mg/L.d, which are shown in a figure 3 and a figure 4.
And the bacterium group obtained by domesticating the sewage of the Hui duck farm in Changle No. 4 is an optimal nitrobacteria bacterium group.
The PCR-DGGE result shows that the nitrifying flora in the Hui-nationality duck farm in Changle No. 4 is stabilized when the 8 th generation is reached, and the figure is shown in figure 5. The 8 th and subsequent generation colonies were subjected to high throughput analysis, and the analysis results showed that Nitrosomonas and Nitrosospira in the colonies were typical ammonia oxidizing bacteria, Paracoccus marcusii was heterotrophic denitrifying bacteria, and Bradyrhizobiae _ unclassified was nitrite oxidizing bacteria, as shown in Table 1.
TABLE 1 high throughput sequencing analysis of the bacterial groups
Figure BDA0002213011090000051
The obtained nitrobacteria flora is preserved by adopting a vacuum freeze drying method, and mixed propagation is carried out during propagation. The culture expanding liquid can be applied to a livestock and poultry breeding sewage treatment tank in a membrane hanging manner, so that the sewage treatment nitration process is effectively shortened, and the ammonia nitrogen in effluent is reduced.
The above examples are only intended to illustrate the technical solution of the present invention, but not to limit it; although the present invention has been described in detail with reference to the foregoing embodiments, it will be apparent to those skilled in the art that various changes may be made and equivalents may be substituted for elements thereof; and such modifications or substitutions do not depart from the spirit and scope of the corresponding technical solutions.

Claims (6)

1. A method for domesticating and evaluating nitrobacteria flora in culture sewage is characterized by comprising the following steps: after enrichment culture is carried out on the culture sewage by adopting an enrichment culture medium, subculturing is carried out for 7-10 times, each generation of acclimation is carried out for 7 days, and high-concentration ammonium salt and nitrite are added during the acclimation period until the composition of the bacteria group is stable.
2. The nitrobacteria flora domestication and evaluation method of claim 1, characterized in that: the enrichment medium comprises the following components: peptone 0.5g/L, yeast powder 1.0g/L, (NH)4)2SO40.05g/L、NaNO20.01 g/L、MgSO4·7H2O 0.25g/L、KH2PO40.25 g/L、NaCl 1.0 g/L、CaCO33 mg/L, pH 8.0 +/-0.2, 20min at 116 ℃; the domestication culture medium comprises the following components: peptone 0.5g/L, yeast powder 1.0g/L, (NH)4)2SO40.5g/L、NaNO21.0g/L、MgSO4·7H2O 0.25g/L、KH2PO40.25 g/L、NaCl 1.0 g/L、CaCO33 mg/L ，pH 8.0±0.2，116℃ 20min。
3. The nitrifying bacteria group domesticated and evaluated method according to claim 1, wherein: and (3) performing flora diversity analysis and evaluation by adopting a PCR-DGGE (polymerase chain reaction-denaturing gradient gel electrophoresis) technology in the passage process, and performing flora composition analysis and evaluation on the obtained flora by adopting a high-throughput sequencing technology.
4. The nitrifying bacteria group domesticated and evaluated method according to claim 1, wherein: the nitrobacteria consists of: halomonas, nitrosomonas, corynebacterium, paracoccus mazei, nitrosospirillum, hydrogenophilum, bradyrhizobiaceae, pseudoxanthomonas, azotobacter, thermomonas.
5. The nitrobacteria group and the evaluation method according to claim 1, characterized in that: adding the bacterium group into the simulated sewage according to the addition amount of 1-5 ml/L.
6. The nitrobacteria group and the evaluation method according to claim 1, characterized in that: the degradation rate of ammonia nitrogen and the degradation rate of nitrite are respectively more than 30mg/L.d and 150 mg/L.d.
CN201910908774.7A 2019-09-24 2019-09-24 Domestication and evaluation method for nitrobacteria flora in culture sewage Pending CN110669715A (en)

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Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN111763632A (en) * 2020-04-01 2020-10-13 浙江省农业科学院 Autotrophic ammonia oxidizing bacteria and screening method thereof
CN113388544A (en) * 2021-06-17 2021-09-14 工大环境股份有限公司 Preparation method and application of solid composite bacterium block for biological sewage treatment
CN114031188A (en) * 2021-11-26 2022-02-11 南京高科环境科技有限公司 Method for biodegrading organic micropollutants

Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN111763632A (en) * 2020-04-01 2020-10-13 浙江省农业科学院 Autotrophic ammonia oxidizing bacteria and screening method thereof
CN113388544A (en) * 2021-06-17 2021-09-14 工大环境股份有限公司 Preparation method and application of solid composite bacterium block for biological sewage treatment
CN114031188A (en) * 2021-11-26 2022-02-11 南京高科环境科技有限公司 Method for biodegrading organic micropollutants
CN114031188B (en) * 2021-11-26 2022-08-23 南京高科环境科技有限公司 Method for biodegrading organic micropollutants

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Application publication date: 20200110