CN116286467A - Heterotrophic nitrification-aerobic denitrification bacterium and application thereof - Google Patents
Heterotrophic nitrification-aerobic denitrification bacterium and application thereof Download PDFInfo
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- CN116286467A CN116286467A CN202211730915.9A CN202211730915A CN116286467A CN 116286467 A CN116286467 A CN 116286467A CN 202211730915 A CN202211730915 A CN 202211730915A CN 116286467 A CN116286467 A CN 116286467A
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Abstract
The invention provides heterotrophic nitrification-aerobic denitrification bacteria and application thereof, and relates to the technical field of environmental microorganisms. The bacteria is Pseudomonas sp.ID4, the strain preservation number is CGMCCNo.24224, the preservation address is China institute of microbiology, national academy of sciences No.3, north Chen West Lu No.1, the Korean area of Beijing, and the preservation date is 2021, 12 months and 30 days. The invention also provides application of Pseudomonas sp.ID4 in sewage treatment, and the bacteria have heterotrophic nitrification-aerobic denitrification, so that the technical problem of nitrite and nitrate accumulation in the existing sewage denitrification process can be effectively solved.
Description
Technical Field
The invention relates to the technical field of environmental microorganisms, in particular to heterotrophic nitrification-aerobic denitrification bacteria and application thereof.
Background
The nitrification and denitrification processes of the traditional denitrification technology are non-uniform in time and space, comprise two independent stages of aerobic and anoxic, have relatively large required capital area and investment cost, are acid-producing processes, and require alkaline substances for neutralization, so that the operation cost is increased. The existing heterotrophic nitrification-aerobic denitrification bacteria can perform nitrification and denitrification processes in one reactor without adding a buffer to adjust the pH of water, so that the facility operation control is simpler and more convenient, the alkali addition amount and the occupied area of a building are greatly reduced, the method has better economy, the method gradually becomes a research hot spot in the field of sewage treatment, and the related research on the heterotrophic nitrification-aerobic denitrification bacteria still needs to be further in depth.
Disclosure of Invention
The first aim of the invention is to provide heterotrophic nitrification-aerobic denitrification bacteria, which can effectively solve the technical problem of nitrite and nitrate accumulation in the existing sewage denitrification process.
The second object of the invention is to provide an application of heterotrophic nitrification-aerobic denitrification bacteria ID4 in sewage treatment.
The third object of the invention is to provide an application of heterotrophic nitrification-aerobic denitrification bacteria ID4 in preparing a sewage treatment reagent.
A fourth object of the present invention is to provide a sewage treatment reagent comprising the heterotrophic nitrification-aerobic denitrification bacterium ID4.
The invention solves the technical problems by adopting the following technical scheme:
in one aspect, the application provides heterotrophic nitrification-aerobic denitrifying bacteria named heterotrophic nitrification-aerobic denitrifying bacteria pseudomonsp.ID4, wherein the preservation number of the heterotrophic nitrification-aerobic denitrifying bacteria pseudomonsp.ID4 is CGMCC No.24224, and the preservation address is China academy of sciences of national institute of sciences of North Chenxi road No.1 in the Korean region of Beijing city, and the preservation date is 2021, 12 months and 30 days. The bacteria are gram negative bacteria, have no flagella and spores, can effectively remove ammonia nitrogen, nitric acid nitrogen and nitrous acid nitrogen in water, can simultaneously remove COD in organic wastewater, and do not produce nitrite and nitrate accumulation in the denitrification process.
In a second aspect, the application also provides an application of the heterotrophic nitrification-aerobic denitrification bacteria ID4 in sewage treatment.
In a third aspect, the application also provides an application of heterotrophic nitrification-aerobic denitrification bacteria ID4 in preparing a sewage treatment reagent.
In a fourth aspect, the present application also provides a sewage treatment reagent comprising the heterotrophic nitrification-aerobic denitrification bacteria ID4.
Compared with the prior art, the invention has at least the following advantages or beneficial effects:
the invention provides heterotrophic nitrification-aerobic denitrification bacteria, which are named heterotrophic nitrification-aerobic denitrification bacteria Pseudomonas sp.ID4, are gram-negative bacteria, have no flagella and spores, can effectively remove ammonia nitrogen, nitric acid nitrogen and nitrous acid nitrogen in water, can remove COD in organic wastewater at the same time, and do not produce nitrite and nitrate accumulation in the denitrification process.
Drawings
In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings that are needed in the embodiments will be briefly described below, it being understood that the following drawings only illustrate some embodiments of the present invention and therefore should not be considered as limiting the scope, and other related drawings may be obtained according to these drawings without inventive effort for a person skilled in the art.
FIG. 1 is a photograph of a culture medium of Pseudomonas sp.ID4 strain of example 1 of the present invention;
FIG. 2 is a photograph of a gram-stained microscope of Pseudomonas sp.ID4 strain of example 1 according to the present invention;
FIG. 3 is a diagram showing the evolutionary tree of the strain Pseudomonas sp.ID4 of example 2 of the present invention;
FIG. 4 is a graph showing the nitrifying performance of Pseudomonas sp.ID4 strain in example 3 according to the present invention;
FIG. 5 is a graph I of denitrification performance of Pseudomonas sp.ID4 strain in example 4 of the present invention;
FIG. 6 is a graph II of denitrification performance of Pseudomonas sp.ID4 strain in example 4 according to the present invention.
Detailed Description
In order to make the objects, technical solutions and advantages of the embodiments of the present invention more clear, the technical solutions of the embodiments of the present invention will be clearly and completely described below. The specific conditions are not noted in the examples and are carried out according to conventional conditions or conditions recommended by the manufacturer. The reagents or apparatus used were conventional products commercially available without the manufacturer's attention.
It should be noted that, in the case of no conflict, the embodiments and features in the embodiments may be combined with each other. The present invention will be described in detail with reference to specific examples.
In one aspect, the application provides heterotrophic nitrification-aerobic denitrifying bacteria named heterotrophic nitrification-aerobic denitrifying bacteria pseudomonsp.ID4, wherein the preservation number of the heterotrophic nitrification-aerobic denitrifying bacteria pseudomonsp.ID4 is CGMCC No.24224, and the preservation address is China academy of sciences of national institute of sciences of North Chenxi road No.1 in the Korean region of Beijing city, and the preservation date is 2021, 12 months and 30 days. The bacteria are gram negative bacteria, have no flagella and spores, can effectively remove ammonia nitrogen, nitric acid nitrogen and nitrous acid nitrogen in water, can simultaneously remove COD in organic wastewater, and do not produce nitrite and nitrate accumulation in the denitrification process.
In a second aspect, the application also provides an application of heterotrophic nitrification-aerobic denitrification bacteria pseudomonasp.ID4 in sewage treatment.
In a third aspect, the application also provides an application of heterotrophic nitrification-aerobic denitrification bacteria pseudomonasp.ID4 in preparing a sewage treatment reagent.
In a fourth aspect, the present application also provides a sewage treatment reagent, including the heterotrophic nitrification-aerobic denitrification bacterium Pseudomonas sp.id4.
The separation and purification method of heterotrophic nitrification-aerobic denitrification bacteria Pseudomonas sp.ID4 comprises the following steps of inoculating activated sludge and SNDM culture solution into a shake flask for enrichment and domestication culture, standing the culture solution, mixing supernatant with the SNDM culture solution, performing domestication culture for 7d, and measuring ammonia nitrogen in the culture solution to basically remove ammonia nitrogen, so that the enrichment of heterotrophic nitrification-aerobic denitrification target flora is improved after the target flora is gradually adapted to become dominant flora, and the purpose of enrichment and domestication is achieved. Taking the enriched and domesticated flora culture solution in the shake flask, sequentially carrying out gradient dilution with sterile water at 10 times concentration, and taking 0.1mL of diluted volume fraction as 10 -6 ～10 -7 Is coated on a BTB screening plate. The temperature of the constant temperature incubator was set at 30℃and cultivation was performed for 20 hours. Before inoculation and culture, the BTB culture medium is yellow-green, and turns blue after 20 hours of inoculation and culture, various bacteria grow at different speeds in the culture process, and the culture stage is observed to select colonies which grow at a high speed and have blue colony positions. And inoculating different efficient aerobic denitrification strains obtained by preliminary screening into HNM culture fluid, and measuring heterotrophic nitrification performance. Before inoculation, 100mL of HNM culture medium is placed in a 250mL shaking flask, and high-temperature high-pressure sterilization is carried out. Sealing with high temperature resistant ventilation filter membrane (0.22 μm) to ensure ventilation of the interior and exterior of shake flask and create aerobic condition for microorganism in shake flask; in addition, can guarantee that after the air exchange, shake the inside air of bottle and keep aseptic state all the time. Inoculating on aseptic console, shake culturing in shake incubator at 25deg.C and rotation speed of 120r/min for 24 hr, and reacting with NH in HNM reaction system 4 + -N and COD are used as indexes, and NH is selected 4 + The strain with higher removal rate of N and COD is determined to have heterotrophic nitrification function. Preferred strains are found in the following detection assaysShort-term storage was performed on slant medium. In addition, the separation and purification method is simple and convenient and is easy to operate. The activated sludge was collected from the beijing poing Zhuang Zhenwu water treatment plants each, which employed the ao+mbr process. The volume ratio of the activated sludge to the SNDM culture medium is 1:9. The proportion interval can ensure that heterotrophic nitrification-aerobic denitrification bacteria Pseudomonas sp.ID4 in the activated sludge can obtain sufficient nutrition in the SNDM culture medium, so that the subsequent separation from other miscellaneous bacteria and the like is facilitated, when the proportion is lower than the proportion interval, the enrichment difficulty of the heterotrophic nitrification-aerobic denitrification bacteria Pseudomonas sp.ID4 is increased, and when the proportion interval is higher than the proportion interval, the growth condition of the heterotrophic nitrification-aerobic denitrification bacteria Pseudomonas sp.ID4 is limited.
The temperature of the enrichment culture is 20-30 ℃, and the rotating speed of the enrichment culture is 100-125 rpm. The temperature interval and the rotating speed interval can ensure constant-temperature growth and large-scale propagation of heterotrophic nitrification-aerobic denitrification bacteria ID4, and when the temperature interval and the rotating speed interval are lower than or higher than the interval, the propagation of the heterotrophic nitrification-aerobic denitrification bacteria ID4 is limited.
The enrichment and domestication culture is to take 20mL of active sludge with different sampling points respectively, inoculate the active sludge in 180mL of SNDM culture solution shaking bacteria bottle, and carry out the enrichment and domestication culture under the conditions of 120r/min and 25 ℃. The culture period is 3d,2d,1d and 1d in turn. After each period, about 180mL of the culture solution in the shaking flask is poured out, and then new SNDM culture solution is added again, and the culture is continued, wherein the domestication process lasts for 7d. The ammonia nitrogen in the culture solution is basically removed through measurement, which shows that the target flora of heterotrophic nitrification-aerobic denitrification is gradually adapted and then the abundance is increased to become dominant flora, thereby achieving the purpose of enrichment and domestication. The culture medium is replaced for a plurality of times to ensure that sufficient propagation conditions can be provided for heterotrophic nitrification-aerobic denitrification bacteria Pseudomonas sp.ID4 in the culture process, so that the heterotrophic nitrification-aerobic denitrification bacteria Pseudomonas sp.ID4 can be propagated in a large scale for enrichment, separation and purification.
The SNDM medium, BTB medium and HNM medium comprise Na 2 HPO 4 ·12H 2 O3g/L、KH 2 PO 4 1g/L、NaCl 0.12g/L、MgSO 4 ·7H 2 O0.1 g/L and 2ml of trace elements, and the SNDM medium also comprises C 6 H 5 Na 3 O 7 ·3H 2 O 6g/L、(NH 4 ) 2 SO 4 0.5g/L and KNO 3 The BTB medium also includes C at 0.2g/L 6 H 5 Na 3 O 7 ·3H 2 O 2.61g/L、KNO 3 1ml of solution of 0.58g/L and BTB, HNM medium further comprising C 6 H 5 Na 3 O 7 2.61g/L and (NH) 4 ) 2 SO 4 The SNDM medium, BTB medium and HNM medium had pH of 6.0 at 0.708 g/L. The heterotrophic nitrification-aerobic denitrification bacteria Pseudomonas sp.ID4 can be enriched and domesticated by the culture of the SNDM culture medium, the strain of the heterotrophic nitrification-aerobic denitrification bacteria Pseudomonas sp.ID4 can be rapidly screened by the characteristic that the BTB culture medium turns blue under alkaline conditions, and the heterotrophic nitrification-aerobic denitrification bacteria Pseudomonas sp.ID4 can be repeatedly hybridized by using the HNM culture medium for separation and purification.
The microelements comprise EDTA 50g/L, (NH) 4 ) 6 Mo 7 O 2 ·4H 2 O 1.10g/L、CaCl 2 5.50g/L、MnCl 2 ·4H 2 O 5.06g/L、CoCl 2 ·4H 2 O 1.61g/L、ZnSO 4 ·7H 2 O 3.92g/L、CuSO 4 ·5H 2 O1.57 g/L and FeSO 4 ·7H 2 O 5.0g/L。
The features and capabilities of the present invention are described in further detail below in connection with the examples.
Example 1
The heterotrophic nitrifying-aerobic denitrifying bacteria Pseudomonas sp.ID4 is inoculated on a solid medium with trisodium citrate as a carbon source, the strain Pseudomonas sp.ID4 is white, as shown in FIG. 1, a microscopic photograph after gram staining is shown in FIG. 2, the bacterial cells are observed to be rod-shaped, the size is about 1 μm×0.4 μm, and no flagellum and spores are found.
Example 2
The 16S rDNA gene was amplified by Polymerase Chain Reaction (PCR) using universal primer 27F/1492R. The PCR reaction conditions are shown in Table 1. Sequence analysis and homology comparisons were performed using the online BLAST program of the NCBI database (http:// www.ncbi.nlm.nih.gov/BLAST. Cgi). In the MEGAX program, a phylogenetic tree is constructed by adopting a neighbor-joining (NJ) method of 1000 bootstrap repeats and a maximum composite likelihood model.
Strain ID4 was sequenced and deposited in GenBank (accession No. OL 655389). A phylogenetic tree was constructed from the 16SrDNA gene sequence of this strain and a part of the phylogenetic-related strain (FIG. 3). The results showed that the strain ID4 had the closest relationship with Pseudomonas sp.strain WM5 of Pseudomonas pseudoalcaligenes (similarity 99.93%). Thus, strain ID4 was identified as a Pseudomonas species.
The 16S rDNA sequence of the strain Pseudomonas sp.ID4 is shown in SEQ ID NO.1, the sequence of the 27F primer is shown in SEQ ID NO.2, the sequence of the 1492R primer is shown in SEQ ID NO.3, and details are shown in Table 2.
TABLE 1PCR conditions
TABLE 2 heterotrophic nitrification-aerobic denitrification bacteria 16SrDNA sequence of Pseudomonas sp.ID4 and primer sequence table used
Example 3
The denitrification performance of the culture medium is studied by taking ammonia nitrogen (HNM), nitrate nitrogen (DM) and nitrite Nitrogen (NDM) as the only nitrogen sources, wherein the DM culture medium comprises common components of SNDM culture medium, BTB culture medium and HNM culture medium and C 6 H 5 Na 3 O 7 2.61g/L and KNO 3 0.722g/L, the NDM medium includes SNDM medium, BTB medium and HNM mediumThe composition also comprises NaNO 2 0.394g/L and C 6 H 5 Na 3 O 7 2.61g/L. The selected strain was inoculated in 5mL of a sterile LB liquid medium and cultured in a shaker at 25℃and 120r/min to logarithmic phase (OD 600 =0.8 to 1.0). Centrifuging the bacterial liquid at 4000r/min for 10min, washing with sterile water for 2-3 times, and preparing into bacterial suspension. The seed solution was sterilized at high temperature and high pressure (103.4 kPa,121.3 ℃) by volume 5% and inoculated into 250mL shake flasks containing 150mL HNM, DM, NDM. In order to ensure the exchange of air in the bottle and external air, a high-temperature-resistant permeable sealing film is adopted to replace a rubber plug for sealing. The experiment was set with a blank control group and no inoculation was performed. Bacterial density (OD) was measured at 6h, 12h, 18h, 24h, 30h, 36h, 42h and 48h, respectively 600 ). Analysis of TN, NH in the supernatant after centrifugation 4 + -N、NO 3 - -N and NO 2 - The growth ability and denitrification performance of the strain were examined for the content of N, and the results are shown in FIG. 4. According to the results shown in FIG. 4, the heterotrophic nitrification ability of strain ID4 was measured in HNM medium using ammonium sulfate as a nitrogen source and sodium citrate as a carbon source. When the strain ID4 is inoculated into a new environment, a period of time (0-6 h) is required to complete the synthesis of various enzymes in the body, the growth speed is slow, and the growth curve is relatively flat (adaptation period). After 6h inoculation, strain ID4 enters the logarithmic growth phase. OD (optical density) 600 The value increased from 0.114 to 1.38 with an average growth rate of 0.11h -1 . The nitrification of the strain ID4 mainly occurs in the period, the ammonia nitrogen concentration is rapidly reduced from 149.2mg/L to 16.32mg/L for 18 hours, and the removal rate is 7.38 mg/L.h. When the removal rate reaches basic stability (stationary phase), the highest removal rate of ammonia nitrogen in 48 hours reaches 97.7 percent. The results indicate that ammonia removal is consistent with cell growth. When the cultivation time is 36h, NH 4 + At this time, bacteria enter an endogenous respiration stage, and nitrogen sources synthesized by dead bacteria cells are decomposed into organic nitrogen due to lack of carbon sources and the like, and released into water again, so that the content of nitrified products is low, and the autolysis speed of degradation bacteria is high, thereby leading to NH 4 + -an increase in N concentration. In the whole process, TN removal trend and NH 4 + The removal trend of N is uniform. To track the strainNH in ID4 denitrification process 4 + The conversion pathway of-N, the nitration intermediate NO was detected experimentally 3 - -N and NO 2 - -a process of change of N. As can be seen from FIG. 4, NO 3 - -N and NO 2 - N does not accumulate during the entire nitration, presumably NH 4 + N is directly assimilated by strain ID4 for cell synthesis and converted to N by heterotrophic nitrification and aerobic denitrification 2 。
Example 4
Based on example 3, denitrification of strain ID4 under aerobic conditions was determined using only nitrate intermediate nitrate nitrogen (DM) and Nitrite (NDM) as nitrogen sources. The results obtained are shown in FIGS. 5 and 6, and strain ID4 was subjected to denitrification under aerobic conditions. NO (NO) 3 - -N and NO 2 - The change rule of the concentration of N is similar, and the rate of the N is closely related to the removal rate of the nitrogen-containing matrix.
According to the results shown in FIG. 5, strain ID4 was inoculated with DM and then, after the adaptation phase, entered the logarithmic phase. NO (NO) 3 - The concentration of N was reduced from 85.94mg/L to 20.98mg/L at the time of incubation (6 h-18 h), with a maximum removal of 84.8%. The highest removal rate was 5.41 mg/L.multidot.h, higher than other HN-AD bacteria reported previously. During this period, NO 2 - There was a short accumulation of N, reaching 2.31mg/L, followed by a decrease below 0.98 mg/L. NO (NO) 2 - An increase in the N concentration may be associated with a higher NO in the initial phase of denitrification 3 - -N conversion is related. With NO 3 - N removal tends to be stable, NO 2 - N is also gradually removed. A small amount of NO is accumulated in the aerobic denitrification process 2 - N, the predominant intermediate, NO 2 - -N can replace NO 3 - N as the primary electron acceptor, inducing nitrite nitrogen reductase to cleave NO 2 - Conversion of-N to N 2 Thereby removing it. NO (NO) 2 - Accumulation of-N tends to be associated with NO 3 - The removal of N is synchronous, NO 2 - The accumulation of N can be considered as a late conversion primerAnd (3) starting. When heterotrophic nitrifying bacteria are treated with NO 3 - NO can occur when denitrification is performed with-N as the sole nitrogen source 2 - Accumulation of N, there is a significant difference in the accumulation of different strains.
As shown in the results of FIG. 6, strain ID4 was expressed as NO 2 - N is the sole nitrogen source and shows good aerobic denitrification performance. The growth curve tended to flatten out 12 hours before inoculation. NO in the logarithmic growth phase (12 h-30 h), system 2 - The N concentration is reduced from 73.61mg/L to 30.43mg/L, and the maximum removal rate is 3.77 mg/L.h. In the whole process, the removal rate of nitrite is 63.6 percent at most. NO (NO) 2 - The average removal rate of N was 1.65 mg/L.h, higher than that of other reported bacteria. After the bacteria enter the decay phase, NO 2 - The N concentration does not vary much. During the test, NO 3 - N has obvious accumulation, and the maximum accumulated amount of 18 hours reaches 12.69mg/L. The reason for this phenomenon may be NO 2 - An initial concentration of N which is too high, a high concentration of NO 2 - -N inhibits nitrate reductase activity resulting in NO 3 - Poor removal of N.
In summary, the embodiment of the invention provides heterotrophic nitrification-aerobic denitrification bacteria and application thereof:
the invention provides heterotrophic nitrification-aerobic denitrification bacteria, which is named as heterotrophic nitrification-aerobic denitrification bacteria ID4, is gram-negative bacteria, has no flagella and spores, can effectively remove ammonia nitrogen, nitric acid nitrogen and nitrous acid nitrogen in a water body, and can simultaneously remove COD in organic wastewater, so that nitrite and nitrate do not accumulate in the denitrification process of sewage.
The embodiments described above are some, but not all embodiments of the invention. The detailed description of the embodiments of the invention is not intended to limit the scope of the invention, as claimed, but is merely representative of selected embodiments of the invention. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.
Claims (4)
1. The heterotrophic nitrification-aerobic denitrifying bacteria are named as heterotrophic nitrification-aerobic denitrifying bacteria Pseudomonas sp.ID4, the preservation number of the heterotrophic nitrification-aerobic denitrifying bacteria Pseudomonas sp.ID4 is CGMCC No.24224, and the preservation address is China academy of sciences of national academy of sciences of North Chenxi No.1 in the Korean of Beijing, and the preservation date is 2021, 12 months and 30 days.
2. Use of heterotrophic nitrification-aerobic denitrification bacteria Pseudomonas sp.id4 as defined in claim 1 in sewage treatment.
3. Use of heterotrophic nitrification-aerobic denitrifying bacteria Pseudomonas sp.id4 as defined in claim 1 in the preparation of a sewage treatment reagent.
4. A sewage treatment reagent comprising the heterotrophic nitrification-aerobic denitrification bacterium Pseudomonas sp.id4 according to claim 1.
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