CN110184223B - Arthrobacter calsium capable of removing ammonia nitrogen in culture sewage and application thereof - Google Patents
Arthrobacter calsium capable of removing ammonia nitrogen in culture sewage and application thereof Download PDFInfo
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- C02—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
- C02F—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
- C02F3/00—Biological treatment of water, waste water, or sewage
- C02F3/34—Biological treatment of water, waste water, or sewage characterised by the microorganisms used
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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- C12N1/00—Microorganisms, 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
- C12N1/20—Bacteria; Culture media therefor
- C12N1/205—Bacterial isolates
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- C—CHEMISTRY; METALLURGY
- C02—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
- C02F—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
- C02F2101/00—Nature of the contaminant
- C02F2101/10—Inorganic compounds
- C02F2101/16—Nitrogen compounds, e.g. ammonia
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- C02—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
- C02F—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
- C02F2103/00—Nature of the water, waste water, sewage or sludge to be treated
- C02F2103/20—Nature of the water, waste water, sewage or sludge to be treated from animal husbandry
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- C12R2001/06—Arthrobacter
Abstract
The invention relates to the field of aerobic advanced treatment of sewage in livestock and poultry farms, and particularly discloses Arthrobacter kaelii capable of removing ammonia nitrogen in culture sewage and application thereof. The applicant screens a heterotrophic nitrification-aerobic denitrification bacterium Arthrobacter Kaisei (Arthrobacter Keysieri) from activated sludge, the strain survives under the condition of ultrahigh ammonia nitrogen concentration (2000mg/L) and has certain denitrification capability, the ammonia nitrogen value in sewage can be greatly reduced when the strain is added into the sewage of a pig farm, indexes of COD, TP, TN and TSS are all reduced, the strain can be used for preparing a commercialized sewage treatment microbial inoculum, the strain is used as an exogenous addition strain of an aerobic treatment process of the sewage of a livestock and poultry farm, the sewage treatment cost is reduced, and the purpose of sewage harmless treatment is achieved.
Description
Technical Field
The invention relates to the field of aerobic advanced treatment of sewage in livestock and poultry farms, in particular to arthrobacter kaisei capable of efficiently removing ammonia nitrogen in the culture sewage and application thereof.
Background
The aquaculture sewage belongs to high-concentration organic sewage rich in a large number of pathogens, is directly discharged into a water body or is not suitable for storage places, is washed by rainwater and enters the water body, and can cause serious deterioration of the quality of surface water or underground water. As the leaching property of the livestock and poultry manure is very strong, the leaching amount of nitrogen, phosphorus, water-soluble organic matters and the like in the manure is very large, and if the treatment is not proper, the manure enters an underground water layer through surface runoff and percolation to pollute underground water. The influence on the surface water is mainly shown in that after a large amount of organic substances enter a water body, the decomposition of the organic substances consumes a large amount of dissolved oxygen in the water, so that the water body smells; after the dissolved oxygen in the water is greatly reduced, a large amount of organic substances can be continuously decomposed under the anaerobic condition, and toxic gases such as methane, hydrogen sulfide and the like can be generated during decomposition, so that a large amount of aquatic organisms die; a large amount of suspended matters in the sewage can make the water body turbid, reduce the photosynthesis of algae in the water, limit the normal activities of aquatic organisms and gradually die the aquatic organisms sensitive to organic pollution, so that the oxygen deficiency at the bottom of the water body is further aggravated and the water body assimilation capability is reduced; nitrogen and phosphorus can cause water body eutrophication, the concentration of nitrate and nitrite in the water body is too high as a result of the eutrophication, people and livestock can be poisoned if drinking for a long time, and a large amount of toxins can be discharged into the water body due to the growth and mass propagation of some toxic algae, so that a large amount of aquatic animals die, and the ecological balance of the water body is seriously damaged; some germs, viruses and the like in the excrement and urine flow with the water, which may cause the spread of some epidemics and the like.
At present, the sewage treatment technology of livestock and poultry farms is mainly divided into two categories of anaerobic treatment and aerobic treatment, the anaerobic processes are many, and the following are commonly used: anaerobic Filter (AF), Upflow Anaerobic Sludge Blanket (UASB), composite anaerobic reactor (UASB + AF), two-stage anaerobic digestion method and upflow sludge blanket reactor (USR); the aerobic treatment process can be divided into a natural aerobic biological treatment method (water body purification and soil purification) and an activated sludge method of artificial aerobic treatment, a biological filter, a biological rotating disc, a biological contact oxidation method, a Sequencing Batch Reactor (SBR) method, an anaerobic/aerobic (A/O) method, an oxidation ditch method and the like.
If the aquaculture sewage after solid-liquid separation is directly treated by adopting an aerobic process, although a large amount of capital construction cost is saved compared with an anaerobic treatment process, the sewage treatment cost is still high due to large power consumed in the aeration process and huge energy cost, so that the sewage treatment cost is a heavy burden for livestock and poultry breeding enterprises, and the problem of reducing the sewage treatment cost needs to be solved urgently.
Arthrobacter calbefaciens belongs to the genus Arthrobacter (Arthrobacter sp.) and is a gram-positive chemoheterotrophic bacterium. Arthrobacter is a ubiquitous bacterium with high genetic adaptability, which is widely present in the environment. There are reports of degrading pollutants by Arthrobacter bacteria, such as Arthrobacter (Arthrobacter sp.) YC-RLl (yan shochun, anyhow, smiths, et al.
Disclosure of Invention
The invention aims to provide an arthrobacter kaiselii capable of efficiently removing ammonia nitrogen in aquaculture sewage, the strain is delivered to a China center for type culture collection for collection in 2019, 1 month and 21 days, and the classification is named as follows: arthrobacter calbefaciens (Arthrobacter Keysieri) A-4, deposit number: CCTCC NO: M2019065, address: wuhan university in Wuhan, China.
Another object of the present invention is to provide the use of Arthrobacter calcoaceticus (Arthrobacter Keysieri) A-4 in sewage treatment.
In order to achieve the above purpose, the invention adopts the following technical measures:
screening of the Arthrobacter calcoaceticus capable of efficiently removing ammonia nitrogen in culture sewage:
the strain sieve is selected from activated sludge at a sewage treatment end of a fine breeding farm of the university of agriculture in Huazhong, the strain with ammonia nitrogen (NH4+ -N) removal rate of more than 70 percent is retained, and after 16SrDNA comparison, the strain is obtained and is sent to a China center for type culture collection for collection in 2019, 1 month and 21 days, and the strain is classified and named: arthrobacter calbefaciens (Arthrobacter Keysieri) A-4, deposit number: CCTCC NO: M2019065, address: wuhan university in Wuhan, China.
The bacterium is gram-positive bacterium, has a short rod shape, and has a light yellow opaque point-shaped colony; the common culture medium LB can be used for shake culture at normal temperature to prepare bacterial liquid for use.
The application of Arthrobacter calbefaciens A-4 in sewage treatment comprises removing ammonia nitrogen content in sewage by using the strain or preparing the sewage treatment agent for removing ammonia nitrogen; can also be used for reducing COD, TSS, TN, TP and the like in the sewage.
The invention has the beneficial effects that:
the screened strain can survive in high-concentration organic sewage generated in the livestock and poultry breeding process; the mixed liquid is added into an aerobic wastewater treatment section which is treated at the front end of the solid-liquid separation process according to a corresponding proportion, so that the content of heterotrophic nitrification-aerobic denitrification denitrogenation bacteria in an aerobic tank is increased, the denitrogenation process is accelerated, the hydraulic retention time is shortened, and the wastewater advanced treatment cost is reduced.
Drawings
FIG. 1 shows the growth pattern and gram staining of Arthrobacter Keysieri A-4 strain.
FIG. 2 is a schematic diagram showing the shapes of the pig farm wastewater with added bacterial liquid before and after reaction;
wherein: o is raw sewage; CT as a control group; d: arthrobacter calcens group.
FIG. 3 is a schematic view of a process flow of advanced treatment of pig farm wastewater.
Detailed description of the preferred embodiments
In the technical scheme of the invention, the reagents are purchased from biochemical shops if not specifically mentioned, and the technical scheme is conventional in the field if not specifically mentioned.
The method for detecting various indexes of sewage in the test comprises the following steps:
COD: heavy chromic acid-base method (GB 11914-89)
NH4 +-N: nashi reagent spectrophotometry (GB 7479-87)
TN alkaline Potassium persulfate digestion ultraviolet spectrophotometry (GB 11894-89)
TP ammonium molybdate spectrophotometry (GB 11893-89)
TSS: gravimetric method (GB 11901-89)
The growth amount of the bacteria: optical density method (OD)600)
The culture medium and the component ratio used in the test are as follows:
1L of nitrification enrichment medium: (NH)4)2SO40.5 g; trisodium citrate 4.08 g; KH (Perkin Elmer)2PO41.0 g; 0.5g of ferrous chloride; magnesium sulfate 1.0 g; adjusting pH to 7.0-7.5, and adding water in balance.
1L Broomthymol blue (BTB) screening medium: KNO31.0 g; l-day1.0g of asparagine; trisodium citrate 8.5 g; KH (Perkin Elmer)2PO41.0 g; 0.05g of ferrous chloride; magnesium sulfate 1.0 g; 0.2g of calcium chloride; 20g of agar; 5mL of a 0.1% bromothymol blue (BTB) solution was diluted with alcohol; adjusting pH to 7.0-7.5, and adding water in balance.
1L heterotrophic ammonia oxidation medium: (NH)4)2SO40.472 g; 4.902g of trisodium citrate; k2HPO40.2 g; magnesium sulfate 0.05 g; 0.01g of ferrous sulfate; 0.01g of manganese sulfate; NaCl 0.12 g; adjusting pH to 7.0-7.5, and adding water in balance.
Example 1:
separation, purification and identification of Arthrobacter calbefaciens (Arthrobacter Keyseri) A-4:
1. sampling, enriching, domesticating and culturing of activated sludge
Selecting a facultative pond at a sewage treatment end of a fine breeding farm of university of agriculture in Huazhong and sampling activated sludge, adding the activated sludge into a special aeration device, adding two to three times of nitrification enrichment culture medium to perform artificial intermittent aeration treatment (stopping for one hour every three hours of aeration), and keeping the dissolved oxygen of a water body not less than 3 mg/L. Continuously culturing for about two months, wherein the nitrification enrichment culture solution (the volume of the culture solution is changed to 1/50 about the initial volume) is changed once every day in the aeration stopping time period until the culture sludge is converted from gray black to earthy yellow, and standing for a period of time after the aeration is stopped to clarify and transparent the water body.
2. Separation and screening of heterotrophic nitrification-aerobic denitrification bacteria
Taking 1g to 50mL of earthy yellow activated sludge in aeration equipment, adding about 30mL of sterile water, and repeatedly and violently shaking to form mixed bacteria liquid. Diluting to 10% by multiple dilution method-1To 10-7And (3) dipping the bacterial liquid of the gradient bacterial suspension by using a sterilization inoculating ring, carrying out lineation on a BTB primary screening solid culture medium, and observing the colony morphology every day by inverting a culture dish in a constant temperature incubator at 30 ℃.
And (3) relatively selecting bacterial colonies which grow well and change the peripheral culture medium of the bacterial colonies into blue to perform streak separation and purification on a BTB re-screening solid culture medium, inverting the culture dish to observe the morphology of the bacterial colonies every day in a 30 ℃ constant-temperature incubator until the bacterial colonies with consistent characteristics are obtained, then selecting the bacterial colonies, repeating the previous step of streak separation and purification culture until the morphological characteristics of the bacterial colonies obtained every time are consistent, and avoiding other mixed bacterial colonies.
3. Preliminary determination of the Performance of the selected strains
The obtained strain is picked out by a sterile inoculating loop and inoculated to a sterilized nitrification culture medium, and the strain is shake-cultured for a period of time until the nitrification culture medium is completely turbid (the effective bacteria content is 10)8-109cfu/mL); adding the culture solution into a heterotrophic ammonia oxidation culture medium according to a certain volume ratio (1: 100), performing shake culture for 48h, and measuring TN and ammonia Nitrogen (NH) of the culture solution4 +-N) value, retention of ammonia Nitrogen (NH)4 +-N) the removal rate reaches more than 70 percent.
4. Identification of strains
Finally obtaining a strain of bacteria with ammonia nitrogen removal function, extracting DNA of the strain, amplifying a 16S rDNA fragment through PCR, and performing BLAST comparison on a DNA sequence in GeneBank after sequencing, wherein the similarity of the DNA sequence comparison of the strain and Arthrobacter calcoaceticus (Arthrobacter Keysteri) reaches 99%.
The bacteria are gram-positive bacteria, have short rod-shaped, and have light yellow opaque dotted distribution (shown in figure 1) and alkali-producing capability. According to the physiological and biochemical characteristics of bacteria which accord with the bacillus bacteria, the bacteria are determined to be the arthrobacter kaiselii according to the growth morphological characteristics of the bacteria by referring to Bergey's manual of bacteriology of systems and ' manual of appraisal of common bacteria systems '.
The strains are sent to China center for type culture Collection in 2019, 1 month and 21 days, and are classified and named: arthrobacter calbefaciens (Arthrobacter Keysieri) A-4, deposit number: CCTCC NO: M2019065, address: wuhan university in Wuhan, China.
Example 2:
the culture or fermentation mode of Arthrobacter calbefaciens (Arthrobacter Keyseri) A-4:
preparing conventional LB culture medium (Tryptone)10g/L, Yeast extract (Yeast extract)5g/L, and sodium chloride (NaCl)10g/L
Adjusting pH to 7.0) and sterilizing with high pressure steam, and placing on a clean benchInoculating Arthrobacter calbefaciens, performing shake culture at 25 deg.C and 120r/min for 48 hr until the culture solution is completely turbid, and the effective bacteria content of the culture solution reaches 108cfu/mL。
Example 3:
the effect of the reaction conditions on the growth of Arthrobacter calbefaciens (Arthrobacter Keysieri) A-4:
1. influence of common factors on growth and denitrification performance of bacteria
Considering that the actual application of the strain may be influenced by the weather temperature, the aeration amount, the pH value of inlet water and the like on the growth performance and the nitrogen removal effect of the arthrobacter kazakii, the test is specially set to test the optimal growth condition of the strain, and a corresponding reference range is provided for the better application of the arthrobacter kazakii in the pig farm wastewater aerobic advanced treatment process. The test set up the influencing factors as follows: designing corresponding test group growth conditions by controlling a variable method at the temperature (20 ℃, 30 ℃, 40 ℃) and the rotating speed (60r/min, 120r/min, 240r/min) and the pH (5, 6, 7, 8, 9); the culture medium used in the test was used to determine the initial ammonia Nitrogen (NH)4 +-N) concentration of about 110.00mg/L, Total Nitrogen (TN) concentration of about 120.0mg/L, pH around 7.00.
TABLE 1 Effect of temperature, rotational speed, pH on growth and Performance of bacteria
Note: ammonia nitrogen and total nitrogen are in mg/L unit, T, R, P respectively represents the influence of three factors of temperature, rotating speed and pH on the growth performance of bacteria, and the lower subscript numbers represent different levels of the factors; the upper capital letters represent different factors in the same column compared to each other and the different letter designations represent significant differences (P < 0.05).
T1-T3, R1-R3 and P1-P5 respectively represent different levels of three factors of temperature (20 ℃, 30 ℃, 40 ℃), rotating speed (60R/min, 120R/min and 240R/min) and pH (5, 6, 7, 8 and 9)
Analysis by experimental data detection we obtained the following results: the optimal growth conditions of the nitrogen removal performance of the Arthrobacter calcoaceticus are respectively that the temperature is 30 ℃, the rotating speed is 120r/min, the pH is 7, the ammonia nitrogen concentration is reduced from 110.0mg/L to 6.4mg/L after the oscillation treatment for 36 hours, and the removal rate can reach 94.2%.
2. Influence of high ammonia nitrogen concentration wastewater on growth performance of bacteria
The pig farm wastewater is high-concentration organic wastewater, and the pig farm wastewater without front-end solid-liquid separation has the typical characteristics of high organic matter concentration, more suspended matters and high ammonia nitrogen content, namely COD (chemical oxygen demand) of 3000-12000 mg.L-1800-2200 mg.L of ammonia nitrogen-1And TSS exceeds dozens of times. Wherein ammonia nitrogen has great influence on the growth of bacteria. The test mainly tests the tolerance of the Arthrobacter calbefaciens to the ammonia nitrogen concentration, and provides a corresponding reference range for better application of the Arthrobacter calbefaciens.
The test sets the ammonia nitrogen concentration as follows: 200mg/L, 400mg/L, 800mg/L, 1600mg/L and 2000mg/L, and collecting culture solution of Arthrobacter calbefaciens (effective bacteria content is 10)8cfu/mL) in a certain volume ratio (1: 100) adding into wastewater of pig farm, shake culturing at normal temperature for 36 hr, and detecting the density (OD) of bacteria growth600) Values, and other indicators.
TABLE 2 influence of Ammonia Nitrogen content at different concentrations on growth and Performance of bacteria
Note: the unit of ammonia nitrogen and total nitrogen is mg/L; n represents the influence of ammonia nitrogen concentration on the growth performance of bacteria, and N represents1～N5Corresponding to pig farm wastewater with ammonia nitrogen concentration of 200mg/L, 400mg/L, 800mg/L, 1600mg/L and 2000mg/L respectively, and the lower subscript numbers represent different levels of all factors; the upper capital letters represent different factors in the same column compared with each other, and the different letter designations represent significant differences (P)<0.05)。
From the data, the Arthrobacter calcoaceticus has strong high ammonia nitrogen concentration resistance, can grow in an environment with the ammonia nitrogen concentration reaching 2000mg/L, has a certain ammonia nitrogen removal rate, and has the ammonia nitrogen removal rate exceeding 70% under the environment with the ammonia nitrogen concentration of 200mg/L-800 mg/L. Example 3:
the actual treatment effect of Arthrobacter calcoaceticus (Arthrobacter Keysieri) A-4 on the wastewater of the pig farm is determined:
1. laboratory bench
Taking culture solution of Arthrobacter calcoaceticus (effective bacteria content is 10)8cfu/mL) in a certain volume ratio (1: 100) adding into wastewater of pig farm, culturing at normal temperature (25-30 deg.C) under shaking (rotation speed of 120 rpm) for 36 hr, and measuring various indexes of wastewater; the same wastewater without added bacterial liquid was used as a control.
The piggery wastewater is taken from the water inlet end of a sewage treatment facility in a breeding farm of the university of agriculture in Huazhong, and the shape of the piggery wastewater is observed by naked eyes before and after treatment as follows:
comparing the forms of the pig farm wastewater before and after the reaction, it is obvious from fig. 2 that the pig farm wastewater (O) before the reaction is grayish-blue opaque, the surface layer has foam, and the bottom layer has impurity precipitation after standing; after inoculating Arthrobacter kazakii and shake culturing for 36h, it can be seen that the pig farm sewage is changed from an opaque gray cyan state into a clear transparent state, the surface layer has no foam, the bottom layer has a large amount of flocculent gray cyan precipitates, and the specific index change is shown in Table 3.
TABLE 3
Note: the data unit in the table is mg/L, and the national standard refers to discharge Standard of pollutants for livestock and poultry Breeding (GB 18596-; the test groups were compared to the starting concentration and the different letter designations indicated significant differences (P < 0.05).
The removal rate of each index of the wastewater in the pig farm with the Arthrobacter calbefaciens added is higher than that of the pig farm without the addition of the bacteria liquid control group, and the ammonia Nitrogen (NH) of the test group is subjected to 36h oscillation treatment4 +the-N) value is reduced to 55.2mg/L from about 225.8mg/L, the reduction rate is as high as about 75 percent, the concentration reduction rate of Total Suspended Solids (TSS) in the wastewater is about 69 percent due to certain flocculation capacity of the Arthrobacter calzakii, and both indexes can reach the national pollutant discharge standard of livestock and poultry breeding (GB 18567-2001).
2. Pig farm sewage A2/O treatment system pilot plant
The test site is a large pig farm in Wuhan city, Hubei province, the sewage treatment process scheme of the farm covers a pretreatment system (a dry-wet separation system), an anaerobic biogas system, a deep A2/O biological treatment system and an oxidation pond aquatic plant purification system, the four systems are assisted with each other, an energy ecological unified utilization circulation mode is constructed, and the reaction flow is shown in figure 3.
Adding a bacteria liquid of the Acetobacter calcoaceticus (added according to the volume ratio of 1: 10000) to a secondary anaerobic pool (micro-aeration) and an aerobic pool at the outer edge, and detecting the water quality (COD and NH) of a water inlet end and a water outlet end through 48-hour reaction4 +-N、TP、TN)。
TABLE 4 Water quality results at the water inlet and outlet ends of a sewage treatment system before exogenous addition of a bacterial liquid
Note: the data units in the table are mg/L.
TABLE 5 Water quality results at water inlet and outlet ends of sewage treatment system after exogenous addition of bacterial liquid
Note: the data units in the table are mg/L.
By comparing the data in tables 3-2 and 3-3, it can be found that the COD and NH of the sewage at the water outlet end of the Arthrobacter calcoaceticus liquid are added by external sources4 +The reduction rate of each index of-N, TP and TN is higher than that before addition, wherein NH4 +the-N removal rate of the added bacteria liquid group is approximately 10% higher than that of the non-added group.
Claims (5)
1. An isolated Arthrobacter calcoaceticus (A)ArthrobacterKeyseri) The Arthrobacter calbefaciens is named as Arthrobacter calbefaciens A-4, and the preservation number is as follows: CCTCC NO: M2019065.
2. Use of Arthrobacter calcoaceticus according to claim 1 for sewage treatment.
3. Use of Arthrobacter calbefaciens according to claim 1 for preparing a sewage treatment agent.
4. The use according to claim 2 or 3, wherein the Arthrobacter calcoaceticus is used for reducing the content of ammonia nitrogen in sewage.
5. Use according to claim 2 or 3, of Arthrobacter calbefaciens for reducing total nitrogen, total phosphorus, chemical oxygen demand or total suspended matter content in wastewater.
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