CN115975845B - Application of salt-resistant/acid-resistant heterotrophic nitrification-aerobic denitrification bacteria in environmental wastewater treatment - Google Patents
Application of salt-resistant/acid-resistant heterotrophic nitrification-aerobic denitrification bacteria in environmental wastewater treatment Download PDFInfo
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Classifications
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02W—CLIMATE CHANGE MITIGATION TECHNOLOGIES RELATED TO WASTEWATER TREATMENT OR WASTE MANAGEMENT
- Y02W10/00—Technologies for wastewater treatment
- Y02W10/10—Biological treatment of water, waste water, or sewage
Abstract
The application discloses application of a strain of salt-tolerant/acid-tolerant heterotrophic nitrification-aerobic denitrification bacteria in environmental wastewater treatment. The application provides application of middle-sized pseudoacidophilic bacteria (Pseudacidovorax intermedius) XH-237 or a microbial inoculum containing the middle-sized pseudoacidophilic bacteria (Pseudacidovorax intermedius) XH-237 in nitrogen-containing wastewater treatment; the middle-sized pseudoacidophilic bacteria (Pseudacidovorax intermedius) XH-237 is ACCC 62209 in China Committee for culture Collection of microorganisms. The strain has the characteristics of acid resistance, salt resistance and heavy metal resistance, and the method for treating environmental wastewater by using the strain is simple to operate, mild in reaction, short in time and good in effect, and has good application value in sewage treatment engineering.
Description
Technical Field
The application relates to the technical field of environmental microorganisms, in particular to application of a salt-tolerant/acid-tolerant heterotrophic nitrification-aerobic denitrification bacterium in environmental wastewater treatment.
Background
Nitrogen is a major component of the nature and is constantly transformed and recycled between compounds of valence-5 to +3 and simple substances in the natural environment. In natural environment by organic nitrogen and NH 4 + -N、NO 2 - -N、NO 3 - -N four forms exist stably in the water environment. Accumulation of certain types of nitrogen does not occur in steady state environments of nitrogen cycles, but in recent years, more and more human production and living activities have destroyed this natural cycle to some extent. Along with the industrial progress, the agricultural development and the improvement of the living standard of residents, the water resource pollution problem is increasingly serious, and the ecological environment and the human beings are subjected to high-rise conditionsHealth poses a threat. The large amount of nitrogen discharged by different sources cannot be effectively treated, and the discharge to the water body can cause serious harm to human health and natural environment. When the nitrate nitrogen and the nitrite nitrogen in water exceed 10mg/L of the standard DB 4403/T60-2020 of the drinking water, the water is accumulated for a long time, so that the water is easy to cause intolerance of infants, and causes blue infant disease and ferrous hemoglobin diseases, and the water is cancerogenic or dead in serious cases. For natural ecological environment, continuous discharge and enrichment of high nitrogen can cause water pollution to be eutrophicated, so that dissolved oxygen in water is greatly reduced, a large amount of autotrophic photosynthetic bacteria and algae are greatly propagated, and finally, vicious circle is changed into 'dead water'. When nitrite nitrogen in the water body is accumulated, the aquatic animals are easy to change the iron valence state in hemoglobin after absorbing the nitrite nitrogen, the oxygen combination ability of red blood cells is damaged, and the asphyxia death of the aquatic animals is finally caused. And the polluted water enters plants along with agricultural irrigation, and finally enriches to human bodies along with the drinking of human beings, so that huge damage is caused to the human bodies.
Biological denitrification mainly utilizes the nitrification and denitrification of denitrification related microorganisms, and converts various nitrogen-containing substances in water into nitrogen to escape under the action of enzyme channels of the microorganisms, so that the nitrogen-containing substances in sewage are reduced or removed. At present, biological denitrification technology is widely applied to denitrification treatment of sewage by matching with related processes such as immobilization and the like. The reaction process is mainly characterized in that organic nitrogen is sequentially converted into ammonia nitrogen, nitrite nitrogen and nitrate nitrogen through ammoniation and nitration under aerobic conditions, and then denitrification is utilized, so that the nitrate nitrogen is reduced into gaseous nitrogen under anoxic conditions and overflows from sewage, and the aim of denitrification is achieved. However, it has the following disadvantages: (1) Nitrifying bacteria are inorganic autotrophic bacteria, and the nutrition metabolism type determines that the nitrifying bacteria grow slowly, have long generation period, lower biomass concentration and poor environmental adaptability. (2) Traditional nitrifying bacteria are all strict aerobic bacteria, and traditional denitrifying bacteria are anaerobic or facultative anaerobic bacteria. The different conditions limit the denitrification rate of the water body to be influenced under the same environment.
Thus, strains having nitrification and denitrification functions have been sought. In 1983, robertson et al succeededSeparating a strain of nitrogen-containing wastewater which can oxidize ammonia nitrogen into hydroxylamine by utilizing organic carbon under the condition of participation of oxygen, then convert the hydroxylamine into nitrite nitrogen and nitrate nitrogen, and finally generate nitrogen to discharge water (NH) 4 + →NH 2 OH→NO 2 - →NO 3 - →NO 2 - →NO→N 2 O→N 2 ) The pantoea (Thiosphaera pantotropha) is revised as paracoccus denitrificans (Paracoccus pantotrophus) to start the screening and research of nitrifying and denitrifying co-functional bacteria. And the bacteria react in an abnormal way under the aerobic condition, therefore Robertson proposes the theory of heterotrophic nitrification-aerobic denitrification HN-AD (Heterotrophic Nitrification-Aerobic Denitrification). Compared with the traditional microbial denitrifying bacteria, the heterotrophic nitrification-aerobic denitrifying bacteria have the advantages of high growth rate, short period, simple denitrification process, high denitrification rate, low requirements on living environment and strong adaptability to temperature, pH and DO. And the strain has the function of removing nitrogen sources (organic nitrogen and NH) with different valence types in the wastewater 4 + -N、NO 2 - -N、NO 3 - -N). The energy consumption is low, and carbon sources are not required to be added in the middle after the organic matter supply of the nitration reaction is started only when bacteria are added initially in the denitrification reaction process. The acid and alkali generated by the reaction can be neutralized in the whole reaction process, so that the pH of the system can be stabilized. However, the reports are more reports on denitrification characteristics of strains, but the reports on heterotrophic nitrification aerobic denitrifying bacteria with weak acid resistance, heavy metal resistance, salt resistance and simultaneous removal of ammonia nitrogen, nitrate nitrogen and nitrite nitrogen in environmental wastewater are not reported.
Disclosure of Invention
The application aims to provide application of a salt-tolerant/acid-tolerant heterotrophic nitrification-aerobic denitrification bacterium in environmental wastewater treatment.
In a first aspect, the application claims the use of a mesophilic pseudoacidophilus (Pseudacidovorax intermedius) XH-237 or a microbial inoculum containing said mesophilic pseudoacidophilus (Pseudacidovorax intermedius) XH-237 in the treatment of nitrogen-containing wastewater. Wherein the middle-sized pseudoacidophilic bacteria (Pseudacidovorax intermedius) XH-237 is ACCC 62209 in China Committee for culture Collection of microorganisms.
Further, the wastewater treatment is to biologically denitrify the water body.
Further, the nitrogen in the wastewater may be ammoniacal nitrogen, nitrite nitrogen, and/or nitrate nitrogen.
Further, the pH of the wastewater is not lower than 5. For example, pH 5-11, and further for example, pH 7-11.
Further, the total salinity in the wastewater is not higher than 10% by mass fraction (i.e., the mass fraction of NaCl in the wastewater is not higher than 10%).
Further, mn in the wastewater 2+ Not higher than 10mmol/L, pb 2+ 、Fe 3+ Not higher than 5mmol/L, zn 2+ 、Cu 2+ 、Co 2+ 、Cd 2+ Not higher than 1mmol/L.
Further, the wastewater C/N ratio is 10+/-0.2.
In a second aspect, the application claims a wastewater treatment process.
The wastewater treatment method claimed by the application can comprise the following steps: the medium-sized pseudoacidophilus (Pseudacidovorax intermedius) XH-237 or the microbial inoculum containing the medium-sized pseudoacidophilic bacteria (Pseudacidovorax intermedius) XH-237 are inoculated to the wastewater to be treated for cultivation. Wherein the middle-sized pseudoacidophilic bacteria (Pseudacidovorax intermedius) XH-237 is ACCC 62209 in China Committee for culture Collection of microorganisms.
Further, the content of the medium-sized pseudoacidophilic bacteria (Pseudacidovorax intermedius) XH-237 in the wastewater system after inoculation may be 1×10 6 CFU/ml-3.0×10 7 CFU/ml, e.g. 1X 10 7 CFU/ml。
Further, the temperature at which the cultivation is carried out may be 28 to 32℃such as 30 ℃.
Further, the cultivation may be performed for 36 to 60 hours, such as 48 hours.
Further, the culture may be shaking culture at 180 rpm.
Further, the wastewater is nitrogen-containing wastewater. Still further, the nitrogen in the wastewater may be ammoniacal nitrogen, nitrite nitrogen, and/or nitrate nitrogen.
Further, the pH of the wastewater is not lower than 5. For example, pH 5-11, and further for example, pH 7-11.
Further, the total salinity in the wastewater is not higher than 10% by mass fraction (i.e., the mass fraction of NaCl in the wastewater is not higher than 10%).
Further, mn in the wastewater 2+ Not higher than 10mmol/L, pb 2+ 、Fe 3+ Not higher than 5mmol/L, zn 2+ 、Cu 2+ 、Co 2+ 、Cd 2+ Not higher than 1mmol/L.
Further, the wastewater C/N ratio is 10+/-0.2.
In a third aspect, the application claims the use of a mesophilic pseudoacidophilus (Pseudacidovorax intermedius) XH-237 or a microbial inoculum containing said mesophilic pseudoacidophilus (Pseudacidovorax intermedius) XH-237 in biological denitrification. Wherein the middle-sized pseudoacidophilic bacteria (Pseudacidovorax intermedius) XH-237 is ACCC 62209 in China Committee for culture Collection of microorganisms.
In a specific embodiment of the present application, the biological denitrification is to inoculate the medium-sized pseudoacidophilic bacteria (Pseudacidovorax intermedius) XH-237 into a heterotrophic nitrification-aerobic denitrification medium for cultivation, thereby completing denitrification; the solvent of the heterotrophic nitrification-aerobic denitrification culture medium is water, and the solute and the concentration are as follows: CH (CH) 3 COONa 3.42g/L，NH 4 Cl 0.0.1284g/L，NaNO 2 0.1643g/L，KNO 3 0.241g/L，K 2 HPO 4 0.2g/L，MgCl 2 0.05g/L，CaCl 2 0.05g/L，ZnSO 4 0.05g/L,0.1% (V/V) trace element solution; pH7.2. The trace element solution solvent is water, and the solute and the concentration are as follows: EDTA 50g/L, caCl 2 5.5g/L，ZnSO 4 2.2g/L，MnCl 2 ·4H 2 O 5.06g/L，FeSO 4 ·7H 2 O 5.0g/L，(NH 4 ) 6 Mo 7 O 24 ·4H 2 O 1.1g/L，CuSO 4 ·5H 2 O 1.57g/L，CoCl 2 ·6H 2 O 1.61g/L；pH7.0。
Further, the medium-sized pseudoacidophilus (Pseudacidovorax intermedius) XH-237 may be contained in the culture system at a ratio of 1X 10 after inoculation 6 CFU/ml-3.0×10 7 CFU/ml, e.g. 1X 10 7 CFU/ml。
Further, the temperature at which the cultivation is carried out may be 28 to 32℃such as 30 ℃.
Further, the cultivation may be performed for 36 to 60 hours, such as 48 hours.
Further, the culture may be shaking culture at 180 rpm.
Further, the step of activating and/or preparing a bacterial suspension of said medium-sized pseudoacidophilic bacteria (pseudoacidophilic bacteria) XH-237 is included before performing said biological denitrification. The method comprises the following steps: inoculating it on LB slant culture medium, and culturing at 35 deg.C for 1-2 days to obtain activated thallus. Then inoculating the activated thalli into 100ml of LB liquid seed culture medium according to the conventional quantity, and culturing for 12 hours at 35 ℃ to obtain the bacterial suspension of the medium-sized pseudoacidophilic bacteria (Pseudacidovorax intermedius) XH-237.
Experiments prove that the medium-sized pseudoacidophilic bacteria (Pseudacidovorax intermedius) XH-237 (ACCC 62209) can grow by taking ammonia nitrogen, nitrate nitrogen or nitrite nitrogen as the sole nitrogen source under the aerobic condition, and simultaneously perform heterotrophic nitrification and aerobic denitrification, and has the removal capability for the ammonia nitrogen, the nitrate nitrogen and the nitrite nitrogen, and the removal capability is 1 multiplied by 10 within 48 hours 7 CFU/ml concentration bacterial load p-nitrate Nitrogen (NO) in heterotrophic nitrification-aerobic denitrification culture medium 3 - -N), nitrite nitrogen (NO 2 - -N), ammoniacal Nitrogen (NH) 4 + -N) removal rates of 25.38%, 17.20% and 83.28%, respectively. And can tolerate a salt concentration of 10% at a pH of 5 for 10mmol/L metal ion Mn, respectively 2+ 5mmol/L metal ion Pb 2+ 、Fe 3+ 1mmol/L metal ion Zn 2+ 、Cu 2+ 、Co 2+ 、Cd 2+ Is tolerant. The method for treating environmental wastewater by using the strain has the advantages of simple operation, mild reaction, short time and good effect, and has good application value in sewage treatment engineering.
Drawings
FIG. 1 shows the results of a genetic evolutionary tree (adjacent method) analysis of Pseudoacidophilus mesoscale (Pseudacidovorax intermedius) XH-237 in a strain according to the application.
FIG. 2 is a single colony morphology map of medium-sized pseudoacidophilic bacteria (Pseudacidovorax intermedius) LB solid plates streaked according to the present application.
FIG. 3 is a gram-type optical microscope morphological and gram-type staining chart of medium-sized pseudoacidophilic bacteria (Pseudacidovorax intermedius) LB liquid culture of the application.
FIG. 4 shows the effect of different carbon sources on the 48h growth of P.mesogenes (Pseudacidovorax intermedius).
FIG. 5 shows the effect of different carbon nitrogen ratios on the 48h growth of Pseudoacidophilus mesoscale (Pseudacidovorax intermedius).
FIG. 6 shows the effect of different pH on 48h growth of medium pseudoacidophilus (Pseudacidovorax intermedius).
FIG. 7 shows the effect of different temperatures on the 48h growth of P.mesogenes (Pseudacidovorax intermedius).
FIG. 8 shows the effect of different salinity on the 48h growth of Pseudoacidophilus mesoscale (Pseudacidovorax intermedius).
FIG. 9 shows the effect of different heavy metal concentrations on the growth of P.mesogenes (Pseudacidovorax intermedius) 48.
FIG. 10 shows the ammonia nitrogen removal effect of medium-sized pseudoacidophilus (Pseudacidovorax intermedius) for 48h.
FIG. 11 shows the effect of medium-sized pseudoacidophiles (Pseudacidovorax intermedius) on nitrite nitrogen removal for 48h.
FIG. 12 shows the effect of medium-sized pseudoacidophiles (Pseudacidovorax intermedius) on removal of nitrate nitrogen for 48h.
FIG. 13 shows the effect of medium-sized pseudoacidophilus (Pseudacidovorax intermedius) on the removal of mixed nitrogen sources for 48h.
Detailed Description
The following detailed description of the application is provided in connection with the accompanying drawings that are presented to illustrate the application and not to limit the scope thereof. The examples provided below are intended as guidelines for further modifications by one of ordinary skill in the art and are not to be construed as limiting the application in any way.
The experimental methods in the following examples, unless otherwise specified, are conventional methods, and are carried out according to techniques or conditions described in the literature in the field or according to the product specifications. Materials, reagents and the like used in the examples described below are commercially available unless otherwise specified.
The middle-sized pseudoacidophilic bacteria (Pseudacidovorax intermedius) XH-237 in the following examples were collected in China Committee for culture Collection of microorganisms (Agricultural Culture Collection of China, ACCC for short, address: guangcun's south Dajie No. 12 in the sea area of Beijing city, post code 100081 of institute of agricultural resources and agriculture, national academy of agricultural sciences) before the application date of the present application, the strain number was ACCC 62209, and the collection date was 2022, 6 months and 28 days, from which the public could obtain the strain from the China Committee for culture collection of microorganisms. The ACCC is provided with a special website, and the website address is as follows: http:// www.accc.org.cn, the public can order the strains directly on the internet. The website of the medium-sized pseudoacidophilic bacteria (Pseudacidovorax intermedius) XH-237 is http:// www.accc.org.cn/column. Asp? Column_id=34929 & model=product_detail & p_id= 1817447492.
Example 1 isolation and identification of Pseudomonas mesophilic (Pseudacidovorax intermedius) XH-237
And (3) taking a sludge sample of a No. 2 sedimentation tank of a sewage treatment plant of a Wang pond in Hefei city of Anhui province, domesticating and enriching a carbon dioxide source and a nitrate nitrogen source, and then performing coating separation to obtain the medium-sized pseudoacidophilic bacteria (Pseudacidovorax intermedius) XH-237.
1. Domestication enrichment, separation culture medium and expansion culture medium
Acclimatization enrichment medium: the solvent is water, and the solute and concentration are as follows: KNO (KNO) 3 0.722g/L，CO 2 Continuously introducing K 2 HPO 4 0.2g/L，MgCl 2 0.05g/L，CaCl 2 0.05g/L，ZnSO 4 0.05g/L,0.1% (V/V) trace element solution; pH7.2. Trace element solution: solvent(s)Is water, the solute and concentration are as follows: EDTA 50g/L, caCl 2 5.5g/L，ZnSO 4 2.2g/L，MnCl 2 ·4H 2 O 5.06g/L，FeSO 4 ·7H 2 O 5.0g/L，(NH 4 ) 6 Mo 7 O 24 ·4H 2 O 1.1g/L，CuSO 4 ·5H 2 O 1.57g/L，CoCl 2 ·6H 2 O 1.61g/L；pH7.0。
Isolation medium: the solvent is water, and the solute and concentration are as follows: CH (CH) 3 COONa 1.0g/L，KNO 3 0.2g/L，K 2 HPO 4 0.2g/L，MgCl 2 0.05g/L，CaCl 2 0.05g/L，ZnSO 4 0.05g/L,0.1% (V/V) trace element solution; pH7.2. The solid medium was additionally supplemented with agar 2% (20 g/L) and 0.1% (V/V) bromomusk thymol indicator solution.
Expansion culture medium: the solvent is water, and the solute and concentration are as follows: 10g/L tryptone, 5g/L, naCl g/L yeast extract; pH7.2.
2. Isolation and purification of Strain XH-237
Sterile water of the domesticated sample subjected to the high-nitrate nitrogen environment is prepared into 100mL solution according to the proportion of 1:9, and the dilution factor of the solution is 10 -1 1mL of the solution is added into a 9mL test tube filled with sterile water, and the solution can be diluted to 10 in sequence by repeating the steps -2 、10 -3 、10 -4 、10 -5 Five gradients total. In a sterile ultra-clean bench, 20 μl of 5 gradient concentration bacterial suspensions were respectively applied to the surface of the prepared solid isolation medium, and uniformly spread with a sterile glass spreading rod, after it was completely infiltrated, the dish was covered with a lid, the dish was sealed, and three concentration gradients were parallel. The plates to be cultivated are inverted and cultivated in an incubator at a cultivation temperature of 30 ℃ for 2-4d. After a clear single colony is grown, single strains to be picked are screened and marked by a marker pen and counted. In a sterile environment, opening a culture dish, dipping a small amount of single colony in a prepared LB culture medium with a sterile inoculating loop, gently streaking to avoid scratching, slowly rotating a plate after one area is full, placing the inoculating loop on an alcohol lamp for burning, and removing micro-particles thereonAnd (3) living things wait until the temperature of the inoculating loop is cooled, and connecting the painted area with the blank area to continue scribing. A plate was divided into 4 areas and scored, and the plate was sealed with a sealing film. The inverted plate was cultured in an incubator at 30℃for 2-3d. If the streaking strain is not a single strain, it is necessary to streak again until streaking is pure cultured as a single strain. Each bacteria was purified more than twice. One of the obtained strains is the strain XH-237.
3. PCR amplification and sequencing of 16S rRNA
The DNA of the obtained XH-237 strain was extracted and purified using a Soxhaust company bacterial DNA extraction D1600 kit (Beijing Soxhaust technology Co., ltd.). Taking 5 mu L of DNA sample, carrying out electrophoresis detection under the conditions of 110V and 20min, and carrying out observation quality inspection under a gel electrophoresis apparatus.
Bacterial universal primers 16S-27F (5'-AGAGTTTGATCCTGGCTCAG-3') and 16S-1492R (5'-CGGTTACCTTGTTACGACTTC-3') were selected for PCR amplification. The PCR reaction conditions were: pre-denaturation at 94℃for 3min; denaturation at 94℃for 30s, annealing at 55℃for 30s, elongation at 72℃for 90s, and cycling for 35 cycles; then extending for 10min at 72 ℃; finally, the mixture is preserved at the temperature of 4 ℃.
The PCR amplification product is sequenced by principal biological engineering Co., ltd after PCR product purification, quality inspection and purification.
The sequence of 16S rDNA of the strain obtained after sequencing was 1422bp in length (SEQ ID No. 1), and the sequence of 16S rRNA of the strain XH-237 of the present application was found to have 99.72% homology with the sequence of 16S rRNA of a NCBI-registered multi-strain Pseudoacidophilus mesogenes (Pseudacidovorax intermedius) by using BLASTN program alignment of the American bioengineering information center (National Center for Biotechnology Information, NCBI). Phylogenetic tree (FIG. 1) was constructed using the phylogenetic analysis (MEGA 11) software orthotopic ligation (Neighbour Joining) method, indicating that the XH-237 strain of the application is closely related to the known M.mesogenes DSM 21353.
4. Morphological characteristics observation of XH-237 strain
Experimental methods for morphological characterization refer to the "handbook for identification of common bacterial systems", edited by Dongxiu beads, cai Miaoying, et al, science Press, 2001, first edition, p353-363.
Morphological characteristics of XH-237 Strain: culturing on LB solid medium, the colony is round, convex, neat in edge, smooth in surface, light yellowish and slightly transparent, as shown in figure 2. Culturing in LB liquid medium, observing under a bacterial strain microscope to obtain bacillus without forming spore, and culturing in straight or slightly bent shape with irregular shape at two ends to obtain gram positive strain, as shown in figure 3.
5. Physiological and biochemical characterization of XH-237 strain
Physiological and biochemical test media and methods of experiments reference BIOLOG GenIII plate BIOLOG, inc., U.S. Pat. No. 5,627,045, incorporated herein by reference.
Physiological and biochemical characteristics of XH-237 strain: gram-positive, aerobic, optimal growth temperature is 30-40 ℃, and fructose, galactose, glycerol, salt tolerance and acid resistance are utilized at 30 ℃; the results of the physiological and biochemical experiments are shown in Table 1.
Table 1, partial physiochemical characteristics of XH-237 Strain biological GenIII plate
Note that: "(+)" grew well or was positive; "(-)" does not grow or is negative; "(W)" grew close to the blank control or was false positive.
In view of the morphological characteristics, physiological and biochemical characteristics and 16S sequencing results performed on the XH-237 strain, the XH-237 strain was identified as a medium-sized pseudoacidophilic bacterium (Pseudacidovorax intermedius) and has been collected in China Commission for culture Collection of microorganisms, also known as China center for agricultural microbiological culture Collection (Agricultural Culture Collection of China, ACCC for short, address: guangcun south street 12 in the sea lake area of Beijing, institute of agricultural resources and agriculture, and post code 100081) at day 28 of 2022.
6. Influence of carbon Source on the growth of pseudo acidophilic bacteria (Pseudacidovorax intermedius)
After the medium-sized pseudoacidophilic bacteria (Pseudacidovorax intermedius) XH-237 suspension cultured in the LB culture medium is resuspended by PBS, glucose, sucrose, sodium acetate, sodium citrate and sodium succinate are respectively added into a substrate universal culture medium as unique carbon sources, 100mg/L of ammoniacal Nitrogen (NH) 4 + -N), 100mg/L nitrite nitrogen (NO 2 - -N), 100mg/L nitrate nitrogen (NO 3 - -N) and 100mg/L of three nitrogen mixed nitrogen sources (33.33 mg/L of three nitrogen sources, respectively) are nitrogen sources, respectively, and the carbon-nitrogen ratio in the medium is controlled to be C/n=10 (basal universal medium: k (K) 2 HPO 4 0.2g/L，MgCl 2 0.05g/L，CaCl 2 0.05g/L，ZnSO 4 0.05g/L,0.1% (V/V) trace element solution; pH7.2. Trace element solution: EDTA 50g/L, caCl 2 5.5g/L，ZnSO 4 2.2g/L，MnCl 2 ·4H 2 O 5.06g/L，FeSO 4 ·7H 2 O 5.0g/L，(NH 4 ) 6 Mo 7 O 24 ·4H 2 O 1.1g/L，CuSO 4 ·5H 2 O 1.57g/L，CoCl 2 ·6H 2 O1.61 g/L; ph 7.0). After culturing at 30℃for 48 hours at 180rpm, the OD600 of the medium-sized pseudoacidophilic bacteria (Pseudacidovorax intermedius) in the system was monitored. The results show that medium-sized pseudoacidophiles (Pseudacidovorax intermedius) XH-237 utilized sodium acetate to grow at the fastest rate, so sodium acetate was chosen as the carbon source for the subsequent experimental media. See in particular fig. 4.
7. Carbon-to-nitrogen ratio Effect on growth of Medium-sized Acidophilic bacteria (Pseudacidovorax intermedius) XH-237
After the medium-sized pseudoacidophilic bacteria (Pseudacidovorax intermedius) XH-237 suspension cultured in LB is resuspended by PBS, sodium acetate is selected as the only carbon source, 100mg/L ammonia Nitrogen (NH) 4 + -N), 100mg/L nitrite nitrogen (NO 2 - -N), 100mg/L nitrate nitrogen (NO 3 - -N) and 100mg/L of three nitrogen sources (33.33 mg/L of three nitrogen sources respectively) are nitrogen sources respectively, and are added into the substrate universal culture medium respectivelyC/N is different amounts of 5, 10, 15, 20, 25. (basal Medium: K) 2 HPO 4 0.2g/L，MgCl 2 0.05g/L，CaCl 2 0.05g/L，ZnSO 4 0.05g/L,0.1% (V/V) trace element solution; pH7.2. Trace element solution: EDTA 50g/L, caCl 2 5.5g/L，ZnSO 4 2.2g/L，MnCl 2 ·4H 2 O 5.06g/L，FeSO 4 ·7H 2 O 5.0g/L，(NH 4 ) 6 Mo 7 O 24 ·4H 2 O 1.1g/L，CuSO 4 ·5H 2 O 1.57g/L，CoCl 2 ·6H 2 O1.61 g/L; pH 7.0), after culturing at 30℃and 180rpm for 48 hours, the OD600 of the pseudoacidophilic bacteria (Pseudacidovorax intermedius) in the system was monitored. The results showed that medium-sized pseudoacidophiles (Pseudacidovorax intermedius) XH-237C/N were shown to grow at the fastest rate of 10, so C/N was selected to be 10 as the carbon-nitrogen ratio of the subsequent experimental media. See in particular fig. 5.
8. Influence of pH on the growth of pseudo acidophilic bacteria (Pseudacidovorax intermedius) XH-237
The medium-sized pseudoacidophilus (Pseudacidovorax intermedius) XH-237 was inoculated into LB medium with pH values of 3, 5, 7, 9 and 11, respectively, and after culturing at 30℃and 180rpm for 48 hours, the OD600 of the medium-sized pseudoacidophilic bacteria (Pseudacidovorax intermedius) was monitored. The results show that the mesophilic pseudoacidophilus (Pseudacidovorax intermedius) XH-237 has better growth rate at pH 7-11. Subsequent experiments were performed with a pH of 7. See in particular fig. 6.
9. Effect of temperature on growth of Medium-sized Acidophilic bacteria (Pseudacidovorax intermedius) XH-237
Medium-sized pseudoacidophilic bacteria (Pseudacidovorax intermedius) XH-237 are respectively inoculated into LB culture medium with the temperature value of 10 ℃,20 ℃,30 ℃, 40 ℃ and 50 ℃ and cultured for 48 hours at the temperature of 30 ℃ and at the speed of 180rpm, and then the OD600 value of the medium-sized pseudoacidophilic bacteria (Pseudacidovorax intermedius) in the system is monitored. The results show that the medium-sized pseudoacidophilus (Pseudacidovorax intermedius) XH-237 has better growth rate at the temperature of 30-40 ℃. The subsequent experiment was performed at a selected temperature of 30 ℃. See in particular fig. 7.
10. Effect of salinity on growth of Pseudoacidophilus mesoscale (Pseudacidovorax intermedius) XH-237
Medium-sized pseudoacidophilus (Pseudacidovorax intermedius) XH-237 is inoculated into LB culture medium with salinity of 0%, 1%, 3%, 5% and 10% (mass fraction of NaCl) respectively, and after culturing for 48h at 30 ℃ and 180rpm, the OD600 value of the medium-sized pseudoacidophilic bacteria (Pseudacidovorax intermedius) in the system is monitored. The results show that medium-sized pseudoacidophiles (Pseudacidovorax intermedius) XH-237 have better growth rates at salinity of 5% and are able to tolerate 10% salinity. See in particular fig. 8.
11. Medium-sized pseudoacidophiles (Pseudacidovorax intermedius) XH-237 resistant to heavy metals
The mesophilic pseudoacidophilus (Pseudacidovorax intermedius) XH-237 is respectively inoculated with Mn with heavy metal concentration of 1mM, 5mM and 10mM 2+ 、Pb 2+ 、Fe 3+ 、Zn 2+ 、Cu 2+ 、Co 2+ 、Cd 2+ 、Hg 2+ 、Ni 2+ 、Cr 6+ After culturing at 30℃and 180rpm for 48 hours, the OD600 of the medium-sized pseudoacidophilic bacteria (Pseudacidovorax intermedius) in the system was monitored. The results show that the mesophilic pseudoacidophilus (Pseudacidovorax intermedius) XH-237 has a specific molecular weight of 10mmol/L metal ion Mn 2+ 5mmol/L metal ion Pb 2+ 、Fe 3+ 1mmol/L metal ion Zn 2+ 、Cu 2+ 、Co 2+ 、Cd 2+ Is tolerant. See in particular fig. 9.
12. Medium-sized pseudoacidophilic bacteria (Pseudacidovorax intermedius) XH-237 removal capability for different types of nitrogen sources
The following methods for measuring ammonia nitrogen, nitrite nitrogen and nitrate nitrogen are respectively as follows: ammoniacal Nitrogen (NH) 4 + -N) determination using "determination of aqueous ammonia nitrogen naloxone reagent spectrophotometry" (HJ 535-2009); nitrite Nitrogen (NO) 2 - -N) determination by means of nitrous coupling spectrophotometry (GB 7493-87) for determination of nitrite nitrogen in water quality; nitrate Nitrogen (NO) 3 - -N) method for measuring nitrate nitrogen using ultraviolet spectrophotometry water quality (HJ 346-2007).
The following media formulations were used:
basic propagation culture medium: the solvent is water, and the solute and concentration are as follows: 10.0g/L tryptone; 5.0g/L yeast powder; naCl 10.0g/L.
Denitrification performance measurement medium: CH (CH) 3 COONa 3.42g/L，KNO 3 0.722g/L，K 2 HPO 4 0.2g/L，MgCl 2 0.05g/L，CaCl 2 0.05g/L，ZnSO 4 0.05g/L,0.1% (V/V) trace element solution; pH7.2.
Nitrite denitrification performance determination medium: CH (CH) 3 COONa 3.42g/L，NaNO 2 0.4928g/L，K 2 HPO 4 0.2g/L，MgCl 2 0.05g/L，CaCl 2 0.05g/L，ZnSO 4 0.05g/L,0.1% (V/V) trace element solution; pH7.2.
Ammonification performance determination of ammonium salts medium: CH (CH) 3 COONa 3.42g/L，NH 4 Cl 0.3853g/L，K 2 HPO 4 0.2g/L，MgCl 2 0.05g/L，CaCl 2 0.05g/L，ZnSO 4 0.05g/L,0.1% (V/V) trace element solution; pH7.2.
Heterotrophic nitrification-aerobic denitrification medium: CH (CH) 3 COONa 3.42g/L，NH 4 Cl 0.1284g/L，NaNO 2 0.1643g/L，KNO 3 0.241g/L，K 2 HPO 4 0.2g/L，MgCl 2 0.05g/L，CaCl 2 0.05g/L，ZnSO 4 0.05g/L,0.1% (V/V) trace element solution; pH7.2.
Microelement solution, EDTA 50g/L, caCl 2 5.5g/L，ZnSO 4 2.2g/L，MnCl 2 ·4H 2 O 5.06g/L，FeSO 4 ·7H 2 O 5.0g/L，(NH 4 ) 6 Mo 7 O 24 ·4H 2 O 1.1g/L，CuSO 4 ·5H 2 O 1.57g/L，CoCl 2 ·6H 2 O 1.61g/L；pH7.0。
Inoculating medium-sized pseudoacidophilus (Pseudacidovorax intermedius) XH-237 on LB plate medium, and culturing at 35 ℃ for 24 hours to obtain activated bacteria.
The prepared activated thalli are inoculated into 100ml of LB liquid seed culture medium according to the proportion of 5 percent according to the conventional quantity, and are cultured for 12 hours at 35 ℃ to obtain the medium-sized pseudoacidophilic bacteria (Pseudacidovorax intermedius) seed suspension.
The prepared seed suspension is resuspended by PBS and inoculated into a corresponding ammonia nitrogen removal capacity determination culture medium, so that the bacterial load in the system is 1 multiplied by 10 7 CFU/ml. Culturing at 30deg.C and 180rpm for 48 hr, and measuring ammonia Nitrogen (NH) every 12 hr 4 + -N), nitrite nitrogen (NO 2 - -N), nitrate nitrogen (NO 3 - -N) index. Determination of Denitrification Performance in 48h nitrate Nitrogen (NO) 3 - -N) removal rate 45.6%; nitrite denitrification Performance determination of nitrite Nitrogen (NO) in culture Medium 2 - -N) removal rate 24.1%; determination of ammonification Performance of ammonium salts Medium Ammonia Nitrogen (NH) 4 + -N) removal rate 44.1%; nitrate Nitrogen (NO) in heterotrophic nitrification-aerobic denitrification culture medium 3 - -N), nitrite nitrogen (NO 2 - -N), ammoniacal Nitrogen (NH) 4 + -N) removal rates were 25.38%, 17.20% and 83.28%, respectively. The results are shown in detail in FIGS. 10 to 13.
The present application is described in detail above. It will be apparent to those skilled in the art that the present application can be practiced in a wide range of equivalent parameters, concentrations, and conditions without departing from the spirit and scope of the application and without undue experimentation. While the application has been described with respect to specific embodiments, it will be appreciated that the application may be further modified. In general, this application is intended to cover any variations, uses, or adaptations of the application following, in general, the principles of the application and including such departures from the present disclosure as come within known or customary practice within the art to which the application pertains. The application of some of the basic features may be done in accordance with the scope of the claims that follow.
Claims (6)
1. Middle-sized pseudoacidophilic bacteriaPseudacidovorax intermedius) XH-237 or a medium-sized pseudoacidophilic bacterium containing the samePseudacidovorax intermedius) Application of XH-237 microbial inoculum in nitrogen-containing wastewater treatment;
the medium-sized pseudoacidophilic bacteriaPseudacidovorax intermedius) XH-237 in ChinaThe number of the agricultural microorganism center of the Committee for conservation of the species is ACCC 62209;
the wastewater treatment is to biologically denitrify the water body;
the nitrogen in the wastewater is ammoniacal nitrogen, nitrite nitrogen and/or nitrate nitrogen.
2. The use according to claim 1, characterized in that: the pH of the wastewater is 5-11.
3. The use according to claim 1, characterized in that: the total salinity in the wastewater is not higher than 10% in mass fraction.
4. The use according to claim 1, characterized in that: mn in the wastewater 2+ Not higher than 10mmol/L, and/or Pb 2+ 、Fe 3+ Not higher than 5mmol/L, and/or Zn 2+ 、Cu 2+ 、Co 2+ 、Cd 2+ Not higher than 1mmol/L.
5. A biological denitrification method for nitrogen-containing wastewater, comprising the steps of: middle-sized pseudoacidophilic bacteriaPseudacidovorax intermedius) XH-237 or a medium-sized pseudoacidophilic bacterium containing the samePseudacidovorax intermedius) Inoculating XH-237 microbial inoculum to the nitrogenous wastewater to be treated for culturing;
the medium-sized pseudoacidophilic bacteriaPseudacidovorax intermedius) XH-237 has a deposit number ACCC 62209 in China Committee for culture Collection of microorganisms;
after inoculation, the medium-sized pseudoacidophilic bacteriaPseudacidovorax intermedius) XH-237 content in the wastewater system is 1×10 6 CFU/ml-3.0×10 7 CFU/ml；
The temperature at which the cultivation is carried out is 28-32 ℃;
the cultivation is carried out for 36-60h;
the nitrogen in the wastewater is ammoniacal nitrogen, nitrite nitrogen and/or nitrate nitrogen.
6. The method according to claim 5, wherein: the culture is 180r/min shake culture.
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| CN110656058A (en) * | 2018-06-29 | 2020-01-07 | 龙岩学院 | Heterotrophic nitrification-aerobic denitrification pseudomonas strain, seed liquid, and preparation method and application thereof |
| CN110655196A (en) * | 2018-06-29 | 2020-01-07 | 龙岩学院 | Method for treating nitrite nitrogen wastewater by using heterotrophic nitrification-aerobic denitrification pseudomonas strain |
| CN110655197A (en) * | 2018-06-29 | 2020-01-07 | 龙岩学院 | Method for treating nitrate nitrogen wastewater by using heterotrophic nitrification-aerobic denitrification pseudomonas strain |
| CN110655199A (en) * | 2018-06-29 | 2020-01-07 | 龙岩学院 | Method for treating ammonia nitrogen wastewater by using heterotrophic nitrification-aerobic denitrification pseudomonas strain |
| CN111172051A (en) * | 2018-11-12 | 2020-05-19 | 中国科学院沈阳应用生态研究所 | Method for expanding culture of rice straw degradation bacterial line |
| WO2021120364A1 (en) * | 2019-12-16 | 2021-06-24 | 南京农业大学 | Method and system for biologically treating acidic mine wastewater while recovering iron ion |
| CN111088200A (en) * | 2020-02-25 | 2020-05-01 | 山东大学 | Heterotrophic nitrification aerobic denitrifying bacterium and application thereof in pig-raising wastewater treatment |
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