CN115975845A - Application of salt-tolerant/acid-tolerant heterotrophic nitrification-aerobic denitrification bacterium in environmental wastewater treatment - Google Patents
Application of salt-tolerant/acid-tolerant heterotrophic nitrification-aerobic denitrification bacterium in environmental wastewater treatment Download PDFInfo
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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 invention discloses an application of a salt-tolerant/acid-tolerant heterotrophic nitrification-aerobic denitrification bacterium in environmental wastewater treatment. The invention provides application of a medium pseudomonad (Pseudomonas intermedia) XH-237 or a microbial inoculum containing the medium pseudomonad (Pseudomonas intermedia) XH-237 in nitrogen-containing wastewater treatment; the number of the pseudomonas mesophila (Pseudomonas intermedia) XH-237 in the agricultural microorganism center of the China Committee for culture Collection of microorganisms is ACCC 62209. The strain has the characteristics of acid resistance, salt resistance and heavy metal resistance, and the method for treating the environmental wastewater by using the strain is simple to operate, mild in reaction, short in time, good in effect and high in application value in sewage treatment engineering.
Description
Technical Field
The invention relates to the technical field of environmental microorganisms, in particular to application of a salt/acid tolerant heterotrophic nitrification-aerobic denitrification bacterium in environmental wastewater treatment.
Background
Nitrogen is a main component of nature, and is continuously converted and circulated between compounds with a valence of-5 to +3 and simple substances in a natural environment. Organic nitrogen and NH in natural environment 4 + -N、NO 2 - -N、NO 3 - the-N four forms exist stably in the water body environment. Accumulation of certain types of nitrogen does not occur in the steady-state environment of the nitrogen cycle, but in recent years, more and more human productive life activities have to some extent destroyed this natural cycle. With the industrial process, the agricultural development and the improvement of the living standard of residents, the problem of water body resource pollution is increasingly serious, and the water body resource pollution is high and still poses threats to the ecological environment and the human health. A large amount of nitrogen discharged from different sources cannot be effectively treated, and the discharge of the nitrogen to a water body can cause serious harm to human health and natural environment. When nitrate nitrogen and nitrite nitrogen in water exceed 'standard for drinking water DB 4403/T60-2020', and are more than 10mg/L, the infant is easy to be intolerant under long-term accumulation, blue infant disease and ferrous hemoglobin disease are caused, and cancer or death can be caused when the infant is serious. For natural ecological environment, continuous discharge and enrichment of high nitrogen can cause water body pollution and eutrophication, 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'. Similarly, when nitrite nitrogen in the water body is accumulated, the iron valence state in hemoglobin of the aquatic animals is easily changed after the aquatic animals absorb the nitrite nitrogen, the oxygen binding capacity of red blood cells is damaged, and finally the aquatic animals are suffocated and die. And the polluted water body enters plants along with agricultural irrigation, and is finally enriched to human bodies along with human drinking, so that the pollution causes great damage to the human bodies.
The biological denitrification mainly utilizes the nitrification and denitrification of denitrification related microorganisms, and various nitrogen-containing substances in the water are finally converted into nitrogen to escape under the action of enzyme passages of the microorganisms, so that the nitrogen-containing substances in the sewage are reduced or removed. At present, the biological denitrification technology is widely applied to the denitrification treatment of sewage by matching with related processes such as immobilization and the like. The reaction process mainly converts organic nitrogen into ammonia nitrogen, nitrite nitrogen and nitrate nitrogen in turn through ammoniation and nitrification under aerobic condition, and reduces the nitrate nitrogen into gaseous nitrogen to overflow from sewage under anoxic condition by using denitrification, thereby achieving the aim of denitrification. However, it has the following disadvantages: (1) Nitrifying bacteria are inorganic chemoautotrophic bacteria, and the nutrient metabolism type determines that the nitrifying bacteria grow slowly, have long generation period, low biomass concentration and poor environmental adaptability. (2) Traditional nitrifying bacteria are strictly aerobic bacteria, and traditional denitrifying bacteria are anaerobic or facultative anaerobic bacteria. The different conditions limit the influence on the denitrification rate of the water body under the same environment.
Thus, the search for strains having nitrification and denitrification functions began. In 1983, robertson et al successfully separated a strain which can oxidize ammonia nitrogen into hydroxylamine by using organic carbon under the condition of oxygen participation, convert the hydroxylamine into nitrite nitrogen and nitrate nitrogen, and finally generate nitrogen to be discharged out of a water body (NH) 4 + →NH 2 OH→NO 2 - →NO 3 - →NO 2 - →NO→N 2 O→N 2 ) The pantococcus erythropolis (Thiosphaera pantotropha) is revised as Paracoccus denitrificans (Paracoccus pantotrophus), so that the screening and the research of the nitrifying and denitrifying co-functional bacteria are started. And the bacteria react in a heterogeneous mode under Aerobic conditions, so that a Heterotrophic Nitrification-Aerobic Denitrification HN-AD (Heterotrophic Nitrification-Aerobic Denitrification) theory is proposed by Robertson. Compared with the traditional microorganism denitrifying bacteria, the heterotrophic nitrification-aerobic denitrifying bacteria have the advantages of high growth rate, short period, simple denitrifying procedure, high denitrifying rate, low requirement on the ecological 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 states in wastewater 4 + -N、NO 2 - -N、NO 3 - -N). The energy consumption is low, and the carbon source does not need to be added midway after the organic matter supply of the nitrification reaction is started while the bacteria are initially added in the denitrification reaction process. In the whole reactionThe acid and alkali generated in the process can be neutralized, and the pH stability of the system can be stabilized. However, most of the related reports are reports on the denitrification characteristics of strains, but the reports on heterotrophic nitrification aerobic denitrifying bacteria which have weak acid resistance, heavy metal resistance and salt resistance and can simultaneously remove ammonia nitrogen, nitrate nitrogen and nitrite nitrogen from environmental wastewater are not found.
Disclosure of Invention
The invention aims to provide application of a salt-tolerant/acid-tolerant heterotrophic nitrification-aerobic denitrification bacterium in environmental wastewater treatment.
In a first aspect, the present invention claims the use of Pseudomonas mesogenes (Pseudomonas intermedia) XH-237 or a bacterial agent comprising said Pseudomonas mesogenes XH-237 in the treatment of nitrogen containing wastewater. Wherein, the number of the pseudomonas mesophila (Pseudomonas intermedia) XH-237 in the agricultural microorganism center of China Committee for culture Collection of microorganisms is ACCC 62209.
Further, the wastewater treatment is to carry out biological denitrification on a water body.
Further, the nitrogen in the wastewater may be ammonia nitrogen, nitrous nitrogen, and/or nitrate nitrogen.
Further, the pH of the wastewater is not less than 5. E.g., pH 5-11, and further e.g., 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 more than 10mmol/L, pb 2+ 、Fe 3+ Not more than 5mmol/L, zn 2+ 、Cu 2+ 、Co 2+ 、Cd 2+ Not higher than 1mmol/L.
Further, the C/N ratio of the wastewater is 10 +/-0.2.
In a second aspect, the invention claims a method for treating wastewater.
The wastewater treatment method claimed by the invention can comprise the following steps: inoculating the pseudomonas mediterrae (Pseudomonadaceae acidovorax intermedia) XH-237 or a microbial inoculum containing the pseudomonas mediterrae (Pseudomonadaceae acidovorax intermedia) XH-237 to-be-treated wastewater for culture. Wherein, the number of the pseudomonas mesophila (Pseudomonas intermedia) XH-237 in the agricultural microorganism center of China Committee for culture Collection of microorganisms is ACCC 62209.
Further, the content of Pseudomonadaceae intermedia (Pseudomonas intermedia) XH-237 in the wastewater system after inoculation can 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 culturing may be carried out for a period of 36-60 hours, such as 48 hours.
Further, the culturing may be shaking culturing at 180 rpm.
Further, the wastewater is nitrogen-containing wastewater. Still further, the nitrogen in the wastewater may be ammoniacal nitrogen, nitrous nitrogen, and/or nitrate nitrogen.
Further, the pH of the wastewater is not less than 5. E.g., pH 5-11, and further e.g., 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 more than 10mmol/L, pb 2+ 、Fe 3+ Not more than 5mmol/L, zn 2+ 、Cu 2+ 、Co 2+ 、Cd 2+ Not higher than 1mmol/L.
Further, the C/N ratio of the wastewater is 10 +/-0.2.
In a third aspect, the invention claims the application of the pseudomonas mesophila (Pseudomonas intermedia) XH-237 or a microbial inoculum containing the same in biological denitrification. Wherein, the number of the pseudomonas mesophila (Pseudomonas intermedia) XH-237 in the agricultural microorganism center of China Committee for culture Collection of microorganisms is ACCC 62209.
In a particular embodiment of the invention, the biological denitrification is the lysis of the Pseudomonas intermedia (Pseudomonas intermedia)ius) XH-237 is inoculated into a heterotrophic nitrification-aerobic denitrification culture medium for culture, thereby completing denitrification; the heterotrophic nitrification-aerobic denitrification culture medium has the solvent of water, and has the following solutes and concentrations: 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) microelement solution; the pH was 7.2. The solvent of the trace element solution is water, and the solutes and the concentrations 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 content of said Pseudomonas intermedia (Pseudomonas intermedia) XH-237 in the culture system after inoculation may be 1X 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 culturing may be carried out for a period of 36-60 hours, such as 48 hours.
Further, the culturing may be shaking culturing at 180 rpm.
Further, the method may further comprise the step of activating and/or preparing a bacterial suspension of the Pseudomonadaceae (Pseudomonas xintermedius) XH-237 prior to the biological denitrification. The method specifically comprises the following steps: inoculating it to LB slant culture medium, and culturing at 35 deg.C for 1-2 days to obtain activated thallus. Then, the activated thallus is inoculated into 100ml LB liquid seed culture medium according to the conventional amount, and cultured for 12 hours at 35 ℃, thus obtaining the bacterial suspension of the pseudomonas mesophila (Pseudomonas intermedia) XH-237.
Experiments prove that the medium-sized pseudoacidophilic bacteria (Pseudomonas intermedia) XH-237 (ACCC 62209) can grow by taking ammonia nitrogen, nitrate nitrogen or nitrite nitrogen as a unique nitrogen source under aerobic conditionsSimultaneously carries out heterotrophic nitrification and aerobic denitrification, has the capability of removing ammonia nitrogen, nitrate nitrogen and nitrite nitrogen within 48h by 1 multiplied by 10 7 The bacterial amount of CFU/ml concentration is used for controlling nitrate Nitrogen (NO) in a heterotrophic nitrification-aerobic denitrification culture medium 3 - -N), nitrous Nitrogen (NO) 2 - -N), ammoniacal Nitrogen (NH) 4 + -N) removal rates were 25.38%, 17.20%, 83.28%, respectively. And can resist 10% of salt concentration and pH 5, respectively to 10mmol/L of metal ions Mn 2+ 5mmol/L of metal ion Pb 2+ 、Fe 3+ 1mmol/L of metal ion Zn 2+ 、Cu 2+ 、Co 2+ 、Cd 2+ Has tolerance. The method for treating the environmental wastewater by using the strain is simple to operate, mild in reaction, short in time, good in effect and high in application value in sewage treatment engineering.
Drawings
FIG. 1 shows the results of analysis of genetic evolutionary tree (neighbor) of Pseudomonas intermedia (XH-237) of the strain of the present invention.
FIG. 2 is a single colony morphology of Pseudomonadaceae intermedia (Pseudomonas intermedia) LB solid plate streaks of the present invention.
FIG. 3 is a 100-fold optical microscope morphology-plus-gram staining diagram of a Pseudomonadaceae (Pseudomonas intermedia) LB liquid culture according to the present invention.
FIG. 4 shows the effect of different carbon sources on the growth of Pseudomonadaceae intermedia (Pseudomonas intermedia) for 48h.
FIG. 5 shows the effect of different carbon nitrogen ratios on the growth of Pseudomonas mesogenes (Pseudomonas intermedia) for 48h.
FIG. 6 shows the effect of different pH on 48h growth of Pseudomonadaceae intermedia (Pseudomonas aeruginosa).
FIG. 7 is a graph showing the effect of different temperatures on the growth of Pseudomonas intermedia (Pseudomonas intermedia) for 48h.
FIG. 8 is a graph showing the effect of different salinity on 48h growth of Pseudomonas mesophila (Pseudomonas intermedia).
FIG. 9 shows the effect of different concentrations of heavy metals on the growth of Pseudomonas intermedia (Pseudomonas intermedia) 48.
FIG. 10 shows the ammonia nitrogen removal effect of Pseudomonas mesophila (Pseudomonas intermedia) for 48h.
FIG. 11 shows the nitrite nitrogen removal effect of Pseudomonas intermedia (Pseudomonas intermedia) for 48h.
FIG. 12 shows the effect of Pseudomonas intermedia (Pseudomonas intermedia) for 48h on nitrate nitrogen removal.
FIG. 13 shows the effect of Pseudomonas mesophila (Pseudomonas intermedia) for 48h on the removal of mixed nitrogen sources.
Detailed Description
The present invention is described in further detail below with reference to specific embodiments, which are given for the purpose of illustration only and are not intended to limit the scope of the invention. The examples provided below serve as a guide for further modifications by a person skilled in the art and do not constitute a limitation of the invention in any way.
The experimental procedures in the following examples, unless otherwise specified, were carried out in a conventional manner according to the techniques or conditions described in the literature in this field or according to the product instructions. Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.
The following examples of the medium-sized Pseudoacidovorax intermedia XH-237 were collected in China microbial cultures Collection center (Agricultural Culture Collection of China, abbreviated as ACCC, address: no. 12 of south China Central China center for Culture Collection of Haematococcus, beijing, institute of Agricultural resources and Agricultural divisions, postal code 100081) by the date of the application of the present application, and the strain number is ACCC 62209, which was collected at 6/28 th 2022 by the date of Collection, and was obtained by the public from the Agricultural microbial cultures center of the China microbial cultures Collection center. The ACCC is provided with a special website, and the website address is as follows: http:// www.accc.org.cn, the public can directly order strains on the internet. The website address of Pseudomonadaceae (Pseudomonas intermedia) XH-237 is http:// www.accc.org.cn/column. Asp? Column _ ID =34929 and model = product _udetail &p _id =1817447492.
Example 1 isolation and identification of Pseudomonadaceae (Pseudomonas intermedia) XH-237
A sludge sample of a No. 2 sedimentation tank of a Pond sewage treatment plant of the Anhui province Hefei city is taken, and is subjected to coating separation after being acclimatized and enriched by a carbon dioxide carbon source and a nitrate nitrogen source, so that the type Pseudoacidovorax intermedia (Pseudomonas acidovorax intermedia) XH-237 is obtained.
1. Domesticating enrichment, separation culture medium and expanding culture medium
Domesticating an enrichment medium: the solvent is water, and the solutes and the concentrations are as follows: 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) microelement solution; the pH was 7.2. Solution of trace elements: the solvent is water, and the solutes and the concentrations 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。
Separating a culture medium: the solvent is water, and the solutes and the concentrations 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) microelement solution; pH7.2. Solid medium was supplemented with agar 2% (20 g/L) and 0.1% (V/V) bromothymol indicator solution.
Expanding culture medium: the solvent is water, and the solutes and the concentrations are as follows: 10g/L of tryptone and 5g/L, naCl of yeast extract; pH7.2.
2. Separation and purification of bacterial strain XH-237
Preparing sterile water for the sample acclimatized in the high-nitrate-nitrogen environment into 100mL of solution according to the proportion of 1:9, wherein the dilution multiple of the solution is 10 -1 1mL of the solution was added to a 9mL test tube containing sterile water,repeating this step can sequentially dilute to 10 -2 、10 -3 、10 -4 、10 -5 For a total of five gradients. In a sterile ultra-clean workbench, 20 mu L of bacterial suspension with 5 gradient concentrations is respectively taken on the surface of the prepared solid separation culture medium, the solid separation culture medium is uniformly coated by a sterile glass coating rod, after the bacterial suspension completely permeates into the solid separation culture medium, a culture dish cover is covered, the culture dish is sealed, and three concentration gradients are parallel. The plate to be cultured is placed upside down in an incubator with the culture temperature of 30 ℃ for culture for 2-4d. After the clear single strains grow out, screening and marking the single strains to be picked by a marker pen and counting. In an aseptic environment, opening a culture dish, dipping a small amount of single bacterial colony in a prepared LB culture medium by using an aseptic inoculating loop, slightly scribing to avoid scratching, slowly rotating a flat plate after an area is full, placing the inoculating loop on an alcohol lamp for burning, removing microorganisms on the inoculating loop, waiting until the temperature of the inoculating loop is cooled, and connecting the painted area and a blank area together to continue scribing. One plate was scribed in 4 zones and the plate was sealed with a sealing film. The plates were inverted and incubated at 30 ℃ in an incubator for 2-3 days. If the streaked strain is not a single strain, then it is necessary to re-streak until the streaked pure culture is a single strain. Each strain was purified twice more. The obtained strain is an XH-237 strain.
3. PCR amplification and sequencing of 16S rRNA
The DNA of the obtained XH-237 strain was extracted and purified using a Solebao bacterial DNA extraction D1600 kit (Beijing Solebao technologies, ltd.). 5 mu L of DNA sample is taken for electrophoresis detection under the condition of 110V and 20min, and quality inspection is carried out under a gel electrophoresis instrument.
Bacterial universal primers 16S-27F (5'-AGAGTTTGATCCTGGCTCAG-3') and 16S-1492R (5'-CGGTTACCTTGTTACGACTTC-3') were selected for PCR amplification. The PCR reaction conditions are as follows: pre-denaturation at 94 ℃ for 3min; denaturation at 94 ℃ for 30s, annealing at 55 ℃ for 30s, extension at 72 ℃ for 90s, and circulating for 35 cycles; then extending for 10min at 72 ℃; finally, the mixture is stored at 4 ℃.
And sequentially carrying out PCR product purification and quality inspection, purifying the product, and sequencing the recovered PCR amplification product by committee of biotechnology, ltd.
The 16S rDNA of the strain obtained after sequencing was 1422bp in length (SEQ ID No. 1), and the gene sequence of the 16S rRNA of the XH-237 strain of the invention was found to have 99.72% homology with the gene sequence of the 16S rRNA of the NCBI-registered multiple Pseudomonas intermedia (Pseudomonas intermedia) strain, when aligned using the program BLASTN of the National Center for Biotechnology Information (NCBI). The phylogenetic tree (FIG. 1) was constructed by using the phylogenetic analysis (MEGA 11) software ortho-ligation (Neighbour Joining) method, which indicates that the XH-237 strain of the invention is closely related to the known Pseudomonas mesophila DSM 21353.
4. Observation of morphological characteristics of XH-237 Strain
The experimental method for morphological feature observation is described in "Manual of identification of common bacteria System", scientific Press, 2001, first edition, p353-363, authored by east elegant bead, cai Miaoying, and the like.
Morphological characteristics of XH-237 Strain: the colony is circular, convex, neat in edge, smooth in surface, light beige and slightly transparent when cultured on an LB solid medium, as shown in figure 2. Culturing in LB liquid culture medium, observing bacillus under strain microscope, forming no spore, making thallus form straight or slightly bent, and making two ends of rod-shaped, gram-positive, as shown in FIG. 3.
5. Physiological and biochemical characteristic identification of XH-237 strain
Physiological and biochemical test media and experimental methods reference is made to BIOLOG GenIII plate BIOLOG, inc., US Patent #5,627,045, bilog, usa.
Physiological and biochemical characteristics of the XH-237 strain: gram stain is positive and aerobic, the optimal growth temperature is 30-40 ℃, and fructose, galactose, glycerol, salt resistance and acid resistance are utilized at the temperature of 30 ℃; the results of the physiological and biochemical experiments are detailed in Table 1.
TABLE 1 Biochemical characteristics of part of the XH-237 Strain biolog GenIII plates
Note: "(+)" grows well or is positive; "(-) -does not grow or is negative; "(W)" growth was near blank or false positive.
In view of the morphological characteristics, physiological and biochemical characteristics and 16S sequencing results of the XH-237 strain, the XH-237 strain was identified as a medium pseudoacidophile (Pseudomonas intermedius) and was collected in the Agricultural microorganism center of China Committee for Culture Collection of microorganisms, also known as the China Agricultural Culture Collection, ACCC (Agricultural Culture Collection of China, ACCC for short) at 28 th 6 th 2022, at the address of No. 12 southern Settlement of Guancun, in the Hakken zone of Beijing, the institute of Agricultural resources and Agricultural zoning, the postal code 100081.
6. Carbon source influences the growth of Pseudomonadaceae intermedia
Suspending a medium-sized pseudoacidophilic bacteria (Pseudomonas intermedia) XH-237 suspension cultured in an LB culture medium by PBS, respectively adding glucose, sucrose, sodium acetate, sodium citrate and sodium succinate in a substrate general culture medium as a unique carbon source, and selecting 100mg/L 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 ratios in the control medium are all C/N =10 (basal universal 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) microelement solution; the pH was 7.2. Solution of trace elements: 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). 30 ℃ and 180rpm cultureAfter 48h of cultivation, the system was monitored for the OD600 value of Pseudomonadaceae (Pseudoacidovorax intermedia). The results show that sodium acetate was selected as the carbon source for the subsequent experimental media, indicating that Pseudomonas mesophila (Pseudomonas intermedia) XH-237 utilizes sodium acetate for the fastest growth rate. See in particular fig. 4.
7. Carbon-nitrogen ratio influences the growth of Pseudomonas mesogenes (Pseudomonas intermedia) XH-237
Suspending the suspension of XH-237 species of Pseudomonadaceae (Pseudomonas intermedia) cultured in LB with PBS, selecting sodium acetate as the sole carbon source, and selecting 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 mixed nitrogen sources (33.33 mg/L of three nitrogen sources, respectively) were nitrogen sources, and different amounts of C/N of 5, 10, 15, 20, 25 were added to the basal general medium, respectively. (substrate general-purpose 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) microelement solution; the pH was 7.2. Solution of trace elements: 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; pH7.0), culturing at 30 ℃ and 180rpm for 48h, and monitoring the OD600 value of Pseudomonacolis (Pseudoacidovorax intermedia) in the system. The results show that the growth rate of the Pseudomonas mediterrae (Pseudomonas intermedia) XH-237C/N is 10 fastest, so that the C/N is 10 selected as the carbon nitrogen ratio of the culture medium in the subsequent experiment. See in particular fig. 5.
8. Effect of pH on growth of Pseudomonadaceae intermedia XH-237
The medium pseudomonad (Pseudomonas intermedium) XH-237 is respectively inoculated into LB culture medium with the pH value of 3, 5, 7, 9 and 11, cultured for 48h at 30 ℃ and 180rpm, and the OD600 value of the medium pseudomonad (Pseudomonas intermedium) in the system is monitored. The results show that the Pseudomonas mesogenes (Pseudomonas intermedia) XH-237 has a good growth rate at a pH of alkaline 7 to 11. Subsequent experiments were performed with a pH of 7. See in particular fig. 6.
9. Temperature Effect on growth of Pseudomonadaceae intermedia XH-237
The medium pseudoacidophilic bacteria (Pseudopseudomonas intermedia) XH-237 is respectively inoculated into LB culture medium with the temperature value of 10 ℃,20 ℃,30 ℃, 40 ℃ and 50 ℃, cultured for 48 hours at 30 ℃ and 180rpm, and the OD600 value of the medium pseudoacidophilic bacteria (Pseudopseudomonas intermedia) in the system is monitored. The results show that the medium-sized pseudoacidophilic bacteria (Pseudomonas intermedia) XH-237 has a good growth rate at a temperature of 30-40 ℃. Subsequent experiments were carried out at a temperature of 30 ℃. See in particular fig. 7.
10. Salinity influence on growth of Pseudomonadaceae (Pseudomonas intermedia) XH-237
The medium-sized pseudoacidophile (Pseudomonas intermedium) XH-237 is respectively inoculated into LB culture medium with the salinity of 0%, 1%, 3%, 5% and 10% (mass fraction of NaCl), cultured for 48h at 30 ℃ and 180rpm, and then the OD600 value of the medium-sized pseudoacidophile (Pseudomonas intermedium) in the system is monitored. The results show that the Pseudomonas mesophila (Pseudomonas intermedia) XH-237 has a good growth rate at salinity of 5% and can tolerate salinity of 10%. See in particular fig. 8.
11. Pseudomonadaceae (Pseudomonas intermedia) XH-237 in tolerance to heavy metals
Respectively inoculating Pseudomonas intermedia (XH-237) into 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 the strain in the LB medium (Amersham pharmacia) at 30 ℃ and 180rpm for 48 hours, the OD600 value of Pseudomonacolis (Pseudoacidovorax intermedia) in the strain was monitored. The results showed that Pseudomonas intermedia (Pseudoacidovorax intermedia) XH-237 was 10mmol/L of the metal ion Mn, respectively 2+ 5mmol/L of metal ion Pb 2+ 、Fe 3+ 1mmol/L of metal ion Zn 2+ 、Cu 2+ 、Co 2+ 、Cd 2+ Has tolerance. See in particular fig. 9.
12. Ability of Pseudomonas intermedia (Pseudomonas intermedia) XH-237 to remove 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) measuring by adopting a 'measuring Nagowski reagent spectrophotometry for ammonia nitrogen in water' (HJ 535-2009); nitrous Nitrogen (NO) 2 - -N) using diazo coupling spectrophotometry for nitrite nitrogen determination in water (GB 7493-87); nitrate Nitrogen (NO) 3 - -N) method for determining nitrate nitrogen using ultraviolet spectrophotometric water quality (HJ 346-2007).
The media formulations used below were as follows:
basic propagation culture medium: the solvent is water, and the solutes and the concentrations are as follows: tryptone 10.0g/L; 5.0g/L of yeast powder; naCl 10.0g/L.
Denitrifying performance determination culture 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) microelement solution; the pH was 7.2.
Nitrite denitrification performance determination culture 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) microelement solution; the pH was 7.2.
Ammonium salt ammonification performance determination culture 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) microelement solution; the pH was 7.2.
Heterotrophic nitrification-aerobic denitrification culture 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) microelement solution; the pH was 7.2.
The trace element solution comprises 50g/L of EDTA and 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 Pseudomonadaceae (Pseudomonas intermedia) XH-237 on LB plate culture medium, and culturing at 35 deg.C for 24h to obtain activated thallus.
The prepared activated thallus is inoculated into 100ml of LB liquid seed culture medium according to the proportion of 5 percent according to the conventional quantity, and cultured for 12 hours at 35 ℃ to obtain the pseudomonad (pseudomonad intercedius) seed suspension.
Re-suspending the prepared seed suspension with PBS, and inoculating to corresponding ammonia nitrogen removal capacity determination culture medium to make the bacterial load in the system be 1 × 10 7 CFU/ml. Culturing at 30 deg.C and 180rpm for 48h, and measuring ammoniacal Nitrogen (NH) in the culture solution every 12h 4 + -N), nitrous Nitrogen (NO) 2 - -N), nitrate Nitrogen (NO) 3 - -N) an index. Determination of nitrate Nitrogen (NO) in culture medium by denitrification performance within 48h 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 ammoniation Performance of ammonium salt Ammonia Nitrogen (NH) in culture Medium 4 + -N) removal rate 44.1%; nitrate Nitrogen (NO) in heterotrophic nitrification-aerobic denitrification culture medium 3 - -N), nitrous Nitrogen (NO) 2 - -N), ammoniacal Nitrogen (NH) 4 + -N) removal rates were 25.38%, 17.20%, 83.28%, respectively. The results are shown in detail in FIGS. 10 to 13.
The present invention has been described in detail above. It will be apparent to those skilled in the art that the invention can be practiced in a wide range of equivalent parameters, concentrations, and conditions without departing from the spirit and scope of the invention and without undue experimentation. While the invention has been described with reference to specific embodiments, it will be appreciated that the invention can be further modified. In general, this application is intended to cover any variations, uses, or adaptations of the invention following, in general, the principles of the invention and including such departures from the present disclosure as come within known or customary practice within the art to which the invention pertains. The use of some of the essential features is possible within the scope of the claims attached below.
Claims (10)
1. The application of the pseudomonas mesophila (Pseudomonas intermedia) XH-237 or a microbial inoculum containing the same in the treatment of nitrogen-containing wastewater;
the number of the pseudomonas mesophila (Pseudomonas intermedia) XH-237 in the agricultural microorganism center of the China Committee for culture Collection of microorganisms is ACCC 62209.
2. Use according to claim 1, characterized in that: the wastewater treatment is to carry out biological denitrification on a water body.
3. Use according to claim 2, characterized in that: the nitrogen in the wastewater is ammonia nitrogen, nitrite nitrogen and/or nitrate nitrogen.
4. Use according to any one of claims 1 to 3, characterized in that: the pH of the wastewater is not lower than 5.
5. Use according to any one of claims 1 to 4, characterized in that: the total salinity in the wastewater is not higher than 10 percent by mass fraction.
6. Use according to any one of claims 1 to 5, characterized in that: mn in the wastewater 2+ Not more than 10mmol/L, and/or Pb 2+ 、Fe 3+ Not more than 5mmol/L, and/or Zn 2+ 、Cu 2+ 、Co 2+ 、Cd 2+ Not higher than 1mmol/L.
7. A method of wastewater treatment comprising the steps of: inoculating a medium-sized pseudoacidophilic bacterium (Pseudomonas intermedium) XH-237 or a microbial inoculum containing the medium-sized pseudoacidophilic bacterium (Pseudomonas intermedium) XH-237 to the wastewater to be treated for culturing;
the medium pseudoacidophilic bacterium (Pseudoacidovorax intermedia) XH-237 has the preservation number of ACCC 62209 in the agricultural microorganism center of China Committee for culture Collection of microorganisms.
8. The method of claim 7, wherein: the content of the pseudomonas mesophila (Pseudomonadaceae intemediate) XH-237 in the wastewater system after inoculation is 1 x 10 6 CFU/ml-3.0×10 7 CFU/ml。
9. The method according to claim 7 or 8, characterized in that: the temperature for the culture is 28-32 ℃; and/or
The time for carrying out the culture is 36-60h; and/or
The culture is shaking culture at 180 r/min.
10. Application of pseudomonas mesophila (Pseudomonas intermedia) XH-237 or a microbial inoculum containing the same in biological denitrification;
the medium-sized pseudoacidophilic bacteria (Pseudomonas intermedia) XH-237 has the preservation number of ACCC 62209 in the agricultural microorganism center of the China Committee for culture Collection of microorganisms.
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