CN112175863B - Preparation method and application of complex microbial inoculant for removing nitrogen-containing pollutants in high-salinity wastewater - Google Patents
Preparation method and application of complex microbial inoculant for removing nitrogen-containing pollutants in high-salinity wastewater Download PDFInfo
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Abstract
The invention relates to a preparation method and application of a composite microbial inoculum for removing nitrogen-containing pollutants in high-salinity wastewater, wherein the composite microbial inoculum is prepared by compounding Rhodococcus erythropolis C1, Leibetococcus rhodochrous, Pseudomonas aeruginosa, Alcaligenes faecalis and Acinetobacter. The composite microbial inoculum has the characteristics of high salt resistance and high nitrogen resistance, and can realize high-efficiency denitrification of high-nitrogen wastewater in an aerobic environment under the high-salt condition through the synergistic effect of five microorganisms.
Description
Technical Field
The invention belongs to the technical field of microorganisms, and particularly relates to a preparation method and application of a composite microbial inoculum for removing nitrogen-containing pollutants in high-salinity wastewater.
Background
Along with the aggravation of the industrialized process of China, the amount of high-salt (salinity is more than 3%) wastewater generated in the production processes of chemical wastewater, pickling wastewater, printing and dyeing wastewater, oil extraction wastewater and the like is also greatly increased, the nitrogen content of most of high-salt wastewater is also higher, the high-nitrogen wastewater is improperly treated and directly discharged into a water body, the phenomena of water body eutrophication and the like are caused, and the quality of the water body is deteriorated.
At present, there are various methods for denitrification treatment of wastewater, such as physical method, chemical method, biological method, etc. The physical and chemical methods have the defects of high operation cost and environmental pollution because a large amount of medicaments are required to be added, so that the treatment cost is higher, and the excessive medicaments easily cause secondary pollution; biological processes are widely used for denitrification of wastewater because of their economical efficiency and high efficiency.
The conventional biological method has great advantages when denitrification treatment is carried out under the condition of low salinity, but when the salinity exceeds 3.5 percent, the concentration of chloride ions in high-salinity wastewater is too high, the cell osmotic pressure of microorganisms can be rapidly changed, so that thallus cells are destroyed, the bacterial growth is inhibited, and particularly, the biological method has extremely strong toxic action on NOB (nitrate-oxidizing bacteria) bacteria, anaerobic ammonium oxidation bacteria and the like, so that the common biological methods for the high-salinity wastewater comprise an activated sludge method, an oxidation pond method, an artificial wetland method and the like, wherein the methods comprise the following steps: the activated sludge process has poor impact load resistance; the oxidation pond method and the artificial wetland method have large occupied area, are greatly influenced by natural factors such as seasons, temperature and the like, and have unstable treatment effect and unobvious effect.
Disclosure of Invention
The invention aims to provide a preparation method and application of a composite microbial inoculum for removing nitrogen-containing pollutants in high-salinity wastewater, aiming at the defects of the prior art. The composite microbial inoculum has the characteristics of high salt resistance and high nitrogen resistance, and can realize high-efficiency denitrification of high-nitrogen wastewater in an aerobic environment under the high-salt condition through the synergistic effect of five microorganisms.
The Total Nitrogen (TN) of the invention is composed of nitrate Nitrogen (NO)3 -) And (5) characterizing.
The technical scheme of the invention is as follows:
a composite microbial inoculum for removing nitrogen content in high-salinity wastewater is prepared by compounding Rhodococcus erythropolis C1, Leibetococcus motilis, Pseudomonas aeruginosa, Alcaligenes faecalis and Acinetobacter.
The concentrations of Rhodococcus erythropolis C1, Leibetococcus motilis, Pseudomonas aeruginosa, Alcaligenes faecalis and Acinetobacter are OD600=1.0 ± 0.2, deep redRhodococcus C1: motilin lepigone: the bacterium pseudomonas aeruginosa: alcaligenes faecalis: the volume ratio of the acinetobacter is 10-15%: 20-30%: 10-20%: 15-20%: 10 to 20 percent.
The preservation number of the rhodococcus erythropolis C1 is CCTCC NO: m2019725, the number of the Planococcus lepigone is CICC 23939, the number of the bacterium Pseudomonas aeruginosa is CICC 20152, the number of the Alcaligenes faecalis is CICC 22642, and the number of the Acinetobacter is CICC 10695.
The preparation method of the composite microbial inoculum comprises the following steps:
1) domestication of strains
Under the condition of high nitrogen, adopting a salinity gradient domestication method to carry out high-salt and high-nitrogen tolerance domestication on each strain;
2) expanded culture of strains
Using carbon-containing compounds as carbon sources, and respectively carrying out amplification culture on the strains domesticated in the step 1) in a CM culture medium with the salinity of 12%;
3) preparation of complex microbial inoculum
Mixing the bacterial liquid obtained in the step 2) according to the proportion to obtain seed liquid, and carrying out enrichment culture on the seed liquid until the seed liquid reaches OD600And (5) keeping the concentration at 1.0 +/-0.2 to obtain the compound microbial inoculum.
The carbon-containing compound in the step 2) is one or more of anhydrous sodium acetate, succinic acid and glucose.
The method for the enlarged culture of each strain in the step 2) comprises the following steps:
rhodococcus erythropolis C1
Taking 10-20 mL of bacterial liquid to a CM culture medium with the salinity of 12%, sealing, and carrying out shake culture for 3d at 37 ℃ and 150 r/min;
leibetococcus sp
Taking 10-20 mL of bacterial liquid to a CM culture medium with the salinity of 12%, sealing, and carrying out shake culture for 3d at 37 ℃ and 150 r/min;
pseudomonas aeruginosa
Taking 10-20 mL of bacterial liquid to a CM culture medium with the salinity of 12%, sealing, and carrying out shake culture for 3d at 37 ℃ and 150 r/min;
alcaligenes faecalis
And (3) taking 10-20 mL of bacterial liquid to a CM culture medium with the salinity of 12%, sealing, and carrying out shake culture for 3d at 37 ℃ at 150 r/min.
Acinetobacter
And (3) taking 10-20 mL of bacterial liquid to a CM culture medium with the salinity of 12%, sealing, and carrying out shake culture for 3d at 37 ℃ at 150 r/min.
The salt of salinity is NaCl.
The composite microbial inoculum is applied to removing the nitrogen content of high-salinity wastewater.
Rhodococcus rubrum C1 (Rhodococcus ruber C1) belongs to the genus Rhodococcus. The colony is round, opaque, orange red, dry, irradiated at the edge, slightly raised, smooth in surface and wet in texture. The diameter is 4-6 μm, the length is 8-12 μm, gram staining is positive, the optimal growth temperature is 37-40 ℃, and the pH is 7.5-8.0.
Leibetococcus (Leibeto) ((R))Planococcus rifietoensis，CICC 23939), belonging to the genus planococcus. The diameter of a bacterial colony is about 1.0-1.2 UM, no spore is produced, aerobic bacteria are generated, the bacterial colony is yellow orange, the thallus is spherical, gram staining is positive, the optimal growth temperature is 27-37 ℃, and the pH value is 7.5-8.2.
Pseudomonas aeruginosa (Pseudomonas aeruginosaCICC 20152) belonging to the genus Pseudomonas, wherein the diameter of a bacterial colony is about 1.5-4.0 UM, spores are not formed, the bacterial colony is semitransparent and protuberant, the edge is neat, the surface is glossy, the color is white and transparent, and the thallus is in a short rod shape; gram staining was negative. The optimal growth temperature is 30-40 ℃, and the pH is 7.5-8.0.
Alcaligenes faecalis (Alcaligenes faecalis CICC 22642) belonging to the genus Alcaligenes faecalis. The diameter is 0.5-1.0 mm, the length is 0.5-2.6 mm, and the single thallus arrangement is usually adopted, so no spores are produced. Gram staining is negative, the optimal growth temperature is 20-37 ℃, and the pH value is 7.5-8.0.
Acinetobacter (Acinetobacter CICC 10695) belongs to the genus Acinetobacter, is gram-negative, has the cell size of 1.5-2.5 um, and has a mucous strain with capsules, no spores, no flagella and no movement. Obligate aerobic, the optimal growth temperature is 20-37 ℃, and the pH is 7.5-8.0.
The Rhodococcus erythropolis C1 is preserved as CTCC NO: m2019725 "(deposited in the China center for type culture Collection, at address: Wuhan university, Wuhan, China, 10/2/2019) was classified and named Rhodococcus ruber C1.
The Leibetococcus sp is purchased from China center for Industrial culture Collection of microorganisms (number CICC 23939).
The bacterium pseudomonas aeruginosa is purchased from China center for culture collection of industrial microorganisms (number CICC 20152).
Alcaligenes faecalis was purchased from the China center for Industrial culture Collection of microorganisms (accession number CICC 22642).
Acinetobacter is purchased from the China center for Industrial culture Collection of microorganisms (accession number CICC 10695).
The composite microbial inoculum capable of efficiently removing nitrogen under the high-salt condition has the following beneficial effects:
(1) the complex microbial inoculum provided by the invention can resist high osmotic pressure and grow rapidly under the condition of high salt (3-15%), and can keep high activity for a long time.
(2) The composite microbial inoculum provided by the invention can efficiently remove nitrogen in a complete aerobic environment under a high-salt condition (3-15%), overcomes the strong toxic action of a hypertonic environment on microorganisms, and simultaneously solves the two-stage biological denitrification limitation of aerobic-anaerobic combination in the traditional biological denitrification process.
(3) The composite microbial inoculum provided by the invention has the advantages that the components of a culture medium required by the amplification culture of each single bacterium are simple, the cost is low, and the large amount of enrichment can be realized in a short time.
(4) The composite microbial inoculum provided by the invention is convenient to use and small in dosage, can be directly put into a water body to form dominant bacteria for efficiently removing nitrogen, has the advantages of high treatment efficiency and low treatment cost, and can be widely popularized and applied.
The composite microbial inoculum can effectively solve the problems that the biomass is extremely low due to the rupture and death of microbial cells in a high-salinity environment, and particularly the biological denitrification effect is extremely low in the high-salinity environment due to the strong inhibition of nitrifying bacteria, denitrifying bacteria and the like by a high-permeability environment.
The present invention will be described in detail below with reference to the accompanying drawings and specific embodiments.
Drawings
FIG. 1 shows the removal of nitrate nitrogen by a complex microbial inoculum at 30 ℃ and a nitrate nitrogen concentration of 490 mg/L;
FIG. 2 shows the removal of nitrate nitrogen by the complex microbial inoculum under different salinity conditions;
FIG. 3 shows the situation of the composite microbial inoculum for removing the total nitrogen in the tuber mustard wastewater at 30 ℃.
Detailed Description
1. Experimental Material
Cm (czapek medium) medium: NaNO3 3g，KH2PO4 1.5 g，MgSO4·7H2O 0.5 g， KCl 0.5 g,，FeSO40.01 g/L, 30 g/L sucrose, adjusting the salinity of the culture medium by NaCl, and adjusting the pH value of the CM culture medium to 8.0.
The rest reagents are commercial analytical pure products.
2. Detection method
And detecting the OD value of the bacterial liquid by using a UV2000 spectrophotometer, wherein the wavelength is 600 nm. As a method for monitoring and analyzing contaminants, reference is made to "Water and wastewater monitoring and analyzing method" (fourth edition, published by environmental science, China 2002).
Example 1:
step 1) domestication of strains
High salt tolerance acclimatization of Rhodococcus erythropolis C1 (CCTCC NO: M2019725): inoculating 2mL of the bacterial solution into a 250 mL triangular flask containing 100 mL of sterilized CM medium (the concentration of nitric acid nitrogen is 490 mg/L), shaking thoroughly, and heating at 30 deg.C for 150 r min-1Culturing under the condition for 3 d. Under the same conditions, NaCl concentrations were increased, and serial subcultures were carried out for 6 passages, corresponding to NaCl concentrations of 3%, 5%, 7%, 10%, 13%, and 15%, respectively.
Zoococcus lepigone (CICC 23939) high salt tolerance acclimation: inoculating 2mL of the bacterial solution into 100 mL of sterilized CM medium (nitric acid nitrogen concentration is 4)90 mg/L) in a 250 mL triangular flask, shaking thoroughly, 30 ℃, 150 r.min-1Culturing under the condition for 3 d. Increasing NaCl concentration (same as increasing NaCl concentration of Rhodococcus erythropolis C1, the same below), and performing continuous subculture for 6 times.
High salt tolerance acclimatization of pseudomonas aeruginosa (cic 20152): inoculating 2mL of the bacterial solution into a 250 mL triangular flask containing 100 mL of sterilized CM medium (the concentration of nitric acid nitrogen is 490 mg/L), shaking thoroughly, and heating at 30 deg.C for 150 r min-1Culturing under the condition for 3 d. NaCl concentration was increased under the same conditions as above, and serial subculture was carried out for a total of 6 passages.
High-salt tolerance domestication of Alcaligenes faecalis (CICC 22642): inoculating 2mL of the bacterial solution into a 250 mL triangular flask containing 100 mL of sterilized CM medium (the concentration of nitric acid nitrogen is 490 mg/L), shaking thoroughly, and heating at 30 deg.C for 150 r min-1Culturing under the condition for 3 d. NaCl concentration was increased under the same conditions as above, and serial subculture was carried out for a total of 6 passages.
High salt tolerance acclimatization of acinetobacter (CICC 10695): inoculating 2mL of the bacterial solution into a 250 mL triangular flask containing 100 mL of sterilized CM medium (the concentration of nitric acid nitrogen is 490 mg/L), shaking thoroughly, and heating at 30 deg.C for 150 r min-1Culturing under the condition for 3 d. NaCl concentration was increased under the same conditions as above, and serial subculture was carried out for a total of 6 passages.
Step 2) expanded culture of strains
Carrying out amplification culture on each strain domesticated in the step 1) in a CM culture medium with the salinity (NaCl) of 12% by taking anhydrous sodium acetate as a carbon source;
method for enlarging culture of Rhodococcus erythropolis C1 (CCTCC NO: M2019725)
Inoculating 10-20 mL of the acclimatized bacteria solution with high nitrogen and high salt tolerance into a 2000 mL triangular flask containing 1000 mL of sterilized CM medium (the same below) with a salinity of 12%, shaking thoroughly, and standing at 30 deg.C for 150 r min-1Culturing under the condition for 3 d.
Method for expanding culture of Leibetococcus (CICC 23939)
Taking 10-20 mL of the mixture subjected to high-nitrogen and high-salt toleranceInoculating the domesticated bacteria solution into 2000 mL triangular flask containing 1000 mL sterilized CM medium with salinity of 12%, shaking thoroughly, 30 deg.C, 150 r.min-1Culturing under the condition for 3 d.
Method for expanding culture of pseudomonas aeruginosa (CICC 20152)
Inoculating 10-20 mL of the high-nitrogen high-salt tolerant acclimatized bacterial liquid into a 2000 mL triangular flask filled with 1000 mL of sterilized CM medium with the salinity of 12%, sufficiently shaking, and carrying out the steps of 30 ℃, 150 r.min-1Culturing under the condition for 3 d.
Method for expanding culture of alcaligenes faecalis (CICC 22642)
Inoculating 10-20 mL of the high-nitrogen high-salt tolerant acclimatized bacterial liquid into a 2000 mL triangular flask filled with 1000 mL of sterilized CM medium with the salinity of 12%, fully shaking, and culturing for 3d at 30 ℃ under the condition of 150 r.min < -1 >.
Method for expanding culture of acinetobacter (CICC 10695)
Inoculating 10-20 mL of the high-nitrogen high-salt tolerant acclimatized bacterial liquid into a 2000 mL triangular flask filled with 1000 mL of sterilized CM medium with the salinity of 12%, fully shaking, and culturing for 3d at 30 ℃ under the condition of 150 r.min < -1 >.
3) Preparation of complex microbial inoculum
Taking 1-1.5 ml of the Rhodococcus erythropolis C1 bacterial liquid obtained in the step 2), 2-3 ml of the Leibetococcus rhodochrous bacterial liquid, 1-2 ml of the Pseudomonas aeruginosa bacterial liquid, 1.5-2 ml of the Alcaligenes faecalis bacterial liquid and 1-2 ml of the Acinetobacter bacteria liquid, and mixing to obtain a seed liquid.
Inoculating 20 mL of seed solution into 2000 mL of sterilized CM medium with salinity of 12%, shaking thoroughly, at 30 deg.C and 150 r min-1Culturing under the condition of 4 d. When OD is reached600After the value is increased to 0.7, the stationary phase is reached, and the composite microbial inoculum is obtained.
Example 2: experiment for efficiently removing nitrogen in artificial wastewater with salinity of 12 percent by using composite microbial inoculum
The formula of the artificial wastewater is as follows: sodium acetate 4g, NaNO3 3g/L，KH2PO4 0.35g/L，NaHCO3 0.56g/L, and the nitrogen concentration in the obtained artificial wastewater is490 mg/L。
Taking 100 mL of artificial wastewater into a 250 mL conical flask, and adding 2mL of complex microbial inoculum (OD) into the flask by using a micropipettor6000.9-1.2), sealing with sealing film, and placing in a shaking table at 30 deg.C and 150 r min-1Culturing under the condition, and measuring OD of bacterial liquid every 24 h600The growth condition of the thalli is determined, and the content of nitrogen (nitrate) in the artificial wastewater is simultaneously measured, so that the nitrogen removal effect is determined. As can be seen from FIG. 1, the removal rate of the complex microbial inoculum to nitrogen (nitrate) in the artificial wastewater is as high as 91.2% under the condition that the salinity is 12%.
Example 3: high-efficiency nitrogen removal experiment of composite microbial inoculum under different salinity
The formula of the artificial wastewater is as follows: sodium acetate 4g, NaNO3 3g/L，KH2PO4 0.35g/L，NaHCO3 0.56g/L, and the nitrogen concentration in the obtained artificial wastewater is 490 mg/L.
Taking 100 mL of artificial wastewater into a 250 mL conical flask, and adding 2mL of complex microbial inoculum (OD) into the flask by using a micropipettor6001-2), sealing with sealing film, setting different conical flasks to different salinity (3%, 5%, 7%, 10%, 13%, 15%), respectively, placing in a shaking table at 30 deg.C and 150 r.min-1Culturing under the conditions, and determining OD of the bacterial liquid600The growth condition of the thalli is determined, and the content of nitrogen (nitrate) in the artificial wastewater is simultaneously measured, so that the nitrogen removal effect is determined.
As shown in FIG. 2, the complex microbial inoculum can realize the high-efficiency removal of nitrogen (nitrate) in the artificial wastewater within the salinity of 3-15%, and the removal rate is 87.7% -98%.
Example 4: experiment for treating preserved szechuan pickle wastewater by using composite microbial inoculum
Taking 100 mL of hot pickled mustard tuber waste water (from Chongqing Fuling national food Co., Ltd.) into a 250 mL conical flask, and adding 2mL of compound microbial inoculum (OD) into the flask by using a micropipettor6001-2), sealing with sealing film, placing in a shaking table at 30 deg.C and 150 r min-1Culturing under the condition, and measuring OD of bacterial liquid every 24 h600Value determination of thallus growth and simultaneous determination of total nitrogen in waste waterAnd (4) determining the removal effect. As shown in FIG. 3, the composite microbial inoculum can efficiently remove the total nitrogen in the tuber mustard wastewater, and the removal rate is as high as 83.5%.
And (4) conclusion: the applicant finds in experiments that the composite bacteria compounded by rhodococcus erythropolis C1, Leibetococcus motilis, pseudomonas aeruginosa, alcaligenes faecalis and acinetobacter can realize high-efficiency removal of total nitrogen under the conditions of high salt and high nitrogen, and the removal rate is as high as 83.5% -98%.
Claims (7)
1. The composite microbial inoculum for removing the nitrogen content in the high-salinity wastewater is characterized by being compounded by rhodococcus erythropolis C1, Leibetococcus motile, pseudomonas aeruginosa, Alcaligenes faecalis and acinetobacter, wherein the preservation number of the rhodococcus erythropolis C1 is CCTCC NO: m2019725, the number of the Planococcus lepigone is CICC 23939, the number of the Pseudomonas aeruginosa is CICC 20152, the number of the Alcaligenes faecalis is CICC 22642, and the number of the Acinetobacter is CICC 10695.
2. The complex microbial inoculant according to claim 1, wherein the concentrations of Rhodococcus erythropolis C1, Leibetococcus rhodochrous, Pseudomonas aeruginosa, Alcaligenes faecalis and Acinetobacter are OD600=1.0 ± 0.2, rhodococcus erythropolis C1: motilin lepigone: pseudomonas aeruginosa: alcaligenes faecalis: the volume ratio of the acinetobacter is 10-15%: 20-30%: 10-20%: 15-20%: 10 to 20 percent.
3. The preparation method of the composite microbial inoculum is characterized by comprising the following steps:
1) domestication of strains
Performing high-salt and high-nitrogen tolerance acclimation on each strain of claim 1 under a high-nitrogen condition by adopting a salinity gradient acclimation method;
2) expanded culture of strains
Using carbon-containing compounds as carbon sources, and respectively carrying out amplification culture on the strains domesticated in the step 1) in a CM culture medium with the salinity of 12%;
3) preparation of complex microbial inoculum
Taking the bacterial liquid obtained in the step 2), mixing the bacterial liquid according to the volume ratio of claim 2 to obtain seed liquid, and carrying out enrichment culture on the seed liquid until the seed liquid reaches OD600And (5) keeping the concentration at 1.0 +/-0.2 to obtain the compound microbial inoculum.
4. The preparation method according to claim 3, wherein the carbon-containing compound in step 2) is one or more of anhydrous sodium acetate, succinic acid and glucose.
5. The method according to claim 3, wherein the step 2) comprises the steps of:
rhodococcus erythropolis C1
Taking 10-20 mL of bacterial liquid to a CM culture medium with the salinity of 12%, sealing, and carrying out shake culture for 3d at 37 ℃ and 150 r/min;
leibetococcus sp
Taking 10-20 mL of bacterial liquid to a CM culture medium with the salinity of 12%, sealing, and carrying out shake culture for 3d at 37 ℃ and 150 r/min;
pseudomonas aeruginosa
Taking 10-20 mL of bacterial liquid to a CM culture medium with the salinity of 12%, sealing, and carrying out shake culture for 3d at 37 ℃ and 150 r/min;
alcaligenes faecalis
Taking 10-20 mL of bacterial liquid to a CM culture medium with the salinity of 12%, sealing, and performing shake culture for 3d at 37 ℃ and 150 r/min;
acinetobacter
And (3) taking 10-20 mL of bacterial liquid to a CM culture medium with the salinity of 12%, sealing, and carrying out shake culture for 3d at 37 ℃ at 150 r/min.
6. The method of claim 5, wherein: the salt of salinity is NaCl.
7. The use of the composite bacterial agent of any one of claims 1-2 for removing nitrogen content in high-salinity wastewater.
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