CN114262679A - Autotrophic denitrifying bacteria and rapid propagation process thereof - Google Patents
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Abstract
The invention provides an autotrophic denitrogenation bacterium and a rapid propagation process thereof, wherein the strain is identified as bacillus albicans (Bacillus subtilis)Bacillus albus) Compared with other autotrophic denitrifying bacteria, the autotrophic denitrifying bacteria have the advantages of low cost, low energy consumption, less bacteria in effluent and good water quality. Under the condition that the initial concentration of ammonia nitrogen is 50mg/L, the removal rate of nitrogen can reach 100% within 48h, and the degradation effect is very obvious. Meanwhile, the rapid propagation process of the strain is also disclosed, and has the advantages of simple operation, short production period, low production cost, practical application value and wide market prospect.
Description
Technical Field
The invention relates to the field of environmental microorganisms, in particular to an autotrophic denitrogenation bacterium and a rapid propagation process thereof.
Background
Nitrogen pollutants are one of the important reasons for causing water eutrophication, and nitrogen pollution control is a water environment problem which needs to be solved urgently in China and even in the world at present. The nitrogen pollutants in the polluted water bodies such as domestic sewage, polluted surface water, industrial sewage and the like are mainly ammonia nitrogen and nitrate. Among the various denitrification techniques, biological denitrification is considered to be classified into heterotrophic denitrification and autotrophic denitrification depending on the carbon source required by bacteria. The heterotrophic denitrification needs to additionally add an organic carbon source as a donor, and the cost generated by the additional donor is high, so that the problem of subsequent pollution also exists; the autotrophic nitrogen removal does not need to add extra carbon source, and the effluent has the characteristics of low bacteria content and good effluent quality. Autotrophic denitrifying bacteria have therefore attracted general attention from researchers in recent years.
Compared with heterotrophic biological denitrification, autotrophic denitrification has the advantages of low cost, low energy consumption, less bacteria in effluent and good water quality, so that the autotrophic denitrification becomes a key point and a hotspot for research in the field of denitrification. Therefore, it is very important to screen strains capable of autotrophic nitrogen removal with high efficiency.
Disclosure of Invention
The invention aims to provide an autotrophic denitrogenation bacterium and a rapid propagation production process thereof, and provides an effective solution for solving the actual sewage treatment problem.
The technical scheme of the invention is as follows:
an autotrophic denitrogenation bacterium, which is classified and named as Bacillus albus (NZHB-Z1), and has been preserved in China general microbiological culture collection management center at 11/19 th 2021, with the address: no. 3 Xilu No. 1 Beijing, Chaoyang, and the preservation number is CGMCC NO. 23933.
The physiological characteristics are: thick and long rod-shaped with blunt ends and thick capsule around the thallus. Spore formation: the wall thickness is oval, the colony is round, colorless, and the ridge is thick, transparent or semitransparent, and the edge is neat; autotrophic aerobic bacteria, gram positive; obvious colonies appeared after 24h of agar solid plate culture, and the colonies were round or oval, neat in edge, white, large and flat, opaque, moist and matt. Gram stain is positive, and the nutrition mode is an autotrophic aerobic type.
The strain NZHB-Z1 is obtained by screening sludge in an anaerobic pool of a certain sewage plant of Jiangsu salt city, and the screening culture medium is an LB culture medium which comprises the following components: 10.0g of peptone, 5.0g of yeast powder, 10.0g of NaCl, 15.0g of agar, 1.0L of distilled water and pH 7.0-7.3. The culture temperature is as follows: aerobic culture is carried out at 37 ℃.
The culture method of the strain comprises the following steps:
(1) and (3) activation: picking a single colony of the white bacillus from the solid plate, transferring the single colony into an LB liquid culture medium, wherein the rotating speed of a shaking table is 200rpm, and carrying out shaking table culture at 37 ℃ for 3-4 h until the logarithmic phase; the LB culture medium comprises the following components: 5g/L yeast extract and 10g/L peptone, and the pH value is kept between 7.0 and 7.3;
(2) transferring: transferring the bacillus albicans liquid activated in the flow 1 in the logarithmic phase to a 50L seeding tank according to the inoculation amount of 5% for culturing, controlling the temperature of the seeding tank to be 30-37 ℃ and the DO to be 4-6mg/L, and culturing for 24-48 h. The culture medium of the seeding tank comprises the following components in percentage by mass: NH (NH)4Cl 0.1％，NaNO2 0.15％，NaHCO3 0.2％，KH2PO4 0.1％，MgSO4 0.05％，CaCl20.02 percent. The pH value is maintained between 7.0 and 7.3.
(3) And (3) rapid propagation: transferring the white bacillus liquid cultured in the seeding tank of the process 2 to a 1000L fermentation tank for amplification culture according to the inoculation amount of 5%, wherein the components of a culture medium of the fermentation tank are the same as those of the seeding tank, and the indexes of physical and chemical parameters are as follows: the temperature is 37 ℃, the rotating speed is 250rpm, DO is controlled to be 4-6mg/L, and the fermentation time is 48-60 h. After fermentation is finished, the effective viable count of the tank body bacterial liquid can reach more than 500/ml, and the white bacillus microbial inoculum can be obtained after the fermentation culture liquid is taken out of the tank and packaged by a plastic barrel.
The application of the autotrophic denitrogenation bacteria NZHB-Z1 in degrading ammonia nitrogen organic matters.
Application of autotrophic denitrogenation bacteria NZHB-Z1 in rapid formation of denitrogenation sludge.
A denitrified sludge is prepared by inoculating Bacillus albus NZHB-Z1 into a reactor filled with sludge, and rapidly expanding culture to obtain denitrified sludge.
The method for quickly forming the denitrified sludge by the autotrophic denitrified bacteria NZHB-Z1 comprises the following steps:
s1, adding inoculated sludge into the reactor, and conveying sewage and NH of the sewage from a bottom device4 +N400-500 mg/L, the temperature in the device is 30-32 ℃, the rotation speed is maintained at 250rpm at 220-;
s2, setting the retention time to be 24h, and adding nutrient elements and trace elements;
the nutrient elements comprise the following components in percentage by weight: NaHCO 23 0～1％，KH2PO40～1％，MgSO 40～1％，CaCl20-1% of yeast extract and 0-0.1% of yeast extract.
The trace elements are as follows: fe. Cu, Mo, Zn, Co, Mn.
The mass ratio of the trace elements is as follows: 0-1 g/L Fe, 0-1 mg/L Cu, 0-1 mg/L Mo, 0-1 mg/L Co, 0-1 mg/L Mn.
Preferably, the addition amount of the nutrient elements is as follows: NaHCO 23 0.2％，KH2PO4 0.1％，MgSO4 0.05％，CaCl20.02 percent, 0.01 percent of yeast extract and the pH value is maintained between 7.0 and 7.3.
Preferably, the dosage of the trace elements is as follows: fe 10mg/L, Cu 0.3mg/L, Mo 0.1mg/L, Co 0.1mg/L, Mn 0.1 mg/L.
S3, adding the white bacillus NZHB-Z1 into the reactor in a one-time adding mode, wherein the temperature in the reactor is 30-32 ℃, and the pH value is controlled to be 7.0-7.3. The bacterial strain is inoculated into the sludge according to the addition amount of 0.1-10%, and the sludge is quickly expanded to obtain denitrified sludge for the subsequent treatment process of the nitrogen-containing wastewater.
The mass ratio of the addition amount of the strain to the sludge inoculation is 0.1-10%.
The ratio H/D of the height to the diameter of the reactor is 4-8.
Autotrophic denitrogenation bacteria NZHB-Z1The application of the bacillus albus NZHB-Z1 in degrading ammonia nitrogen organic matters is to ferment the bacillus albus NZHB-Z1, and the microbial inoculum obtained by fermentation is added into the solution containing NH according to the volume ratio of 10 percent4 +And (4) carrying out anaerobic treatment on the-N wastewater to degrade ammonia nitrogen organic matters in the wastewater.
The autotrophic denitrogenation bacteria NZHB-Z1 provided by the invention are easy to degrade ammonia nitrogen organic matters, no additional carbon source is needed, and the degradation rate is high. Compared with other autotrophic denitrifying bacteria, the method has the advantages of low cost, low energy consumption, less bacteria in effluent and good water quality. Under the condition of laboratory shaking culture, the initial concentration of NH of 50mg/L within 48h can be realized by NZHB-Z14 +-N is completely removed. Meanwhile, the practical treatment effect of the autotrophic denitrogenation bacteria NZHB-Z1 on the low-concentration nitrogen-containing wastewater is verified. Adding the bacterial agent fermented by NZHB-Z1 into NH containing 20mg/L according to the volume ratio of 10 percent4 +NH in the-N wastewater after anaerobic treatment for 24h4 +The degradation rate of-N can reach 98 percent, and the treatment effect is very ideal.
Has the advantages that:
the expanding culture method can be used for expanding culture on a project site, and the cultured bacteria liquid can be immediately added and used on a use site, so that the transportation cost and the time cost are saved, and the negative influence caused by long-term maintenance can be avoided.
The method for expanding culture of the invention realizes effective on-site expanding culture of bacteria at project sites by providing nutrient substances, oxygen and space required by proliferation for the strains, ensures effective connection of expanding culture and application, and ensures the number of viable bacteria of effective microorganisms, the concentration of the autotrophic ammonia-oxidizing bacteria liquid expanded and cultured by the method of the invention is about 500 bacteria in per milliliter of the liquid, and the transportation cost and the time cost can be greatly reduced.
The invention uses autotrophic denitrogenation bacteria NZHB-Z1 to treat NH in low-concentration nitrogen-containing wastewater for 24h after anaerobic treatment4 +The degradation rate of-N can reach 98%, the treatment effect is very ideal, the operation is simple, the investment cost is low, and the practicability is strong.
Drawings
FIG. 1 shows the growth curve of strain NZHB-Z1 and degradation experiment of ammonia nitrogen;
FIG. 2 shows the degradation rate of the strain NZHB-Z1 in actual nitrogen-containing wastewater.
Detailed Description
The industrial wastewater treatment plant nitrified sludge described in the examples originates from: a secondary sedimentation tank of a pesticide chemical plant in Jiangsu salt city.
Example 1 screening of autotrophic anammox bacteria
The autotrophic denitrogenation bacteria of the invention are obtained by the inventor from sludge at the bottom of a certain lake of Nanjing through an inorganic salt culture medium (NH)4Cl 0.1％，NaNO2 0.15％，NaHCO3 0.2％，KH2PO4 0.1％，MgSO4 0.05％，CaCl20.02 percent of the culture medium is subjected to shaking culture at the temperature of 30 ℃) under 160r/min, and then a single colony is obtained by screening after streak culture. The morphological characteristics of the strain of the invention are as follows: gram staining is positive, the nutrition mode is autotrophic aerobic type, the bacterial strain is thick and long rod-shaped, the two ends are blunt and round, and thick capsules are arranged around the bacterial strain. Spore formation: wall thickness, round colony, colorless, bump, sticky colloid, transparent or translucent, and neat edge.
Example 2 method for culturing autotrophic anammox bacteria NZHB-Z1
The culture method of the autotrophic denitrogenation bacteria NZHB-Z1 comprises the following steps:
(1) and (3) activation: and (3) selecting a single colony of the white bacillus from the solid plate, transferring the single colony into an LB liquid culture medium, and carrying out shake culture at 37 ℃ for 3-4 h until the logarithmic phase at the rotation speed of a shaker of 200 rpm. The LB culture medium comprises the following components: 5g/L yeast extract and 10g/L peptone, and the pH value is kept between 7.0 and 7.3.
(2) Transferring: transferring the bacillus albicans liquid activated in the logarithmic phase in the step 1 to a 50L seeding tank according to the inoculation amount of 5% for culturing, controlling the temperature of the seeding tank to be maintained at 30 ℃ and controlling DO to be 5mg/L, and culturing for 36 h. The culture medium of the seeding tank comprises the following components in percentage by mass: NH (NH)4Cl 0.1％，NaNO2 0.15％，NaHCO3 0.2％，KH2PO4 0.1％，MgSO40.05％，CaCl20.02 percent. The pH value is maintained between 7.0 and 7.3.
(3) And (3) rapid propagation: transferring the white bacillus liquid cultured in the seeding tank in the step 2 into a 1000L fermentation tank for amplification culture according to the inoculation amount of 2-5%, wherein the components of a culture medium of the fermentation tank are the same as those of the seeding tank, and the indexes of physical and chemical parameters are as follows: the temperature is 37 ℃, the rotating speed is 250rpm, DO is controlled to be 4-6mg/L, and the fermentation time is 72. After fermentation is finished, the effective viable count of the tank body bacterial liquid can reach more than 500/ml, and the white bacillus microbial inoculum can be obtained after the fermentation culture liquid is taken out of the tank and packaged by a plastic barrel.
EXAMPLE 3 growth Curve of Strain NZHB-Z1 and degradation experiment on Ammonia Nitrogen
Selecting a single colony of purified bacillus albicans NZHB-Z1 in a shake flask of an inorganic salt culture medium containing 100mg/L ammonia nitrogen, wherein the inorganic salt culture medium comprises the following components in percentage by weight: NH (NH)4Cl 0.1％，NaNO2 0.15％，NaHCO3 0.2％，KH2PO4 0.1％，MgSO4 0.05％，CaCl20.02 percent. The rotation speed of the shaker is 180rpm, the temperature is 37 ℃, and the shaking culture is carried out. The bacterial body concentration OD600 and the ammonia nitrogen concentration are respectively sampled and measured at 0h, 6h, 12h, 18h, 24h, 30h, 36h, 42h, 48h, 60h and 72 h. OD600 is measured by a visible light spectrophotometer, and the ammonia nitrogen concentration is measured by liquid chromatography. The data are shown in figure 1, under the condition that the initial concentration of ammonia nitrogen is 100mg/L, the strain NZHB-Z1 grows slowly within 12h, so that the degradation of ammonia nitrogen is slow, the degradation speed of ammonia nitrogen is obviously improved along with the increase of the concentration of bacteria, and the removal rate of ammonia nitrogen reaches 50% after 24 h. When the culture is carried out for 48 hours, the ammonia nitrogen achieves the effect of complete degradation, the thalli grow and lose the nitrogen source along with complete degradation of the ammonia nitrogen, so that the thalli tend to be stable, and the OD600 value is stabilized to be about 1.2. The data show that the strain NZHB-Z1 has good degradation effect on ammonia nitrogen, and the thallus culture is very convenient and fast.
EXAMPLE 4 application of Strain NZHB-Z1 in actual Nitrogen-containing wastewater
In order to further verify the application effect of the bacillus albicans NZHB-Z1 in actual nitrogen-containing wastewater, the ammonia nitrogen-containing production wastewater is obtained from a chemical plant sewage station which takes ammonia nitrogen as a main production raw material. Through measurement, the concentration of ammonia nitrogen in the wastewater is 200mg/L and is higher. The bacillus albicans NZHB-Z1 bacterial liquid obtained by culturing in example 2 is directly added into ammonia nitrogen production wastewater according to the inoculation amount of 10%, and the pH of the system is adjusted to 7.2 so as to meet the requirement of normal growth of microorganisms. Under the condition of aeration at room temperature, sampling and monitoring the change of ammonia nitrogen in the system at regular time according to 0h, 2h, 6h, 10h, 12h, 18h and 24 h. The ammonia nitrogen degradation data are shown in the attached figure 2: the ammonia nitrogen in the wastewater is degraded within 8 hours, the ammonia nitrogen concentration is reduced from 200mg/L to 150g/L, the degradation effect reaches 25%, the aniline concentration is reduced to 50mg/L after 12 hours, and the degradation rate is 75%. After 24 hours, the ammonia nitrogen concentration is reduced to 4mg/L, and the degradation rate is up to 98%. The experimental data show that the Bacillus albicans NZHB-Z1 has a good treatment effect in the actual ammonia nitrogen-containing wastewater, and has a wide application prospect.
Example 5
Sludge inoculation: taking nitrified sludge of industrial wastewater treatment plant as inoculated sludge and initial NH of wastewater4+The N is 150mg/L, the temperature of the reactor is 31 ℃, the pH is controlled at 7.3, the VSS of the inoculated sludge is 15g/L, the SS is 35g/L, and the retention time is 24 h.
And (3) nutrient elements: NaHCO 23 0.2％，KH2PO4 0.1％，MgSO4 0.05％，CaCl20.02% and 0.01% of yeast extract.
Trace elements: the added trace elements are Fe 10mg/L, Cu 0.3mg/L, Mo 0.1mg/L, Co 0.1mg/L and Mn 0.1 mg/L.
The reactor configuration: a reactor manufactured by Wuhan Hengmega Happy laboratory facilities Ltd is used, and the ratio H/D of the height of the reactor to the diameter of the reactor is 6.
Time to sludge formation: and (4) 120 days.
Ammonia nitrogen removal: 90 percent.
Example 6
Sludge inoculation: taking nitrified sludge of industrial wastewater treatment plant as inoculated sludge and initial NH of wastewater4+N is 50mg/L, the temperature of the reactor is 31 ℃, the pH is controlled to be 7.3, the VSS of the inoculated sludge is 15g/L, the SS is 35g/L, and the retention time is 24 h.
Adding a small amount of white bacillus (NZHB-Z1) into a reactor filled with inoculated sludge, wherein the adding amount of the bacillus is estimated by the sludge inoculation amount, and the mass ratio of the bacillus to the sludge is as follows: 1 percent.
And (3) nutrient elements: NaHCO 23 0.3％，KH2PO4 0.1％，MgSO4 0.05％，CaCl20.02% and 0.01% of yeast extract.
Trace elements: the added trace elements are Fe 10mg/L, Cu 0.3mg/L, Mo 0.1mg/L, Co 0.1mg/L and Mn 0.1 mg/L.
The reactor configuration: a reactor manufactured by Wuhan Hengmega Happy laboratory facilities Ltd is used, and the ratio H/D of the height of the reactor to the diameter of the reactor is 6.
Time to form granular sludge: for 100 days.
Ammonia nitrogen removal: 80 percent.
Example 7
Inoculating sludge with digested sludge of industrial wastewater treatment plant as the initial NH of the inoculated sludge and wastewater4+N is 50mg/L, the temperature of the reactor is 31 ℃, the pH is controlled to be 7.3, the VSS of the inoculated sludge is 15g/L, the SS is 35g/L, and the retention time is 24 h.
Adding a small amount of white bacillus (NZHB-Z1) into a reactor filled with inoculated sludge, wherein the adding amount of the bacillus is estimated by the sludge inoculation amount, and the mass ratio of the bacillus to the sludge is as follows: 5 percent.
And (3) nutrient elements: NaHCO 23 0.3％，KH2PO4 0.1％，MgSO4 0.05％，CaCl20.02 percent of yeast extract and 0.01 percent of yeast extract;
trace elements: none;
the reactor configuration: adopting a reactor produced by Wuhan Henghaxing experimental equipment Limited company, wherein the ratio H/D of the height to the diameter of the reactor is 6;
time to form granular sludge: 150 days;
ammonia nitrogen removal: 75 percent.
Claims (10)
1. An autotrophic denitrogenation bacterium, which is classified and named as Bacillus albus (NZHB-Z1), has been preserved in China general microbiological culture Collection center with the preservation number of CGMCC NO. 23933.
2. The denitrified sludge is characterized in that bacillus albicans NZHB-Z1 is inoculated into a reactor added with sludge, and rapid propagation is carried out to obtain the denitrified sludge.
3. The use of the autotrophic anammox bacteria of claim 1 for degrading ammonia-nitrogen containing organic compounds.
4. The application of claim 3, wherein the bacillus albicans NZHB-Z1 is cultured and then added into the wastewater to be treated to degrade ammonia nitrogen organic matters; the culture method of the bacillus albicans NZHB-Z1 comprises the following steps: and (3) selecting a single colony to an LB liquid efficient culture medium containing ammonia nitrogen, transferring to a seeding tank for culturing for 24-48 h after the culture is carried out to a logarithmic phase, and finally inoculating to a fermentation tank for amplification culture for 48-72 h to obtain a large amount of autotrophic denitrifier enriched bacterial liquid.
5. A rapid propagation process of autotrophic denitrogenation bacteria is characterized in that: inoculating bacillus albicans NZHB-Z1 into a reactor added with sludge, and adding trace elements Fe, Cu, Mo, Zn, Co and Mn into the reactor.
6. The process of claim 4, wherein the culture medium comprises: the mass ratio of the trace elements is as follows: 0-1 g/L Fe, 0-1 mg/L Cu, 0-1 mg/L Mo, 0-1 mg/L Co, 0-1 mg/L Mn.
7. The rapid propagation process of autotrophic anammox bacteria according to claim 4, wherein: the mass ratio of the addition amount of the strain to the sludge inoculation is 0.1-10%.
8. The rapid propagation process of autotrophic anammox bacteria according to claim 4, wherein: the reactor delivers the sewage, NH of sewage, from the bottom unit4+400-500 mg/L of-N, the temperature in the device is 30-32 ℃, and the pH is controlled to be 7.0-7.3.
9. The method of claim 4The method for quickly forming the denitrified sludge is characterized by comprising the following steps: adding nutrient elements into the reactor, wherein the proportion of the nutrient elements is as follows: NaHCO 23 0~1%， KH2PO40~1%，MgSO40~1%，CaCl20-1% of yeast extract, 0-0.1% of yeast extract and pH value maintained between 7.0-7.3.
10. Use of the strain of claim 1 for the rapid formation of denitrified sludge.
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