CN110157639B - High-salt-tolerance denitrifying bacterium and preparation method and application of microbial inoculum thereof - Google Patents
High-salt-tolerance denitrifying bacterium and preparation method and application of microbial inoculum thereof Download PDFInfo
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Abstract
The invention discloses a high-salt-tolerance denitrifying bacterium and a preparation method and application of a microbial inoculum thereof, belonging to the field of environmental microorganisms, wherein the bacterium is named as Pseudomonas stutzeri, belongs to an anaerobic denitrifying bacterium strain, and has a preservation number of CGMCC No. 15310. The pseudomonas stutzeri has high salt-tolerant denitrification characteristic, the denitrification rate reaches more than 99 percent, and the high-concentration nitrate nitrogen in the water body can be obtained under the condition that the salt content is less than or equal to 6 percent
Description
The technical field is as follows:
the invention belongs to the field of environmental microorganisms, and particularly relates to a salt-tolerant denitrifying bacterium, and a preparation method and application thereof.
Background art:
along with the development of industrial and agricultural production and the improvement of living standard of people, the discharge amount of nitrogen-containing pollutants in China is rapidly increased, and the nitrogen-containing pollutants are one of the main sources of water eutrophication. According to rough statistics, about 3000 thousands of people in China drink high-nitrate saline water, and nitrate pollution becomes one of the main environmental factors for cancer occurrence in China. After being taken into human and animal bodies, part of nitrate is reduced into nitrite. Nitrite can oxidize hemoglobin in blood into methemoglobin which does not have the capacity of combining oxygen, and when the methemoglobin content in blood is increased, the oxygen conveying capacity of the blood is reduced, and serious patients cause purpura of human tissues, which is clinically called as methemoglobinemia. At present, three-stage treatment is generally adopted in national urban sewage treatment, the nitrogen and phosphorus removal effect is good, the effluent of most sewage plants can reach the first-level A discharge standard, and the continuous deterioration of water quality pollution is effectively restrained.
However, the high-concentration nitrate waste liquid generated in industrial activities has the characteristics of complex water quality components, organic pollutants, high salinity, strong biological toxicity and the like, and increases the difficulty of denitrification of the waste liquid. Waste liquid discharged from some industrial activities such as chemical fertilizer manufacturing, gunpowder manufacturing, metal surface pickling, electroplating, circuit printing plate etching and the like contains high-concentration nitrate and nitrite. For example, the content of nitrate in the waste liquid discharged by a potassium nitrate production plant reaches 2640mg/L and the content of nitrite in the waste liquid reaches 640 mg/L. For example, the concentration of nitrate contained in waste liquid generated when the rack is deplated in the electroplating industry is as high as 100000 mg/L.
For the denitrification of waste liquid, the conversion of nitrate nitrogen into nitrogen by the denitrification of biological method is the most economic and thorough treatment technology. However, the high salinity waste liquid discharged from industrial production can cause the osmotic pressure of the biological membrane of the microorganism in the conventional biological treatment system to be higher and the cell membrane of the microorganism to be broken; microbial enzyme activity is inhibited; the sludge settling effect becomes poor, etc. Therefore, the conventional biological denitrification process cannot effectively perform denitrification under the condition of high salt, and the processes such as ion exchange, evaporative crystallization, reverse osmosis, catalytic reduction and the like are commonly adopted in the industry for treating the high-concentration nitrate waste liquid at present, but the methods have the problems of pollution transfer, secondary pollution and the like, and the final treatment of pollutants cannot be realized. Therefore, screening of high-efficiency strains with salt tolerance is one of effective ways for solving the denitrification and denitrification of high-concentration nitrate under the high-salt condition.
For example, the invention patent with the publication number of CN 108118008A discloses a high-efficiency salt-resistant microbial agent, a preparation method and application thereof, and a complex microbial agent is utilized: at least one of Paracoccus FSTB-2, Microbacterium beijerinckii FSTB-4 and Pseudomonas stutzeri FSTB-5, simultaneously contains Marcroaker F S D N-A and Staphylococcus cohnii FSDN-C, and contains at least one of Arthrobacter FDN-1 and Flavobacterium aquatile FDN-2. The invention also discloses a preparation method and application of the microbial inoculum. The microbial agent can be directly used for treating total nitrogen and COD in wastewater with high salt content, and can also be added into various biochemical reaction structures to improve the microbial composition, optimize the salt resistance of a microbial system in wastewater treatment and improve the removal rate of the total nitrogen and COD of the system. But also can see that the invention utilizes the combination of the microbial inoculum to treat the waste liquid, thereby improving the cost of the raw material of the microbial inoculum and the time and labor cost for preparing the microbial inoculum invisibly.
The invention content is as follows:
the invention provides a denitrifying bacterium which is efficient, tolerant to high salt and capable of degrading high-concentration nitrate, and a preparation method and application thereof, and aims to solve the problems that in the prior art, the high-concentration nitrate waste liquid is not thoroughly treated and has secondary pollution, and the problems that the conventional denitrification process is poor in salt tolerance effect, complex in treatment process, high in cost and the like.
A high-salt-tolerance denitrifying bacterium is Pseudomonas stutzeri (Pseudomonas stutzeri), and the preservation number is CGMCC No. 15310.
The pseudomonas stutzeri is preserved in the common microorganism center of China general microbiological culture Collection center in 2018, 1 month and 25 months, the preservation number is CGMCC No.15310, and the preservation address is as follows: western road No.1, north chen west road, north kyo, chaoyang, institute of microbiology, china academy of sciences, zip code 100101.
The pseudomonas stutzeri is obtained by taking sludge in an industrial waste liquid anaerobic pool, domesticating for 400 days, separating and screening, and gradually increasing the salinity and nitrate concentration of an anaerobic sludge substrate in the domestication process for 400 days.
The pseudomonas stutzeri is characterized in that: gram negative, the color of the bacterial colony is light yellow, the bacterial colony is a single convex bacterial colony, the surface of the bacterial colony is wrinkled, and the edge of the bacterial colony is irregular; the shape of the thallus is rod-shaped, straight or bent into arc shape when observed under a projection electron microscope.
Preferably, the suitable culture temperature of the anaerobic denitrifying bacteria is 20-40 ℃, and the pH value is 6.6-8.0.
The concentration of tolerance sodium chloride salt of the pseudomonas stutzeri in the waste liquid is 1-6%, and the tolerance nitrate ion is
The concentration is 100-30000 mg/L.
The invention also aims to provide the microbial inoculum for resisting the high-salt denitrifying bacteria, which is specifically dry microbial inoculum or microbial inoculum with the microbial inoculum activity of 103-104cfu/L bacterial suspension.
The invention also aims to provide a preparation method of the high-salt-resistant denitrifying bacteria agent, which comprises the following steps: the method comprises the following specific steps:
(1) fermentation culture: activating the pseudomonas stutzeri, inoculating the activated pseudomonas stutzeri into a fermentation culture medium according to the inoculation amount of 3% -10%, and controlling the fermentation temperature: 20-40 ℃, dissolved oxygen <0.2mg/L, pot pressure: shaking and culturing at 0.03-0.10Mpa and 30-50rpm for 72-96h to obtain fermentation broth; the fermentation medium comprises the following components: 6g/L-12g/L peptone, 3g/L-6g/L yeast extract, 8-12g/L sodium chloride, and the balance of distilled water, with pH of 7.5, sterilizing at 121 deg.C for 20 min;
(2) and (2) centrifuging and freeze-drying the fermentation liquor obtained in the step (1) to obtain the thallus dry powder or centrifuging and diluting the thallus to obtain the thallus suspension.
Preferably, in the step (1), the activating method of pseudomonas stutzeri comprises the following steps: inoculating the pseudomonas stutzeri in a seed culture medium, controlling the culture temperature to be 30-38 ℃, and performing shake culture at 30-50rpm for 48-72 h.
Preferably, the seed liquid culture medium consists of: KNO3 2g/L-4g/L，MgSO4·7H2O 0.2g/L-0.4g/L，K2HPO40.5-0.8 g/L, 20-30 g/L of sodium potassium tartrate, 8-12g/L of sodium chloride and the balance of water, the pH value is 7.2, and the sterilization is carried out for 20min at 121 ℃.
Preferably, in the step (2), the centrifugation conditions are as follows: centrifugation temperature: the rotation speed is 6000-7500rpm at 4 ℃, and the centrifugation time is 10-15 min.
Preferably, in the step (2), the bacterial suspension is obtained by diluting the bacterial precipitate obtained by fermentation with the fermentation medium or with saline water with sodium chloride ion strength such as the fermentation medium.
And the diluting with the equal sodium chloride ion strength specifically comprises the step of diluting the thallus precipitate by using saline with the sodium chloride concentration of 8-12g/L to obtain a bacterial suspension.
More preferably, in the step (2), the bacterial suspension is specifically obtained by diluting the bacterial precipitate obtained by fermentation with the fermentation medium.
Another object of the present invention is to provide the use of the above-mentioned high-salinity-tolerant denitrifying bacteria in the denitrification treatment of waste liquid.
Preferably, the concentration of sodium chloride salt in the waste liquid is 1-6%, and nitrate ions are tolerated
The concentration is 100-30000 mg/L.
Preferably, the amount of the liquid waste is, in the liquid waste,
is (0.7-2.5): 1.
more preferably, in the waste liquid
Is (1-2.5): 1.
preferably, the waste liquid treatment temperature is 20-40 ℃, and the pH of the waste liquid is 4.5-8.0.
More preferably, the waste liquid treatment temperature is 25-35 ℃ and the pH is 6.5-8.0.
Preferably, the method for applying the pseudomonas stutzeri in the denitrification and denitrification treatment of the waste liquid is as follows:
diluting the pseudomonas stutzeri after fermentation to obtain the pseudomonas stutzeri with the bacterial activity of 103-104cfu/L bacterial suspension, mixing the bacterial suspension with sludge in a volume ratio of 5-15:100 to serve as an inoculum, and adding the inoculum into waste liquid; the hydraulic retention time of the waste liquid is 12-22 h.
Wherein the dilution method of the dry thallus powder is the same as the preparation method of the suspension,the dry powder of the thallus is diluted by a fermentation culture medium or saline with the concentration of sodium chloride of 8-12g/L to obtain the thallus vitality of 103-104cfu/L bacterial suspension.
More preferably, the pseudomonas stutzeri is applied to the denitrification and denitrification treatment of the waste liquid by the following specific steps:
diluting the pseudomonas stutzeri after fermentation to obtain the pseudomonas stutzeri with the bacterial activity of 103-104The method comprises the following steps of mixing cfu/L bacterial suspension with sludge in a volume ratio of 5-15:100, adding the bacterial suspension into a reactor, carrying out mixed reaction for 6-12 hours, injecting waste liquid into the reactor, wherein the hydraulic retention time of the waste liquid is 12-22 hours, and the denitrification rate is more than 99%.
More preferably, the pseudomonas stutzeri is applied to the denitrification and denitrification treatment of the waste liquid by a specific method comprising the following steps:
the waste liquid adopts a two-stage anaerobic treatment method, firstly, the waste liquid is acidified by using an anaerobic acidification flora, the organic matter which is difficult to degrade is rapidly subjected to ring opening and chain scission to convert the organic matter into VFA with better biodegradability, the anaerobic acidification reaction time is controlled to be 4-8h, the internal circulation ratio is controlled to be 100-. The denitrification rate is more than 99 percent, and the pseudomonas stutzeri suspension and sludge mixture is obtained by mixing and reacting for 6-12 hours according to the preparation method.
The internal circulation ratio is the ratio of the flow of the backflow waste liquid to the flow of the inflow waste liquid.
In the waste liquid
Is the concentration ratio.
It is noted that the sludge is conventional excess sludge obtained by a common wastewater biochemical treatment system.
Has the advantages that:
compared with the prior art, the pseudomonas stutzeri and the application thereof have the following advantages:
(1) the remarkable strain characteristics are as follows: the pseudomonas stutzeri has strong tolerance to high salt, can realize denitrification under the condition of high salt, solves the problem of limitation of high salt on conventional biological treatment, and ensures that the high-concentration nitrate waste liquid has an economic, efficient, stable and thorough treatment method.
The pseudomonas stutzeri has higher salt-tolerant denitrification characteristic, the denitrification rate reaches more than 99 percent, and the high-concentration nitrate nitrogen in the water body can be treated under the condition that the salt content is less than or equal to 6 percent
The nitrogen is reduced into harmless nitrogen without nitrite accumulation, and the method is applied to a denitrification reactor for efficiently and stably denitrifying without secondary pollution and can shorten the start-up period.
(2) The method can complete the synchronous removal of carbon and nitrogen, and has better economic benefit and environmental protection benefit for removing nitrate from the high-concentration organic waste liquid.
(3) The utilization characteristics of the strains on nitrogen and carbon sources are as follows:
the strain can utilize various carbon sources and has good adaptability.
In the invention, the pseudomonas stutzeri can effectively utilize nitrate in the waste liquid as a nitrogen source to decompose the nitrate with high efficiency.
The pseudomonas stutzeri can effectively utilize fermentation products VFA (volatile fatty acid) and the like produced after fermentation and acidification by anaerobic acidification flora as electron donors to carry out denitrification reaction, thereby achieving the effects of denitrification and removal of organic carbon in organic waste liquid and waste liquid. (4) The microbial inoculum prepared by the culture medium through fermentation culture is added into an anaerobic denitrification reactor, so that the acclimatization starting efficiency of treating the high-salt nitrate waste liquid can be improved.
(5) The denitrification principle of the strain is as follows: the pseudomonas stutzeri provided by the invention can be used under the condition of high salt contentThe organic matter is an electron donor,
it is an electron acceptor, reduced to nitrogen. The strain is used for treating waste liquid, and has the advantages of simple process, thorough denitrification, stable effect and operation cost saving. In practical application, the bacterial strain can be placed in high-salt high-nitrate waste liquid to realize the aim of denitrification.
(6) And (3) denitrification parameter setting: (ii) the present invention
The proportion setting effectively ensures the organic electron donor required by the complete denitrification of the microbial inoculum and effectively controls BOD/NO3 ->0.6, the denitrification efficiency is prevented from being reduced.
Secondly, the temperature and pH parameter control in the invention can effectively ensure the normal operation of the metabolic reaction of the bacterial strain and maintain the obvious microbial activity, and when the temperature and pH parameter control exceeds the range, the functional microbial activity can be inhibited, and the treatment effect can be deteriorated.
The preferred scheme of the invention adds the mixed sludge of the microbial inoculum into the wastewater, and the pure bacteria are attached to the sludge and grow, so that on one hand, the loss of the pure bacteria from the reactor is avoided, on the other hand, the pure bacteria in the reactor are started successfully to become dominant bacteria, and the structures of other microbial populations in the sludge are evolved to form a cooperative metabolic symbiotic relationship with the pure bacteria, thereby enhancing the metabolic rate of functional microorganisms.
The control of the mixing time of the microbial inoculum and the sludge effectively ensures the effective attached growth of the strains on the sludge; in addition, the hydraulic retention time in the invention has a remarkable improvement effect on the denitrification rate.
Description of the drawings:
FIG. 1 is a phylogenetic tree of Pseudomonas stutzeri;
FIG. 2 is a transmission electron micrograph of Pseudomonas stutzeri
The specific implementation mode is as follows:
the invention is described below by means of specific embodiments. In addition, the embodiments should be considered illustrative, and not restrictive, of the scope of the invention, which is defined solely by the claims. It will be apparent to those skilled in the art that various changes or modifications in the components and amounts of the materials used in these embodiments can be made without departing from the spirit and scope of the invention. The present invention will be further described with reference to the following examples, but the present invention is not limited to the following examples.
Example 1: separation and identification of the strain pseudomonas stutzeri:
(1) the enrichment culture medium, the separation culture medium and the expanding culture medium are composed of:
enrichment culture medium: potassium nitrate 2g, magnesium sulfate heptahydrate 0.2g, dipotassium hydrogen phosphate 1.76g, potassium sodium tartrate 20g, 7-hydrate ferrous sulfate 0.005g, calcium chloride 0.02g, ammonium chloride 0.63g, NaCl 5%, distilled water 1000mL, pH 7.5.
Separating a culture medium: potassium nitrate 2g, magnesium sulfate heptahydrate 0.2g, dipotassium hydrogen phosphate 0.5g, sodium potassium tartrate 20g, NaCl 5%, distilled water 1000mL, pH 7.0-7.2, agar 1.5% and 1mL of bromothymol blue solution as solid medium.
Fermentation medium: 8g of peptone, 4g of yeast extract, 10g of sodium chloride and 1000mL of distilled water, wherein the pH value is 7.5.
(2) Separation and purification of pseudomonas stutzeri:
weighing 5g of domesticated denitrification sludge, placing the domesticated denitrification sludge in a 250mL anaerobic bottle, adding 100mL of sterilization enrichment culture medium, blowing nitrogen to discharge oxygen, sealing, placing the domesticated denitrification sludge in a 30 ℃ constant temperature incubator for culturing for 4 days, then taking 2mL of mixed bacteria liquid, inoculating the mixed bacteria liquid in an anaerobic glove box to a selective culture medium, and repeating the operation for 3 times. Then 1mL of the suspension was aspirated from the selection medium to 9mL of a dilution (sterile, oxygen-free water) to give 10-2Diluting the suspension to 10 times by 10 times dilution method-7Thus, each dilution of bacterial suspension was prepared.
0.1mL of each dilution suspension is respectively sucked in an anaerobic glove box until the dilution suspension is evenly coated on a solid culture medium for denitrifying bacteria separation. Then, the plate is inverted and placed in a constant temperature incubator at 30 ℃ to be cultured until obvious bacterial colonies grow. Then, a single strain was picked up and the Pseudomonas stutzeri of the present invention was streaked on a plate several times to purify it.
(3) PCR amplification and sequencing of 16S rRNA:
the DNA of the Pseudomonas stutzeri is extracted and purified by a Smart LabAssist-16 and MO-BIO PowerSoil DNA extraction kit (Taiwan dot nanotechnology Co., Ltd.) of a full-automatic magnetic bead nucleic acid extractor. Taking 5 mu L of DNA sample, carrying out electrophoresis detection under the conditions of 120V and 20-25min, and observing quality under a gel electrophoresis apparatus.
The bacterial universal primers F16S-27(5 '-AGAGTTTGATCCTGGCTCAG-3') and R16S-1492(5 '-CGGTTACCTTGTTACGACTTC-3') are selected for PCR amplification. The PCR reaction conditions are as follows: pre-denaturation at 94 ℃ for 3min, denaturation at 94 ℃ for 30s, annealing at 54 ℃ for 30s, extension at 72 ℃ for 90s, and circulation for 30 cycles; then extending for 10min at 72 ℃; finally, the mixture is stored at 4 ℃.
The PCR amplification product after being recovered is ordered to sequence by Beijing Olympic Gene science and technology Limited.
Obtaining a sequence with the length of 16S rDNA of the strain being 1430bp after sequencing, submitting the sequence to Genbank for comparison with other strains, constructing a phylogenetic tree (refer to attached figure 1) by utilizing a phylogenetic analysis (MEGA3.1) software ortho-position connection (Neighbour Joining) method to find that the strain is closest to the evolutionary distance of Pseudomonas sp, determining that the strain belongs to the genus Pseudomonas stutzeri and is named as Pseudomonas stutzeri, and referring to figure 2, the sequence is a transmission electron microscope photo of the Pseudomonas stutzeri.
(4) Denitrification test of Pseudomonas stutzeri
100mL of sterilized enrichment medium with the nitrate concentration of 2000mg/L and the salt content of 3% is filled into a 250mL triangular flask, inoculated into seed bacterial liquid according to the inoculation amount of 10%, and subjected to standing culture. Within the range of 25-35 ℃, the pseudomonas stutzeri can remove more than 85% of total nitrogen within three days, and the total nitrogen removal rate can reach as high as 99%. The bacterial strain of the invention has nitrite accumulation phenomenon in the first 48 hours of culture, then the nitrite concentration is gradually reduced, and the total nitrogen is reduced to below 20 mg/L.
Example 2: preparation of Pseudomonas stutzeri bacterial agent
Will be Schoenberg falseAfter activated culture, the monad is inoculated into a fermentation medium according to the inoculum size of 5 percent, and the fermentation temperature is controlled: dissolved oxygen at 30 deg.C<0.2mg/L, pot pressure: shaking at 35rpm under 0.03-0.10Mpa for 72 hr to obtain fermentation liquid, centrifuging at 4 deg.C and 7500rpm for 10min, removing supernatant, diluting the precipitate with sterile fermentation medium to obtain 10-strain with bacterial activity3-104cfu/L bacterial suspension, and filling the bacterial suspension to form a liquid Pseudomonas stutzeri agent product for denitrification treatment of high-salt high-concentration nitrate waste liquid.
The fermentation medium comprises the following components: 10g/L of peptone, 5g/L of yeast extract, 10g/L of sodium chloride and the balance of distilled water, wherein the pH value is 7.5, the sterilization is carried out at 121 ℃ for 20min, and the sterilization is carried out and then the cooling is carried out to the room temperature.
Example 3: preparation of pseudomonas stutzeri microbial inoculum
Inoculating the pseudomonas stutzeri in a seed culture medium, controlling the culture temperature to be 32 ℃, and performing shake culture at 35rpm for 52 hours to obtain a seed solution.
Inoculating the pseudomonas stutzeri seed solution into a fermentation culture medium according to the inoculation amount of 10%, wherein the fermentation culture conditions are as follows: culturing at 35 deg.C under dissolved oxygen of less than 0.2mg/L and pot pressure of 0.03-0.10Mpa for 72 h;
placing the fermentation liquid in a refrigerated centrifuge at 4 deg.C and 7500rpm, centrifuging and concentrating for 10min, discarding supernatant, diluting the precipitate with sterile saline water with sodium chloride concentration of 10g/L to obtain bacteria activity of 103cfu/L bacterial suspension, and filling the bacterial suspension to form a liquid Pseudomonas stutzeri agent product for denitrification treatment of high-salt high-concentration nitrate waste liquid.
The seed liquid culture medium comprises the following components: KNO3 3g/L，MgSO4·7H2O 0.3g/L，K2HPO40.6g/L, 25g/L of potassium sodium tartrate, 10g/L of sodium chloride and the balance of water, the pH value is 7.2, and the sterilization is carried out for 20min at the temperature of 121 ℃.
The fermentation medium comprises the following components: 8g of peptone, 4g of yeast extract, 10g of sodium chloride, 1000mL of distilled water, 7.5 of pH value, sterilizing at 121 ℃ for 20min, and cooling to room temperature after sterilization.
Example 4 preparation of the inoculum for Pseudomonas stutzeri
Inoculating the pseudomonas stutzeri in a seed culture medium, controlling the culture temperature to be 30 ℃, and performing shake culture at 30rpm for 60 hours to obtain a seed solution.
Inoculating pseudomonas stutzeri seed liquid into a fermentation culture medium according to the inoculation amount of 3%, and controlling the fermentation temperature: 20 ℃, dissolved oxygen <0.2mg/L, tank pressure: shaking and culturing at 0.03-0.10Mpa and 30rpm for 72-96h to obtain fermentation liquid.
Fermenting and culturing the pseudomonas stutzeri, centrifuging fermentation liquor at low temperature, removing supernatant to obtain thallus precipitate, diluting the thallus precipitate with fermentation culture medium to obtain the thallus vitality of about 104And (5) filling cfu/L of bacterial suspension to finally obtain a microbial inoculum product.
The centrifugation conditions were: centrifugation temperature: the rotation speed is 7500rpm at 4 ℃, and the centrifugation time is 10 min.
The seed liquid culture medium comprises the following components: KNO3 24g/L，MgSO4·7H2O 0.2g/L，K2HPO40.5g/L, 20g/L of sodium potassium tartrate, 10g/L of sodium chloride and the balance of water, the pH value is 7.2, and the sterilization is carried out for 20min at the temperature of 121 ℃.
The fermentation medium comprises the following components: 6g/L of peptone, 3g/L of yeast extract, 10g/L of sodium chloride and the balance of distilled water, wherein the pH value is 7.5, the sterilization is carried out at 121 ℃ for 20min, and the sterilization is carried out and then the cooling is carried out to the room temperature.
Example 5 preparation of the inoculum for Pseudomonas stutzeri
Inoculating the pseudomonas stutzeri in a seed culture medium, controlling the culture temperature to be 38 ℃, and performing shake culture at 50rpm for 48 hours to obtain a seed solution.
Inoculating the pseudomonas stutzeri seed solution into a fermentation culture medium according to the inoculation amount of 10%, and controlling the fermentation temperature: 40 ℃, dissolved oxygen <0.2mg/L, pot pressure: shaking and culturing at 50rpm under 0.10Mpa for 72h to obtain fermentation liquid.
Fermenting and culturing the pseudomonas stutzeri, centrifuging the fermentation liquor at low temperature, removing the supernatant to obtain a thallus precipitate, diluting the thallus precipitate with an aseptic fermentation culture medium to obtain the thallus with the activity of 5 multiplied by 103And (5) filling cfu/L of bacterial suspension to finally obtain a microbial inoculum product.
The centrifugation conditions were: centrifugation temperature: the rotation speed is 7500rpm at 4 ℃, and the centrifugation time is 10 min.
The seed liquid culture medium comprises the following components: KNO3 4g/L，MgSO4·7H2O-0.4g/L，K2HPO40.8g/L, 30g/L of sodium potassium tartrate, 10g/L of sodium chloride and the balance of water, the pH value is 7.2, and the sterilization is carried out for 20min at the temperature of 121 ℃.
The fermentation medium comprises the following components: peptone 12g/L, yeast extract 6g/L, sodium chloride 10g/L, and distilled water in balance, with pH 7.5, sterilizing at 121 deg.C for 20min, sterilizing, and cooling to room temperature.
Example 6 preparation of the inoculum for Pseudomonas stutzeri
Inoculating the pseudomonas stutzeri in a seed culture medium, controlling the culture temperature to be 36 ℃, and performing shake culture at 40rpm for 48 hours to obtain a seed solution.
Inoculating the pseudomonas stutzeri seed solution into a fermentation culture medium according to the inoculation amount of 5%, and controlling the fermentation temperature: 35 ℃, dissolved oxygen <0.2mg/L, pot pressure: shaking and culturing at 35rpm under 0.03-0.10Mpa for 72 hr to obtain fermentation liquid.
Fermenting and culturing the pseudomonas stutzeri, centrifuging the fermentation liquor at low temperature, removing the supernatant to obtain a thallus precipitate, diluting the thallus precipitate with an aseptic fermentation culture medium to obtain the thallus with the activity of 8 multiplied by 103And (5) filling cfu/L of bacterial suspension to finally obtain a microbial inoculum product.
The centrifugation conditions were: centrifugation temperature: the rotation speed is 7500rpm at 4 ℃, and the centrifugation time is 10 min.
The seed liquid culture medium comprises the following components: KNO3 2g/L-4g/L，MgSO4·7H2O 0.2g/L-0.4g/L，K2HPO40.5g/L-0.8g/L, 20g/L-30g/L of sodium potassium tartrate, 10g/L of sodium chloride and the balance of water, the pH value is 7.2, and the sterilization is carried out for 20min at 121 ℃.
The fermentation medium comprises the following components: 10g/L of peptone, 4g/L of yeast extract, 11g/L of sodium chloride and the balance of distilled water, wherein the pH value is 7.5, the sterilization is carried out at 121 ℃ for 20min, and the sterilization is carried out and then the cooling is carried out to the room temperature.
Example 7 preparation of the inoculum for Pseudomonas stutzeri
Performing fermentation culture on pseudomonas stutzeri according to the strain culture method in the embodiment 6 to obtain pseudomonas stutzeri fermentation liquor, performing centrifugation at 4 ℃ and the rotating speed of 7500rpm for 10min, discarding supernatant to obtain thallus precipitate, and performing freeze-drying to obtain the microbial inoculum dry powder.
Example 8 Pseudomonas stutzeri for treatment of high salt and high nitrate waste liquid
The microbial inoculum product prepared in the embodiment 3 of the invention: 1.5g/L of bacterial suspension, and the ratio of the volume of the bacterial suspension to the volume of sludge in a conventional denitrification reactor is 1:10, stopping feeding the waste liquid, avoiding the loss of the microbial inoculum, ensuring the retention time of the microbial inoculum in the reactor to be 7 hours, then feeding the waste liquid, and controlling the reactor to feed the waste liquid
Controlling the reaction treatment temperature at 30-35 ℃, controlling the pH at 6.5-7.5, gradually increasing the concentration of the salt and the nitrate radical of the inlet water in equal steps, finally increasing the concentration of the sodium chloride in the inlet water from 0.5% to 6%, increasing the concentration of the nitrate radical ion from 500mg/L to 20000mg/L, keeping the hydraulic retention time of the waste liquid for 16 hours, and leaving the water without water
And (4) accumulating. After treatment, the nitrate nitrogen in the effluent waste liquid is reduced to 4.5mg/L, and the effluent is discharged
The concentration is kept below 20 mg/L.
Example 9 Pseudomonas stutzeri for treatment of high salt and high nitrate waste liquid
The microbial inoculum product prepared in the embodiment 4 of the invention is mixed with the sludge in the conventional denitrification reactor according to the volume ratio of 1:10 and then is added into the reactor, water feeding is stopped to avoid microbial inoculum loss, the microbial inoculum stays in the reactor for 12 hours, then waste liquid is discharged, and the water feeding of the reactor is controlled to be waste liquid
Controlling the reaction treatment temperature at 35 ℃, controlling the pH value to be 5.5-6.0, gradually increasing the concentrations of sodium chloride salt and nitrate radical in the inlet water in equal steps, finally increasing the concentration of sodium chloride salt from 0.5% to 6%, increasing the concentration of nitrate radical ions from 100mg/L to 30000mg/L, keeping the hydraulic retention time of the waste liquid for 22 hours, and leaving no effluent
And (4) accumulating. After treatment, the nitrate nitrogen in the waste liquid in the effluent is reduced to 20mg/L, and the effluent is discharged
The concentration is kept below 100 mg/L.
Example 10 Pseudomonas stutzeri for treatment of high salt and high nitrate waste liquid
The microbial inoculum product prepared in the embodiment 5 of the invention is mixed with the sludge in the conventional denitrification reactor according to the volume ratio of 15:100 and then is added into the reactor, water feeding is stopped to avoid microbial inoculum loss, the microbial inoculum stays in the reactor for 8 hours, then waste liquid is fed, and the water feeding of the reactor is controlled to be waste liquid
Controlling the reaction treatment temperature at 30-35 ℃, controlling the pH value at 7.5-8.0, gradually increasing the concentrations of sodium chloride salt and nitrate radical in the inlet water in equal steps, finally increasing the concentration of sodium chloride salt from 0.5% to 6% in the inlet water, increasing the concentration of nitrate radical ions from 100mg/L to 25000mg/L in the inlet water, controlling the hydraulic retention time of the waste liquid to be 17.5 hours, and leaving water without water
And (4) accumulating. After treatment, nitrate nitrogen in the effluent is reducedTo 15mg/L, yielding water
The concentration is kept below 50 mg/L.
Example 11 Pseudomonas stutzeri for treatment of high salt and high nitrate waste liquid
The microbial inoculum product prepared in the embodiment 6 of the invention is mixed with the sludge in the conventional denitrification reactor according to the volume ratio of 12:100 and then is added into the reactor, water feeding is stopped to avoid microbial inoculum loss, the microbial inoculum stays in the reactor for 9 hours, then waste liquid is fed, and the water feeding of the reactor is controlled to be waste liquid
Controlling the reaction treatment temperature at 35-40 ℃, controlling the pH at 4.5-5.5, gradually increasing the concentrations of sodium chloride salt and nitrate radical in the inlet water in equal steps, finally increasing the concentration of sodium chloride salt from 0.5% to 6%, increasing the concentration of nitrate radical ions from 100mg/L to 28000mg/L, and keeping the hydraulic retention time of the waste liquid for 12 hours without water
And (4) accumulating. After treatment, the nitrate nitrogen in the waste liquid is reduced to 18mg/L, and the water is discharged
The concentration is kept below 70 mg/L.
Example 12 Pseudomonas stutzeri for treatment of high salt and high nitrate waste liquid
The microbial inoculum dry powder prepared in the embodiment 7 of the invention is diluted by an aseptic fermentation medium until the bacterial activity is 104cfu/L bacterial suspension, mixing and adding bacterial dilution liquid and sludge in a volume ratio of 15:100 into a reactor, stopping water inflow to avoid loss of the bacterial agent, ensuring that the bacterial agent stays in the reactor for 9 hours, then discharging waste liquid, and controlling the water inflow of the reactor to the waste liquid
Controlling the reaction treatment temperature at 35 ℃, controlling the pH value at 5.0-5.5, gradually increasing the concentrations of sodium chloride salt and nitrate radical in the inlet water in equal steps, finally increasing the concentration of sodium chloride salt from 0.5% to 6%, increasing the concentration of nitrate radical ions from 100mg/L to 30000mg/L, keeping the hydraulic retention time of the waste liquid for 12 hours, and leaving water without water
And (4) accumulating. After treatment, the nitrate nitrogen in the waste liquid is reduced to 20mg/L, and the water is discharged
The concentration is kept below 100 mg/L.
Example 13 Pseudomonas stutzeri for treatment of high salt high nitrate waste liquid
Diluting Pseudomonas stutzeri with sterile fermentation medium until the bacterial activity is 104cfu/L bacterial suspension, mixing and adding the bacterial dilution liquid and the sludge in a volume ratio of 1:10 into a reactor, stopping water inflow to avoid loss of the microbial inoculum, and ensuring that the microbial inoculum stays in the reactor for 9 hours.
The waste liquid adopts a two-stage anaerobic treatment method, firstly, anaerobic acidification flora is utilized to carry out acidification treatment on the waste liquid, organic matters which are difficult to degrade are rapidly subjected to ring opening and chain scission to convert the organic matters into VFA with better biodegradability, the anaerobic acidification reaction time is controlled to be 4-8h, the internal circulation ratio is controlled to be 100-300%, after the reaction is finished, the treated waste liquid is mixed with the nitrate waste liquid, the mixture is discharged into a reactor containing the pseudomonas stutzeri thallus and sludge mixture to carry out an anaerobic denitrification stage after the water quality is adjusted, and the water of the waste liquid entering the reactor is controlled to be waste liquid water
The reaction treatment temperature, pH, influent sodium chloride salt and nitrate maximum concentration, and hydraulic retention time of the waste liquid were the same as in example 12.
Pseudomonas stutzeri takes nitrate nitrogen as an electron acceptor and VFA generated by anaerobic acidification as an electron donor to carry out anaerobic treatmentThe denitrification reaction is used for treating sewage, and the internal circulation ratio is controlled to be 100-300%. Reducing nitrate nitrogen in the waste liquid to 20mg/L, discharging water
The concentration is kept below 100 mg/L.
Comparative example 1 treatment of high-salt high-nitrate waste liquid with conventional sludge
The liquid waste treatment was carried out according to the treatment method of example 8, the same amount of sludge was finally added to the reactor, and the operation was carried out according to the procedure of example 8, which is different from example 8 in that: the sludge is not externally inoculated with a microbial inoculum, and the difference is that in the final inlet water, the concentration of sodium chloride salt is improved from 0.5 percent to 2 percent, the concentration of nitrate ion is improved from 500mg/L to 5000mg/L, the hydraulic retention time of the waste liquid is also 16 hours, and finally the outlet water is measured
At a concentration of 50mg/L, and
the accumulation is obvious, and the accumulation is obvious,
at a concentration of 580mg/L, the ORP increased to +150mv, resulting in a continuous deterioration of the treatment effect. According to the experimental effect data, the waste liquid which can continuously improve the concentration of sodium chloride salt and the concentration of nitrate ions through conventional sludge treatment can not achieve good denitrification effect.
Claims (8)
1. A salt-tolerant denitrifying bacterium, which is characterized in that: the salt-tolerant denitrifying bacteria is specifically Pseudomonas stutzeri (Pseudomonas stutzeri)Pseudomonas stutzeri) The preservation number is CGMCC No. 15310.
2. A microbial agent comprising the salt-tolerant denitrifying bacteria of claim 1, characterized in that: the microbial inoculum is specifically dry thallus powder or the activity of the microbial inoculum is 103-104cfu/L bacterial suspension.
3. The method for preparing the salt-tolerant denitrifying bacteria agent of claim 2, characterized in that: the method comprises the following specific steps:
(1) fermentation culture: activating the pseudomonas stutzeri, inoculating the activated pseudomonas stutzeri into a fermentation culture medium according to the inoculation amount of 3% -10%, and controlling the fermentation temperature: 20-40 ℃, dissolved oxygen <0.2mg/L, pot pressure: shaking and culturing at 0.03-0.10Mpa and 30-50rpm for 72-96h to obtain fermentation broth; the fermentation medium comprises the following components: 6g/L-12g/L peptone, 3g/L-6g/L yeast extract, 8-12g/L sodium chloride, and the balance of distilled water, with pH of 7.5, sterilizing at 121 deg.C for 20 min;
(2) and (2) centrifuging and freeze-drying the fermentation liquor obtained in the step (1) to obtain thallus dry powder or centrifuging and diluting the thallus to obtain a thallus suspension.
4. The method for preparing the salt-tolerant denitrifying bacterial agent of claim 3, wherein:
the activating step of the pseudomonas stutzeri comprises the following steps: inoculating the pseudomonas stutzeri in a seed culture medium, controlling the culture temperature to be 30-38 ℃, and performing shake culture at 30-50rpm for 48-72 h.
5. The method for preparing salt-tolerant denitrifying bacteria according to claim 4, wherein: the seed liquid culture medium comprises the following components: KNO3 2g/L-4g/L，MgSO4·7H2O 0.2g/L-0.4g/L，K2HPO40.5-0.8 g/L, 20-30 g/L of sodium potassium tartrate, 8-12g/L of sodium chloride and the balance of water, the pH value is 7.2, and the sterilization is carried out for 20min at 121 ℃.
6. Use of the salt-tolerant denitrifying bacteria of claim 1 in the denitrification of waste streams.
7. The use of the salt-tolerant denitrifying bacteria of claim 6 in the denitrification of waste liquid, characterized in that: the concentration of sodium chloride salt in the waste liquid is 1-6 percent, the concentration of nitrate ions is 100-30000mg/L, BOD/NO3⁻ is (0.7-2.5): 1, the treatment temperature of the waste liquid is 20-40 ℃, and the pH value is 4.5-8.0.
8. The use of the salt-tolerant denitrifying bacteria of claim 7 in the denitrification of waste liquid, characterized in that: diluting the pseudomonas stutzeri after fermentation to obtain the pseudomonas stutzeri with the bacterial activity of 103-104cfu/L bacterial suspension, mixing the bacterial suspension with sludge in a volume ratio of 5-15:100 to serve as an inoculum, and adding the inoculum into waste liquid; the hydraulic retention time of the waste liquid is 12-22 h.
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