CN101348772B - Comamonas aquatica LNL3 and use thereof in waste water biological denitrification - Google Patents
Comamonas aquatica LNL3 and use thereof in waste water biological denitrification Download PDFInfo
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- CN101348772B CN101348772B CN2008100206494A CN200810020649A CN101348772B CN 101348772 B CN101348772 B CN 101348772B CN 2008100206494 A CN2008100206494 A CN 2008100206494A CN 200810020649 A CN200810020649 A CN 200810020649A CN 101348772 B CN101348772 B CN 101348772B
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Abstract
The invention belongs to the bioengineering technical field, in particular relating to an aquatic Comamonas sp. strain and application of the aquatic Comamonas sp. strain in short-range nitration-denitrification denitrogenation during the biological denitrogenation process of waste water. The aquatic Comamonas sp. strain is Comamonas aquatica LNL3 and preserved in 'China General Microbiological Culture Collection Center of the China Committee of Culture Collection for Microorganisms' on January 24th, 2008, with a collection number of CGMCC No.2361. The strain is collected from a hydrophyte root zone in a water body in Jinshan Lake of Zhenjiang, a open water zone in a water body in the Jinshan Lake and deposits of the Jinshan Lake, is separated, and can be grown in a basal culture medium which takes CO2 as a carbon source and energy or CO2 and an organic substance as a mixed carbon source and the energy and takes ammonia nitrogen or nitrate nitrogen as a nitrogen source. Bacteria solution, a dormant cell and a fixed strain of the Comamonas aquatica LNL3 can decompose the ammonia nitrogen into nitrite nitrogen and simultaneously can convert the ammonia nitrogen into nitrogen, thereby the Comamonas aquatica LNL3 not only can be taken as a denitrogenation microorganism to convert the ammonia nitrogen into the nitrite nitrogen but also can convert the ammonia nitrogen into the nitrogen, so as to realize the short-range nitration-denitrification. The strain has the advantages that the strain has the function of nitration-denitrification on the nitrogenous waste water, has quick degradation speed on the ammonia nitrogen and is suitable for processing nitrogenous industrial waste water and nitrogenous sanitary sewage.
Description
One. technical field
The invention belongs to technical field of bioengineering.Be specifically related to aquatic Comamonas bacterial strain and the short distance nitration of nitrogenous trade effluent and nitrogenous sanitary sewage-denitrification denitrogenation is handled and used.
Two. background technology
Along with the development of industrial and agricultural production and the raising of living standards of the people, the quantity discharged of nitrogenous compound sharply increases, and has become the primary pollution source of environment.The environmental hazard that the water body nitrate pollution causes is serious day by day, and topmost consequence is to cause body eutrophication, causes algae and water transition breeding and causes algal bloom to break out.The water body nitrate pollution also can cause aquatic ecosystem and to the harmful effect to the healthy aspect of biology, the most direct influence is that ammonia is to hydrobiological murder by poisoning, the ammonia nitrogen that enters water body is under the effect of nitrifier, oxidable one-tenth nitrite and nitrate, consume a large amount of oxygen, reduce the water body quality, serious harm aquatic ecosystem safety.In addition, nitrite and nitrate also are the objectionable impuritiess of serious threat human health.
Short distance nitration-denitrification biological denitrogenation technology has the minimizing reaction volume than the traditional biological denitride technology, saves initial cost, reduces advantages such as aeration rate and carbon source, has become one of focus of biological denitrificaion area research.At present, the nitrosification control techniques mainly is to go out ammonia oxidizing bacteria by controlled temperature, DO, pH value, the concentration of free ammonia conditional filterings such as (FA), eliminates nitrite-oxidizing bacteria, realizes nitrite accumulation.But also there is control condition shortcoming harsh and that transform to complete nitrification easily simultaneously in these methods, thereby have limited to its application.
Three. summary of the invention
The problem that the present invention need solve is that the biological treatment for nitrogenous effluent provides short distance nitration-denitrifying bacteria preparation efficiently, strengthens the denitrogenation processing of nitrogenous effluent.From the aquatic Comamonas bacterium of the isolated strain of occurring in nature, can be with CO
2For carbon source and energy or with CO
2With organism be that mixed carbon source and energy, ammonia nitrogen or nitric nitrogen are nitrogenous source growth, by ammonia nitrogen is converted into nitrite nitrogen, again nitrite nitrogen is converted into nitrogen and realizes short distance nitration-denitrification, nitrogenous effluent is had denitrogenation efficiently.In the nitrogenous effluent denitrogenation, be with a wide range of applications.
Technical solution of the present invention is that aquatic Comamonas bacterial strain provided by the invention is Comamonas aquatica LNL
3, be preserved in " China Committee for Culture Collection of Microorganisms's common micro-organisms " center ", its preserving number CGMCC No.2361 on January 24th, 2008.
This aquatic Comamonas bacterial strain is that collection from Lake Water Body waterplant root district, Kingsoft, Zhenjiang, the spacious pool of Kingsoft Lake Water Body and Kingsoft lacustrine sediments, separation obtain.
Concrete screening step is as follows:
(15cm * 1.5cm), sediment sampler are from the water body of the following 30cm～50cm in lake water plane, Kingsoft, Zhenjiang with the needle tubing of sterilization in advance, waterplant root district, top layer, lake, Kingsoft bed mud etc. is located, gather the microorganism sample in water body, roots of plants district and the bed mud respectively, seal immediately after the sampling, separate enrichment culture as the bacterium source.Under aseptic condition, water sample is added to (the ammonia oxidizing bacteria medium component is (NH in the ammonia oxidizing bacteria enrichment medium
4)
2SO
40.2g, K
2HPO
40.1g, MgSO
40.05g, NaCl 0.2g, FeSO
40.04g, CaCO
30.5g, H
2O 100ml, pH value 7.2, wherein substratum uses behind 121 ℃ of moist heat sterilization 20min), place on the shaking table and under 28 ℃ of conditions, cultivate 48h.And then forward in the solid medium of ammonia oxidizing bacteria, the solid culture based component is (NH
4)
2SO
40.2g, K
2HPO
40.1g, MgSO
40.05g, NaCl 0.2g, FeSO
40.04g, CaCO
30.5g, agar 2g, H2O 100ml, pH value 7.2 is carried out the plate streaking initial gross separation with the bacterial classification after spreading cultivation, and is dull and stereotyped at 28 ℃ of cultivation 48h.According to the single microbial that grows on the flat board, select wherein growing way to carry out enrichment culture once more preferably.On solid medium, carry out plate streaking at last repeatedly and separate, isolate a strain has the efficient degradation ability to ammonia nitrogen and nitrite nitrogen aquatic Comamonas bacterial strain.
This aquatic Comamonas bacterial strain is cultivated in pH value 6.5～8.5 substratum media, and the growth temperature that is fit to is 25～35 ℃; Its bacteriology morphological specificity is: Gram-negative bacteria, be of a size of 0.5～1.0 * 1～4 μ m, and amphitrichous, aerobic or anaerobism, the catalase positive, oxidase positive is with CO
2For carbon source and energy or with CO
2With organism be that mixed carbon source and energy, ammonia nitrogen or nitric nitrogen are nitrogenous source growth.
This bacterium on solid medium 28 ℃ cultivated 15～20 days, bacterium colony is little, and is rounded, the edge is smooth, smooth surface, thalline presents colourless, very easily provokes.
Learn and molecular biological analysis according to this strain morphology, can identify that it is the bacterial strain of aquatic Comamonas.By the 16S rDNA of this bacterial strain that increases, having obtained length is the 16S rDNA complete sequence of 1415bP.The PCR primer adopts universal primer fD1 (5 '-AGAGTTTGATCCTGGCTCAC-3 ') and the rD1 (5 '-AAGGAGGTGATCCAGCC-3 ') of bacterial 16 S rDNA.(GeneAmp, PCRsystem 9700) carries out amplified reaction with the PCR instrument.PCR reaction system cumulative volume is 40 μ L: 10 * PCR reaction buffer, 4 μ L wherein, the dNTP solution 4 μ L of 2.5mmol/L, each 4 μ L of the primer fD1 of 10pmol/ μ L and rD1,2 units of Taq enzyme, DNA masterplate 40ng.Get 2 μ L in reaction system, 94 ℃ of pre-sex change 5min after the DNA dilution of extracting; 94 ℃ of sex change 1min, 55 ℃ of annealing 30s, 72 ℃ are extended 1min, 33 circulations; 72 ℃ are extended 7min.1.5% sepharose, EB dyeing back ultraviolet detection.Carry out homology relatively by the Blast program, the similarity that shows this bacterial strain and Comamonas aquatica up to more than 99%, repeatability 100%.
Aquatic Comamonas bacterial strain of the present invention both can be grown in the eutrophy substratum, also can be with CO
2For carbon source and energy or with CO
2With organism be that mixed carbon source and energy, ammonia nitrogen or nitric nitrogen are to grow in the basic medium of nitrogenous source.
This aquatic Comamonas bacterial strain is extremely strong to the degradation capability of ammonia nitrogen, and after this bacterial strain effect, ammonia nitrogen disappears at visible region maximum absorption band (412nm), thereby reduces the ammonia nitrogen concentration in the waste water.
This aquatic Comamonas bacterial strain makes nitrite nitrogen disappear at visible region maximum absorption band (540nm) by nitrite nitrogen is converted into nitrogen, thereby reduces the nitrite nitrogen in the waste water.
Aquatic Comamonas bacterial strain of the present invention can be with the bacterial classification or the immobilization bacterial strain carries out degradation treatment to ammonia nitrogen in vegetative period of fresh culture.
Aquatic Comamonas bacterial strain of the present invention, can be with the bacterial classification or the immobilization bacterial strain earlier is converted into nitrite nitrogen with ammonia nitrogen in vegetative period of fresh culture, then nitrite nitrogen is converted into nitrogen, by short distance nitration-denitrification nitrogenous effluent is carried out efficient degradation and handle.
The beneficial effect that the present invention reached is: aquatic Comamonas bacterial strain provided by the invention has stronger conversion capability to ammonia nitrogen and nitrite, and degradation speed is fast, and degradation rate is up to more than 90%.This bacterial strain can be used as free biological bacteria preparation or immobilization bacterial strain, be added in the existing Waste Water Treatment, nitrogenous effluent is carried out short distance nitration-denitrification to be handled, improve the reaction efficiency of former processing system, cut down the consumption of energy, shorten the reaction times, strengthen the processing power and the efficient of former processing system, in Treatment of Industrial Water and sanitary sewage disposal, have wide application potential.
Four. description of drawings
Fig. 1. aquatic Comamonas bacterial strain is removed the ammonia nitrogen ability ,-▲-influent ammonium concentration (mg/L) ,--water outlet ammonia nitrogen concentration (mg/L) ,-◆-ammonia nitrogen removal frank (100%)
Fig. 2. aquatic Comamonas bacterial strain is removed the nitrite ability ,-▲-water inlet nitrite concentration (mg/L) ,--water outlet nitrite concentration (mg/L),
Nitrite clearance (100%)
The aquatic Comamonas bacterial strain of Fig. 3 is nitrated-and denitrification removes the ammonia nitrogen ability
-■-influent ammonium concentration (mg/L) ,-▲-water outlet ammonia nitrogen concentration (mg/L) ,-△-water outlet nitrite concentration (mg/L) ,-●-ammonia nitrogen removal frank (100%)
Fig. 4. experiment reaction unit, (1). inlet chest; (2). dosage pump; (3). reactor; (4). water outlet; (5). air compressor machine; (6). gas meter; (7). water circulating pump; (8). cyclic water tank; (9). heating unit and temperature sensor
Ammonia nitrogen removal frank and nitrosification rate under the different ammonia nitrogen loading situations of Fig. 5
-■-influent ammonium concentration ,-▲-the water outlet ammonia nitrogen concentration ,-△-nitrosification rate ,-zero-ammonia nitrogen removal frank
Nitrite clearance under the different nitrite influent concentrations of Fig. 6
-△-water outlet nitrate concentration ,-◆-water inlet nitrite concentration ,-■-water outlet nitrite concentration ,-▲-the water inlet nitrate concentration ,-●-nitrite clearance
Fig. 7 system handles contains ammonia nitrogen and glucose wastewater ability
-■-influent ammonium concentration,
The water outlet ammonia nitrogen concentration ,-zero-water outlet nitrate concentration ,-△-ammonia nitrogen removal frank ,-●-nitrogen removal rate
Five. embodiment
The screening step of embodiment 1 aquatic Comamonas bacterial strain provided by the invention:
(15cm * 1.5cm), sediment sampler are from the water body of the following 30cm～50cm in lake water plane, Kingsoft, Zhenjiang with the needle tubing of sterilization in advance, waterplant root district, top layer, lake, Kingsoft bed mud etc. is located, gather the microorganism sample in water body, roots of plants district and the bed mud respectively, seal immediately after the sampling, separate enrichment culture as the bacterium source.Under aseptic condition, water sample is added to (the ammonia oxidizing bacteria medium component is (NH in the ammonia oxidizing bacteria enrichment medium
4)
2SO
40.2g, K
2HPO
40.1g, MgSO
40.05g, NaCl 0.2g, FeSO
40.04g, CaCO
30.5g, H
2O 100ml, pH value 7.2, wherein substratum uses behind 121 ℃ of moist heat sterilization 20min), place on the shaking table and under 28 ℃ of conditions, cultivate 48h.And then forward that (the solid culture based component is: (NH in the solid medium of ammonia oxidizing bacteria to
4)
2SO
40.2g, K
2HPO
40.1g, MgSO
40.05g, NaCl 0.2g, FeSO
40.04g, CaCO
30.5g, agar 2g, H2O 100ml, pH value 7.2), the bacterial classification after spreading cultivation is carried out the plate streaking initial gross separation, dull and stereotyped at 28 ℃ of cultivation 48h.According to the single bacterial strain that grows on the flat board, select wherein growing way to carry out enrichment culture once more preferably.Carrying out plate streaking at last on solid medium repeatedly separates, isolate a strain has the efficient degradation ability to ammonia nitrogen and nitrite nitrogen aquatic Comamonas bacterial strain, and this bacterial strain is preserved in " China Committee for Culture Collection of Microorganisms's common micro-organisms " center ", its preserving number CGMCC No.2361 on January 24th, 2008.
This aquatic Comamonas bacterial strain is cultivated in pH value 6.5～8.5 substratum media, and the growth temperature that is fit to is 25～35 ℃; Its bacteriology morphological specificity is: gram negative bacillus, be of a size of 0.5～1.0 * 1～4 μ m, and amphitrichous, aerobic or anaerobism, the catalase positive, oxidase positive is with CO
2For carbon source and energy or with CO
2With organism be that mixed carbon source and energy, ammonia nitrogen or nitric nitrogen are nitrogenous source growth.
The 16S rRNA amplification of aquatic Comamonas bacterial strain provided by the invention:
By the 16S rDNA of this bacterial strain that increases, having obtained length is the 16S rDNA complete sequence of 1415bP.The PCR primer adopts universal primer fD1 (5 '-AGAGTTTGATCCTGGCTCAC-3 ') and the rD1 (5 '-AAGGAGGTGATCCAGCC-3 ') of bacterial 16 S rDNA.(GeneAmp, PCRsystem 9700) carries out amplified reaction with the PCR instrument.PCR reaction system cumulative volume is 40 μ L: 10 * PCR reaction buffer, 4 μ L wherein, the dNTP solution 4 μ L of 2.5mmol/L, each 4 μ L of the primer fD1 of 10pmol/ μ L and rD1,2 units of Taq enzyme, DNA masterplate 40ng.Get 2 μ L in reaction system, 94 ℃ of pre-sex change 5min after the DNA dilution of extracting; 94 ℃ of sex change 1min, 55 ℃ of annealing 30s, 72 ℃ are extended 1min, 33 circulations; 72 ℃ are extended 7min.1.5% sepharose, EB dyeing back ultraviolet detection.Carry out homology relatively by the Blast program, the similarity that shows this bacterial strain and Comamonas aquatica up to more than 99%, repeatability 100%.
The cell liquid culture for preparing aquatic Comamonas bacterial strain:
Aquatic Comamonas bacterial strain list bacterium colony on the picking solid medium is in the liquid nutrient medium that sterilization is housed, in 28 ℃, pH 7.5, and 80r/min carries out aerobic cultivation 48h, getting cultured bacterium liquid 1ml is seeded in and is equipped with in the 250ml Erlenmeyer flask of basic medium that 100ml contains ammonia nitrogen (100mg/L), 28 ℃, pH7.5,80r/min, carry out aerobic cultivation 48h, be the cell liquid culture of aquatic Comamonas bacterial strain.
The aquatic comamonas of preparation immobilization:
Adopting wetting ability vitreous state monomer methacrylic acid β hydroxyl ethyl ester (HEMA), Hydroxyethyl acrylate (HEA) to mix according to certain volume ratio with distilled water, under-63 ℃～-78 ℃ temperature condition, is 1 * 10 with dosage
3Gy～1 * 10
6The high-energy ray irradiation of Gy forms biocompatibility immobilization multipolymer porous support, add activated cultivation and enter the aquatic Comamonas bacterium liquid of logarithmic phase, make it to be adsorbed in the fixation support surface and enter the inner immobilization that realizes of porous support by propagation.
To the application of ammonia nitrogen degradation research, its step is as follows in the eutrophy substratum in the present invention:
[1] in the Erlenmeyer flask of 250ml, adds 50ml eutrophy substratum (ammonia nitrogen concentration is 50mg/L).
[2] the cell liquid culture of the aquatic Comamonas bacterial strain that embodiment 1 is made adds in the Erlenmeyer flask of above-mentioned [1], and 28 ℃, 80r/min, aerobic cultivation.Every 2 hours sampling and measuring.Fig. 1 is aquatic Comamonas bacterial strain degradation of ammonia nitrogen situation in enrichment medium.As can be seen from Figure 1, a spot of growth of thalline is enough to make ammonia nitrogen degradation, inoculates after 6 hours, and the degradation rate of ammonia nitrogen is near 100%.
The present invention is with glucose and CO
2Be mixed carbon source, nitrite is the application of in the nitrogenous source substratum nitrite degradation being studied, and its step is as follows:
[1] substratum among the embodiment 1 is changed to glucose and CO
2Be mixed carbon source, nitrite is the basic medium (nitrite concentration is 50mg/L) of nitrogenous source
The cell liquid culture of the aquatic Comamonas bacterial strain strain that [2] embodiment 1 is made adds in the Erlenmeyer flask of above-mentioned [1], and 28 ℃, 80r/min, anaerobism is cultivated.Every 2 hours sampling and measuring.Fig. 2 is aquatic Comamonas bacterial strain degrading nitrite situation in enrichment medium.As can be seen from Figure 2, a spot of growth of thalline is enough to make nitrite degradation, inoculates after 12 hours, and the degradation rate of nitrite is near 100%.
Embodiment 4
The present invention is being a mixed carbon source with glucose and COX, and ammonia nitrogen is the application of in the nitrogenous source substratum ammonia nitrogen degradation being studied, and its step is as follows:
[1] substratum among the embodiment 1 is changed to glucose and CO
2Be mixed carbon source, ammonia nitrogen is the basic medium (ammonia nitrogen concentration is 50mg/L) of nitrogenous source
[2] the cell liquid culture of the aquatic Comamonas bacterial strain that embodiment 1 is made adds in the Erlenmeyer flask of above-mentioned [1], and 28 ℃, 80r/min, aerobic-anaerobism is cultivated.Every 2 hours sampling and measuring.Fig. 3 is aquatic Comamonas bacterial strain degradation of ammonia nitrogen situation in enrichment medium.As can be seen from Figure 3, a spot of growth of thalline is enough to make ammonia nitrogen degradation, inoculates after 12 hours, and the ammonia nitrogen concentration in the water outlet is very low, and nitrite concentration is also low, and system's denitrification percent reaches 65.8%, realizes short distance nitration-denitrification, reaches the purpose of denitrogenation.
To the application of nitrogen-containing wastewater degraded, its step is as follows in the laboratory simulation fluidized-bed reactor in the present invention:
[1] Fig. 4 is the fluidized-bed reactor of laboratory simulation.The immobilization bacterial strain of the aquatic Comamonas bacterial strain of embodiment 1 preparation is dropped in the reactor, and the filling ratio of carrier is 10% in the reactor.
[2] will contain respectively in the waste water adding reactor that ammonia nitrogen concentration is 100mg/L, 150mg/L and 200mg/L, at 30 ℃, aeration rate is to react under the 250ml/min condition, sampling and measuring after 24 hours.Fig. 5 is the immobilization bacterial strain of aquatic Comamonas bacterial strain is handled nitrogen-containing wastewater in the simulation fluidised bed system a situation.From curve as can be seen, after carrying out reacting in 24 hours, the ammonia nitrogen concentration in the water outlet is very low, and ammonia nitrogen removal frank is near 100%, and the ammonia nitrogen of degraded almost is converted into nitrite nitrogen, and the nitrosification rate maintains more than 90%, and the short distance nitration effect is obvious.
Embodiment 6
To containing the application of nitrite nitrogen waste water degraded, its step is as follows in the laboratory simulation fluidized-bed reactor in the present invention:
[1] Fig. 4 is the fluidized-bed reactor of laboratory simulation.The immobilization bacterial strain of the aquatic Comamonas bacterial strain of embodiment 1 preparation is dropped in the reactor, and the filling ratio of carrier is 10% in the reactor.
[2] will contain respectively in the waste water adding reactor that nitrite concentration is 30mg/L, 60mg/L and 90mg/L and a certain amount of glucose (TOC=100mg/L), at 30 ℃, aeration rate is to react sampling and measuring after 24 hours under 60ml/min (the guaranteeing certain mixing effect) condition.Fig. 6 handles the situation that contains nitrite nitrogen waste water for the immobilization bacterial strain of aquatic Comamonas bacterial strain in the simulation fluidised bed system.From curve as can be seen, after carrying out reacting in 24 hours, the nitrite nitrogen concentration in the water outlet is very low, and the nitrite nitrogen removal efficiency has been realized short-cut denitrification near 100%, reaches the purpose of denitrogenation.
To containing the application of ammonia nitrogen and glucose wastewater degraded, its step is as follows in the laboratory simulation fluidized-bed reactor in the present invention:
[1] Fig. 4 is the fluidized-bed reactor of laboratory simulation.The immobilization bacterial strain of the aquatic Comamonas bacterial strain of embodiment 1 preparation is dropped in the reactor, and the filling ratio of carrier is 10% in the reactor.
[2] will contain in the waste water adding reactor that ammonia nitrogen concentration is 100mg/L and a certain amount of glucose (TOC=100mg/L), at 30 ℃, aeration rate is to react under the 250ml/min condition, sampling and measuring after 24 hours.Fig. 7 handles the situation that contains ammonia nitrogen and glucose wastewater for the immobilization bacterial strain of aquatic Comamonas bacterial strain in the simulation fluidised bed system.From curve as can be seen, after carrying out reacting in 24 hours, the ammonia nitrogen concentration in the water outlet is very low, and nitrite concentration is also low, and system's denitrification percent reaches 75.2%, has realized short distance nitration-denitrification, reaches the purpose of denitrogenation.
Claims (3)
1. an aquatic Comamonas bacterial strain is characterized in that this bacterial strain is aquatic comamonas (Comamonas aquatica) LNL
3, its bacteriology morphological specificity is a gram negative bacillus, is of a size of 0.5～1.0 * 1～4 μ m, amphitrichous, and aerobic or anaerobism, the catalase positive, oxidase positive is with CO
2For carbon source and energy or with CO
2With organism be that mixed carbon source and energy, ammonia nitrogen or nitric nitrogen are to grow in the basic medium of nitrogenous source, the pH value of substratum is 6.5～8.5, the growth temperature that is fit to is 25～35 ℃; This bacterial strain is that waterplant root district, Kingsoft Lake Water Body from Kingsoft, Zhenjiang Lake Water Body opens pool and Kingsoft lacustrine sediments and gather, separation obtains; This bacterial strain is preserved in " China Committee for Culture Collection of Microorganisms's common micro-organisms " center ", its preserving number CGMCC No.2361 on January 24th, 2008.
2. the application of aquatic Comamonas bacterial strain according to claim 1 in wastewater biological denitrificaion.
3. the application of aquatic Comamonas bacterial strain according to claim 1 in the short distance nitration-denitrification of wastewater biological denitrificaion.
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| CN102443556B (en) * | 2011-12-14 | 2013-04-24 | 首创爱华(天津)市政环境工程有限公司 | Bacterium Rhodanobacter sp. for removing ammonia nitrogen in sewage at low temperature and separation culture method |
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| CN117025457B (en) * | 2023-07-31 | 2024-03-29 | 湘湖实验室(农业浙江省实验室) | Comamonas aquatica and application thereof in seaweed fermentation |
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