CN103966138A - Paracoccus bengalensis N74-1 and application thereof in aquaculture water purification - Google Patents
Paracoccus bengalensis N74-1 and application thereof in aquaculture water purification Download PDFInfo
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- CN103966138A CN103966138A CN201410204027.2A CN201410204027A CN103966138A CN 103966138 A CN103966138 A CN 103966138A CN 201410204027 A CN201410204027 A CN 201410204027A CN 103966138 A CN103966138 A CN 103966138A
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- nitrogen
- aquaculture
- secondary coccus
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- MYPYJXKWCTUITO-LYRMYLQWSA-N vancomycin Chemical compound O([C@@H]1[C@@H](O)[C@H](O)[C@@H](CO)O[C@H]1OC1=C2C=C3C=C1OC1=CC=C(C=C1Cl)[C@@H](O)[C@H](C(N[C@@H](CC(N)=O)C(=O)N[C@H]3C(=O)N[C@H]1C(=O)N[C@H](C(N[C@@H](C3=CC(O)=CC(O)=C3C=3C(O)=CC=C1C=3)C(O)=O)=O)[C@H](O)C1=CC=C(C(=C1)Cl)O2)=O)NC(=O)[C@@H](CC(C)C)NC)[C@H]1C[C@](C)(N)[C@H](O)[C@H](C)O1 MYPYJXKWCTUITO-LYRMYLQWSA-N 0.000 description 1
- 229960003165 vancomycin Drugs 0.000 description 1
- MYPYJXKWCTUITO-UHFFFAOYSA-N vancomycin Natural products O1C(C(=C2)Cl)=CC=C2C(O)C(C(NC(C2=CC(O)=CC(O)=C2C=2C(O)=CC=C3C=2)C(O)=O)=O)NC(=O)C3NC(=O)C2NC(=O)C(CC(N)=O)NC(=O)C(NC(=O)C(CC(C)C)NC)C(O)C(C=C3Cl)=CC=C3OC3=CC2=CC1=C3OC1OC(CO)C(O)C(O)C1OC1CC(C)(N)C(O)C(C)O1 MYPYJXKWCTUITO-UHFFFAOYSA-N 0.000 description 1
Classifications
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02W—CLIMATE CHANGE MITIGATION TECHNOLOGIES RELATED TO WASTEWATER TREATMENT OR WASTE MANAGEMENT
- Y02W10/00—Technologies for wastewater treatment
- Y02W10/10—Biological treatment of water, waste water, or sewage
Landscapes
- Purification Treatments By Anaerobic Or Anaerobic And Aerobic Bacteria Or Animals (AREA)
- Farming Of Fish And Shellfish (AREA)
Abstract
The invention provides newly isolated paracoccus bengalensis N74-1, wherein the preservation number of the strain is CGMCC No.9148. Furthermore, the invention provides application of the strain N74-1 in aquaculture water purification. The paracoccus bengalensis N74-1 disclosed by the invention has heterotrophic nitrification and aerobic denitrification functions, and can simultaneously remove ammoniacal nitrogen and nitrite nitrogen in water; compared with a complex bacterium production process, the paracoccus bengalensis is simpler in preparation process, compatible with nitrification and denitrification functions, and applicable to industrial circulating aquaculture water treatment.
Description
Technical field
The present invention relates to the biologic treating technique field of ammonia-state nitrogen and nitrite nitrogen in aquaculture, specifically, relate to the secondary coccus N74-1 of Bangladesh and the application in aquaculture water purifying thereof.
Background technology
In recent years, along with China's culture fishery development, early stage extensive style, half intensive culture industry are gradually to factory's formula, highly intensive future development.Yet meeting people to the requirement of various fishery products simultaneously, high-density breeding water body, also due to the input of a large amount of bait, change water and the use of sterilization and antimicrobial drug in a large number frequently, increases the weight of water environment pollution load day by day, and disease frequently occurs.In breeding process, the accumulation of the ight soil of aquatic animal, secretory product and residual bait, the concentration of ammonia-state nitrogen and nitrite nitrogen in the water that can raise, causes easily morbidity or dead of Aquatic farming animals, total product of aquaculture and per unit area yield is declined, and improved aquaculture cost.For fear of this situation, occur, what needs were timely, a large amount of changes water with the concentration of dilution toxic substance, has not only wasted water resources but also has additionally increased energy consumption.
The biological treatment of aquiculture waste water can be removed the toxic pollutants such as ammonia-state nitrogen in waste water and nitrite nitrogen, make breeding water obtain recycle, can meet intensive Production requirement and avoid again environmental pollution, and water saving, the requirement of culture fishery Sustainable development met.In aquaculture organisms treatment process, generally adopt biological filter, rely on and be attached to the microorganism place to go pollutent on filtration supports.In culture environment of aquatic products, organic content is few, microbial film film forming in biological filter on carrier is slower, and the nitrobacteria doubling time of autotrophy is long, poor growth, because the biofilm efficiency of whole bio-reactor is low, from starting to normal operation, often need the two weeks time.By adding the purification of water quality microbiobacterial agent of external source, can cross film by enhancement vector, shorten start time, but aquaculture microbiobacterial agent is mainly the microecological microbial agent for cultivation at present, photosynthetic bacterium etc. wherein, be applicable to the bacterial strain of pond culture, can not in industrial circulating water cultivating, play a role well.
Summary of the invention
The object of this invention is to provide the secondary coccus N74-1 of Bangladesh and the application in aquaculture water purifying thereof.
In order to realize the object of the invention, the present invention isolates the secondary coccus that a strain can be removed ammonia-state nitrogen and nitrite nitrogen in aquaculture system from active sludge, secondary coccus (Paracoccus bengalensis) N74-1 of Gai Junwei Bangladesh, now be preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center, No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City, address, Institute of Microorganism, Academia Sinica, postcode 100101, deposit number CGMCC NO.9148, preservation date on May 12nd, 2014.
The 16S rDNA sequence of the secondary coccus N74-1 of Bangladesh is as shown in Seq ID No.1.
The physio-biochemical characteristics of this bacterium are: nitrate reductase, and indole reaction is negative, does not utilize glucose to produce acid; Urase, glucuroide, proteolytic enzyme, galactoside enzyme reaction is feminine gender; Enough utilize carbon source to have glucose, pectinose, seminose, N.F,USP MANNITOL, sorbyl alcohol, arabitol, N-Acetyl-D-glucosamine, maltose, trehalose, sucrose, gluconate, oxysuccinic acid, Sodium.alpha.-hydroxypropionate, Trisodium Citrate and toluylic acid; Do not utilize dextrin, cellobiose, gentiobiose, stachyose, raffinose, lactose, melibiose, 3-formyl glucose, fructose, rhamnosyl, inosine, Serine, aspartic acid, capric acid, hexanodioic acid, oxysuccinic acid; Rifomycin, lincomycin, Guanidinium hydrochloride can be tolerated, troleomycin, vancomycin can not be tolerated.
The observations of N74-1 bacterial strain under electron microscope as shown in Figure 1.
The present invention also provides the microbial inoculum of being prepared by the secondary coccus N74-1 of Bangladesh.
The present invention also provides a kind of water quality cleansing agent, and its effective constituent is above-mentioned microbial inoculum.Rapidly, degraded ammonia-state nitrogen and nitrite nitrogen efficiency are high in bacterial strain N74-1 growth, can remove the organism in water simultaneously, reach the cleaning action to aquaculture system, meet aquaculture needs.
The present invention also provides the fermention medium of the secondary coccus N74-1 of Bangladesh Bangladesh, and described culture medium prescription is: extractum carnis 2-4g/L, peptone 8-10g/L and sodium-chlor 3-5g/L, pH value 7.0-7.5, prepares with water.Preferably, described culture medium prescription is: extractum carnis 3g/L, peptone 10g/L and sodium-chlor 5g/L, pH value 7.0-7.5, prepares with water.
The present invention further provides the application of bacterial strain N74-1 in aquaculture water purifying.
Particularly, get the secondary coccus N74-1 of Bangladesh seed liquor, be seeded in the fermention medium of this bacterium, in 28-30 ℃, 200rpm is cultured to total count and reaches 1 * 10
8individual/mL, gained fermented liquid is added in aquiculture waste water to be clean by the amount of 1-3v/v ‰, add every day 1 time, continuous adding 5-7 days, then by the amount of 1-3v/v ‰, add 1 time weekly, to reduce ammonia-state nitrogen, nitrite nitrogen and the organic content in water body, realize the recycle of aquaculture system.
Preparation method and the using method of the N74-1 liquid bacterial agent relating in the present invention are as follows:
(1) actication of culture: under aseptic condition, picking-80 ℃ cryopreservation, in the N74-1 of glycerine pipe bacterial strain 1-2 ring, at the fresh flat lining out of beef extract-peptone solid medium, is cultivated 2-4 days for 28 ℃-30 ℃.
(2) culture of picking one ring (1) is to 28-30 ℃ of shaking table shaking culture in 50-150ml beef extract-peptone liquid bacterial substratum, cultivates and reaches 1 * 10 to bacterium number in 24-30 hour
7individual/ml order of magnitude, obtains seed liquor.
(3) by above-mentioned seed liquor, by the volume ratio of 2-5%, be seeded in the beef extract-peptone liquid nutrient medium of fresh sterilizing, 28 ℃ of fermentation culture, reach 1 * 10 to total count
8individual/ml order of magnitude, stops fermentation, and gained zymocyte liquid is microorganism purification of water quality microbial inoculum.
Using method: at the use initial stage, bacterium liquid is carried out to interpolation every day by the amount of the 1-3v/v of water processing reactor ‰, require continuous adding 5-7 days, then add 1 time by the amount of 1-3v/v ‰ weekly, until biological treatment device runs well in aquaculture system.
Wherein, the preparation method of beef extract-peptone liquid nutrient medium is: take extractum carnis 3g, peptone 10g and sodium-chlor 5g, add distilled water 1L, with the NaOH aqueous solution of 1mol/L, adjust pH to 7.0-7.5,121 ℃, sterilizing 20min.
The preparation method of beef extract-peptone solid medium is: on the basis of aforesaid liquid substratum, add 1.5%-2% agar, and 121 ℃, sterilizing 20min.
The salient features of bacterial strain N74-1:
1, bacterial strain N74-1 has heterotrophic nitrification ability, can be under organism existence condition Fast Growth, ammonia-state nitrogen is oxidized to nitric nitrogen, and there is no the residual of nitrite nitrogen, be conducive to reduce ammonia nitrogen concentration poisonous to cultivated animals in aquaculture system.Advantage is by heterotrophism, to be cultivated and to be obtained a large amount of thalline rapidly, thus the start-up course of strengthening aquatic products aquaculture water treatment reactor.
2, bacterial strain N74-1 has aerobic denitrification function, can under aerobic condition, remove nitrite nitrogen, reduces the nitrite content in water body, reduces the harm of nitrite nitrogen to aquaculture; Also can subdue the total nitrogen in water body, reach the object of subduing pollutant emission simultaneously.
Microbial inoculum by bacterial strain N74-1 fermentation preparation provided by the invention can be removed the key pollutants in aquaculture system simultaneously: ammonia-state nitrogen and nitrite nitrogen, and there is the ability of certain degraded COD.In addition, N74-1 can be on heterotrophism substratum Fast Growth, thereby be different from Autotrophic nitrification bacterium, can obtain fast a large amount of thalline, after interpolation, can improve rapidly the ability that reactor is removed ammonia-state nitrogen and nitrite nitrogen.
The secondary coccus N74-1 of Bangladesh provided by the invention has heterotrophic nitrification and aerobic denitrification function, can remove ammonia-state nitrogen and nitrite nitrogen in water body simultaneously, compare with the production process of composite fungus agent, preparation technology is more simple, have nitrification and denitrification dual-use function concurrently, be applicable to industrial circulating water cultivating water treatment.
Accompanying drawing explanation
Fig. 1 is the observations of bacterial strain N74-1 of the present invention under electron microscope.
Fig. 2 is that in the embodiment of the present invention 2, bacterial strain N74-1 removes the ability of ammonia-state nitrogen under different carbon source conditions.
Fig. 3 is the heterotrophic nitrification efficiency of bacterial strain N74-1 under different carbon source conditions in the embodiment of the present invention 2.
Fig. 4 is the aerobic denitrification efficiency of bacterial strain N74-1 in the embodiment of the present invention 3.
Fig. 5 is the reactor schematic diagram relating in the embodiment of the present invention 4.
Fig. 6 is the removal effect of N74-1 microbial inoculum to ammonia-state nitrogen and nitrite nitrogen in the embodiment of the present invention 4.
Embodiment
Following examples are used for illustrating the present invention, but are not used for limiting the scope of the invention.If do not specialize, the conventional means that in embodiment, technique means used is well known to those skilled in the art, the raw materials used commercial goods that is.
Embodiment 1 microorganism purification of water quality microbial inoculum and preparation method thereof
In 200ml water, prepare beef-protein medium, add extractum carnis 0.6g, peptone 2g, sodium-chlor 1g, adjusts pH to 7.0, packing 250ml triangular flask, each 100ml, 121 ℃, sterilizing 20 minutes with the 1mol/L NaOH aqueous solution.Two bottles of the N74-1 bacterial strains that inoculation activates through solid plate, 28 ℃ 200 revs/min, shaking table is cultivated 24h, as seed liquor.In 7L small-sized fermentation tank, add 5L water, and add extractum carnis 15g, peptone 50g, yeast extract paste 15g, NaCl15g, adjusts pH to 7.0,121 ℃, sterilizing 20min with the 1mol/L NaOH aqueous solution.When substratum temperature is down to 28 ℃, by 2 bottles of seed liquor combined inoculations in fermentor tank, 28 ℃ of temperature, air flow 5L/ minute, mixing speed 150-200rpm, adds 2.5ml bubble enemy as defoamer, every two hours sampling once, is surveyed the absorbancy under 600nm after dilution, reach 1 * 10 to bacteria concentration
8individual/during mL, to stop fermentation, fermented liquid is microorganism purification of water quality microbial inoculum.
The heterotrophic nitrification characteristic test of embodiment 2 bacterial strain N74-1
1, materials and methods
(1) substratum
Heterotrophic nitrification substratum: glucose 5g, NH
4cl0.76g, NaCl0.3g, yeast extract paste 0.5g, K
2hPO
41g, MgSO
47H
2o0.3g, distilled water 1L, 1mol/L hydrochloric acid is adjusted pH to 7.5,121 ℃, sterilizing 20min.
(2) carbon source experiment
1. on the basis of heterotrophic nitrification substratum, use respectively sucrose, sodium acetate, Soduxin, each 5g place of glucose of Trisodium Citrate, prepare the heterotrophic nitrification substratum of different carbon sources.
2. actication of culture: glycerine pipe bacterial strain one ring of picking-80 ℃ preservation, in the fresh flat lining out of beef extract-peptone solid medium, cultivate two days for 28 ℃, each picking one encircles to 5ml sterilizing beef extract-peptone liquid nutrient medium again, 28 ℃, 200rpm cultivates, incubation time 12 hours.
3. inoculation: the bacterium liquid of activation is seeded in the ratio of 2% (v/v) in the heterotrophic nitrification substratum of different carbon sources of above-mentioned preparation of 100ml sterilizing, 28 ℃, 200rpm cultivation 24 hours.Sampling in every 24 hours once, is got 1ml sample and-20 ℃ of freezing preservations, finishes rear survey detect an ammonia nitrogen content every 10h sampling to experiment.
4. the detection method of ammonia nitrogen concentration: nessler reagent light-intensity method (< < water and waste water determination method > > the 4th edition), detection reagent is purchased from Lian Hua Science and Technology Ltd..
5. nitrite nitrogen concentration detection method: molecular absorption light-intensity method (GB7493-87).
2, experimental result and analysis
The ability that bacterial strain N74-1 removes ammonia-state nitrogen under different carbon source conditions as shown in Figure 2.The heterotrophic nitrification efficiency of bacterial strain N74-1 under different carbon source conditions as shown in Figure 3.Bacterial strain N74-1, when sodium acetate and Soduxin are carbon source, utilizes ammonia-state nitrogen ability the strongest, and within 35 hours, energy utilizes about 320mg/L ammonia-state nitrogen more than 75%, result shows that small molecules acids is easier to be utilized by bacterial strain N74-1, for heterotrophic nitrification, after testing, nutrient solution does not have nitrite nitrogen accumulation.In aquaculture, ammonia nitrogen concentration maintains below 5mg/L substantially, well below experimental concentration, so this bacterium can be applicable to the purification of aquaculture system well.
The aerobic denitrification characteristic test of embodiment 3 bacterial strain N74-1
1, materials and methods
(1) aerobic denitrification substratum: glucose 5g, NaNO
20.78g, NaCl0.3g, yeast extract paste 0.5g, K
2hPO
41g, MgSO
47H
2o0.3g, distilled water 1L, 1mol/L hydrochloric acid is adjusted pH to 7.5,121 ℃, sterilizing 20min.
(2) actication of culture: glycerine pipe bacterial strain one ring of picking-80 ℃ preservation, in the fresh flat lining out of beef extract-peptone solid medium, cultivate two days for 28 ℃, each picking one encircles to 5ml sterilizing beef extract-peptone liquid nutrient medium again, 28 ℃, 200rpm cultivates, incubation time 12h.
(3) inoculation: the bacterium liquid of activation is seeded in the ratio of 2% (v/v) in the aerobic denitrification substratum of 100ml sterilizing, and 28 ℃, 200rpm cultivates 24 hours.Sampling in every 6 hours once, is got 1ml sample and-20 ℃ of freezing preservations, finishes rear sampling detect nitrite nitrogen and nitrate nitrogen content to experiment.
(4) nitrite nitrogen concentration detection method: molecular absorption light-intensity method (GB7493-87).
(5) nitrate detection method: ultraviolet spectrophotometry (< < water and waste water determination method > > the 4th edition).
2, experimental result and analysis
The aerobic denitrification efficiency of bacterial strain N74-1 as shown in Figure 4.In initial nitrite nitrogen concentration, be under 160mg/L condition, this bacterium can utilize rapidly nitrite nitrogen in heterotrophism substratum, after 85 hours, nitrite nitrogen concentration can be reduced to below 10mg/L, illustrate that this bacterium has good nitrite nitrogen tolerance and aerobic denitrifying capacity, also can reduce the nitrate in substratum simultaneously.Aquaculture water Central Asia nitrate is well below experimental concentration (below 1mg/L), so this bacterium can be applicable to the removal of nitrite nitrogen and nitre nitrogen in aquaculture system.
Embodiment 4 utilizes the experiment of microorganism purification of water quality microbial inoculum strengthening moving-bed biofilm
1, materials and methods
Simulation aquatic products aquaculture water: peptone 20mg, NH
4cl38.2mg, NaHCO
371.4mg, NaHPO
418.85mg, tap water 1L.
Environment simulating culturing pool volume: 8L, moving-bed volume: 2L, floating stuffing: 600ml.
Reactor operation method: sequencing batch operation, ammonia-state nitrogen adds NH by 38.2mg/L after being down to 1mg/L again
4cl.
Microbial inoculum addition means: be initially aquaculture system (from culture of ornamental fish base, Tongzhou, Beijing) in experimental group and control group reactor, move the 1st, 8 days, experimental group is added the microorganism purification of water quality microbial inoculum (being N74-1 microbial inoculum) of preparing in the embodiment 1 of 2 ‰ (v/v).
As shown in Figure 5, cyclic water tank is simulation aquaculture pond to reactor schematic diagram, by concentrated liquid pool, pumps into water distribution to provide enough ammonia nitrogen concentration for simulating aquaculture system.Simulation aquatic products aquaculture water pumps into reactor by peristaltic pump, through being attached to microbial process on filler, removes the pollutents such as ammonia nitrogen in water body, nitrite nitrogen, then by rising pipe, flows back to cyclic water tank.
2, result and analysis
N74-1 microbial inoculum to the removal effect of ammonia-state nitrogen and nitrite nitrogen as shown in Figure 6.As can be seen from Figure 6, the interpolation of microbial inoculum has significantly improved the removal efficiency of experimental group ammonia-state nitrogen, reduces the generation of nitrite nitrogen, and in 20 days, ammonia-state nitrogen and nitrite nitrogen concentration are reduced in aquaculture demand, reactor start-up success.And the control group nitrite nitrogen that does not add microbial inoculum accumulates for a long time, although after the later stage reduces, can not degrade rapidly after again adding ammonia-state nitrogen, cause ammonia nitrogen concentration accumulation in culturing pool, reactor fluctuation of service.This experiment shows to add the startup that N74-1 microbial inoculum can shorten moving-burden bed reactor, can fast and stable provide cyclic water outlet for aquaculture.
Although above the present invention is described in detail with a general description of the specific embodiments, on basis of the present invention, can make some modifications or improvements it, this will be apparent to those skilled in the art.Therefore, these modifications or improvements, all belong to the scope of protection of present invention without departing from theon the basis of the spirit of the present invention.
Claims (7)
1. secondary coccus (Paracoccus bengalensis) N74-1 of Bangladesh, is characterized in that, its deposit number is CGMCC No.9148.
2. the microbial inoculum of being prepared by the secondary coccus N74-1 of Bangladesh described in claim 1.
3. a water quality cleansing agent, is characterized in that, its effective constituent is microbial inoculum claimed in claim 2.
4. the fermention medium of the secondary coccus N74-1 of Bangladesh described in claim 1, is characterized in that, described culture medium prescription is: extractum carnis 2-4g/L, peptone 8-10g/L and sodium-chlor 2-5g/L, pH value 7.0-7.5, prepares with water.
5. fermention medium according to claim 4, is characterized in that, described culture medium prescription is: extractum carnis 3g/L, peptone 10g/L and sodium-chlor 5g/L, pH value 7.0-7.5, prepares with water.
6. the application of the secondary coccus N74-1 of Bangladesh in aquaculture water purifying described in claim 1.
7. application according to claim 6, is characterized in that, gets the secondary coccus N74-1 of Bangladesh seed liquor, is seeded in the fermention medium described in claim 4 or 5, and in 28-30 ℃, 200rpm is cultured to total count and reaches 1 * 10
8individual/mL, gained fermented liquid is added in aquiculture waste water to be clean by the amount of 1-3v/v ‰, add every day 1 time, continuous adding 5-7 days, then by the amount of 1-3v/v ‰, add 1 time weekly, to reduce ammonia-state nitrogen, nitrite nitrogen and the organic content in water body, realize the recycle of aquaculture system.
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| CN104877916A (en) * | 2015-06-01 | 2015-09-02 | 云南省烟草农业科学研究院 | Paracoccus limosus, bacterium thereof, method for preparing bacterium and application thereof |
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| CN107760626A (en) * | 2017-11-13 | 2018-03-06 | 中国科学院成都生物研究所 | One plant of Bangladesh's pair meningitidis strains and its application |
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| CN111378603A (en) * | 2020-03-10 | 2020-07-07 | 中国水产科学研究院南海水产研究所深圳试验基地 | Paracoccus angularis LFPH1 for purifying inorganic nitrogen and phosphorus in seawater pond culture water body and application thereof |
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