CN101724594A - Pseudomonas stutzeri CY003 for efficiently removing trite nitrogen, nitrate nitrogen and ammonia nitrogen from water and application thereof - Google Patents
Pseudomonas stutzeri CY003 for efficiently removing trite nitrogen, nitrate nitrogen and ammonia nitrogen from water and application thereof Download PDFInfo
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Abstract
The invention belongs to the technical field of water environmental biological treatment and relates to a separating screen of a bacterial strain CY003 for efficiently degrading trite nitrogen, nitrate nitrogen and ammonia nitrogen, method for testing safety and preparing bacterial solution, and application for treating domestic sewage and cultivating sewage. The bacterial strain CY003 of the invention is preserved in Chinese Type Culture Collection Center and the preserved number is CCTCC No.: M209196. According to morphological observation, physiologic biochemical analysis and 16S rDNA homology analysis, the bacterial strain CY003 is Pseudomonas stutzeri which can rapidly degrade the trite nitrogen, nitrate nitrogen and ammonia nitrogen in water and is not toxic to the animals such as silver carp, crucian, freshwater shrimp, small white mouse, and the like. At normal temperature, the Pseudomonas stutzeri can excellently degrade the nitrite and the ammonia nitrogen in actual sewage. After culturing 75 h, the Pseudomonas stutzeri can degrade 94.7% and 92.7% of trite nitrogen and ammonia nitrogen in cultivating sewage and can degrade 96.2% and 94.6% of trite nitrogen and ammonia nitrogen in domestic sewage. The invention has wide application prospect.
Description
Technical field
The invention belongs to water surrounding microbial treatment technical field, be specifically related to screening and application that the Pseudomonas stutzeri CY003 of nitrite nitrogen, nitric nitrogen and ammonia nitrogen in the water body is removed in a strain.
Technical background
At present, a lot of work have been done by China aspect water prevention and cure of pollution, but aquaculture water and natural water body eutrophication problem are still very severe.Investigation shows (metallographic is bright etc., 1995): the most of lake of China, reservoir have reached eutrophication or have surpassed the eutrophication degree." eutrophication " of water body makes water body become stench unpleasant, fish and other biological mass mortality, and blue-green algae breaks out, a lot of local anhydrous drinks of high temperature season.It is the major cause (Rffollety and Jlhatfield, 2002) that causes Fu Yinghua that nutrient salts elemental nitrogen, the phosphorus that contains in the water body exceeds standard.And along with the fast development of China intensification high-density aquiculture, ammonia nitrogen in the aquaculture water, nitrite nitrogen content overproof phenomenon are very serious, and the nitrite nitrogen in the water body, ammonia nitrogen are to cause one of morbific main root of aquatic animal.The a communique of uniting issue according to the Ministry of Agriculture and State Environmental Protection Administration recently shows that 2003 because the natural fishery resources economy loss of calculating that environmental pollution causes reaches 36.36 hundred million yuan.Therefore the removal of nitrogen in the water body all has great importance for protection environment, development fishery economic and guarantee human health.How economical, remove research emphasis and the focus that nitrogen in the water body has become the water pollution control field efficiently.
Nitrogen is present in the water body with organonitrogen and two kinds of forms of inorganic nitrogen.Organonitrogen has protein-n, polypeptide, amino acid and urea etc., and they derive from the residual bait of feed, sanitary sewage, agricultural wastes (straw, excrement of animals etc.) and trade effluent (as wool processing, process hides, printing and dyeing, food-processing etc.).Inorganic nitrogen refers to mainly with NH
3, NH
4 +, N
2, N
2O, NO, NO
2 -, NO
2, NO
3 -There are (Zheng's equality, 2004) in equivalent state, many studies have shown that, and except molecular nitrogen, all inorganic nitrogen intercycle product accumulation all can produce detrimentally affect to human and environment.Ammonia nitrogen, nitric nitrogen and nitrite nitrogen are the main existence forms of inorganic nitrogen in the water body.
Nitrogen mainly is to rely on action of microorganisms in the conversion of water body, biological denitrificaion (biological nitrogenremoval, BNR) method is meant that nitrification and denitrification by microorganism is used for removing the nitrate pollution (Prakasam and Loehr, 1972) in the water body.Nitrification is meant that nitrifier is oxidized to ammonia nitrogen the process of nitrate nitrogen, comprise two steps: the first step is by Nitrosomas (Nitrosobacter) ammonia nitrogen to be converted into nitrite, and second step was from nitrite is converted into nitrate by Nitromonas (Nitrobacter).Denitrification is meant that denitrifying bacterium changes nitric nitrogen (salt), nitrite nitrogen (salt) and other oxynitride into the biological procedures (Kim et al, 2005) of other gaseous oxidation thing of nitrogen or nitrogen.Denitrifying bacterium is that all can be with NO
3 -Be final electron acceptor(EA), HNO
3Be reduced to N
2The bacterium general name, on kind, do not have special monoid, be scattered in 10 different bacterium sections.Under the general condition, denitrifying bacterium all is to utilize oxygen as electron acceptor(EA), and can utilize in nitrate and the nitrite oxygen to breathe under the very low or anoxybiotic condition in oxygen level, and discharges N
2Most of denitrifying bacteriums are heterotroph facultative anaerobic bacterias, can utilize multiple organism as electron acceptor(EA).The most general denitrification Pseudomonas of occurring in nature is Rhodopseudomonas (Pseudomonas), secondly is Alkaligenes (Alcaligenes).
Traditional theory thinks that the denitrification of bacterium is the anaerobic process of a strictness.Denitrifying bacteria preferentially uses DO (dissolved oxygen) to breathe, and has stoped nitrate or nitrite as final electron acceptor(EA).This becomes the bottleneck that the research of restriction traditional biological denitride technology is used: on the one hand, Nitrite bacteria is a chemoautotrophic bacteria, and growth velocity is slow, and it is long to cause reacting start time, and Nitrite bacteria is to the change of sewage moiety, pH and temperature etc. all responsive (Mobarry et al, 1996).On the other hand, nitrifying process is finished in aerobic environment, and denitrification carries out in the anoxic process.This makes that whole denitrification process must be through aerobic and two links of anoxic (Joo et al, 2005).Yet Robertson and Kuenen observe in the laboratory under the condition that oxygen exists denitrification phenomenon have taken place, and the discovery of Bacterial Aerobic Denitrification has broken through the understanding of traditional theory, for bio-denitrification technology provides a kind of brand-new thinking.People under various environment such as soil, irrigation canals and ditches, the pond, active sludge, settling etc. have all been isolated some aerobic denitrifying bacterias.Lot of domestic and international investigator finds and isolates aerobic denitrifying bacteria, roughly can be classified as: Alcaligenes (Alcaligenes), paracoccus (Paracoccus), Rhodopseudomonas (Pseudomonas) and Rhod (Rhodococcus) etc.These microorganisms all can be with nitrate or nitrite reduction under aerobic condition.
Both at home and abroad to the improvement pay attention to day by day of polluted by nitrogen, bio-denitrification technology gains great popularity safely rapidly because of it in recent years.Have more following unique advantage and compare with traditional bacterium anaerobic denitrifying as the Bacterial Aerobic Denitrification of one of bio-denitrification technology:
1. bacterium can carry out denitrification under aerobic conditions, makes nitrification and denitrification to carry out simultaneously.Nitrated product can be directly as the substrate of denitrification, has avoided the inhibition of the accumulation of nitric acid, nitrous acid to nitration reaction, has quickened nitrated-denitrification process.And the alkali that the OH-that denitrification discharges can partly compensate nitration reaction and consumed can make in the system pH value relatively stable.
2. different with traditional chemoautotrophy nitrifier, aerobic denitrifying bacteria (majority also is a nitrification bacteria) can directly change into gaseous product with ammonia under aerobic condition, and reaction can be finished by one step of single reaction vessel.This has reduced operation easier and running cost.
3. most of aerobic denitrifying bacteria can change in fine adaptation anaerobism (or anoxic) cycle, has ecological growth vigor when aerobic/anoxic replaces.Its fast growth, output height, the dissolved oxygen concentration of requirement is lower, can grow in the slant acidity environment.The microbial culture less investment, and speed of response is fast, and denitrification is thorough, is fit to administer big area polluted by nitrogen waters.
4. the dissolved oxygen of aquaculture water and natural water body is generally more than 4mg/L, and the anaerobe denitrogenation can't realize, and aerobic denitrifying bacteria can be under high dissolved oxygen condition fast denitrogenation, growth and breeding.
Summary of the invention
The objective of the invention is to overcome the defective of prior art, the denitrifying bacterium of screening one strain energy high-efficiency aerobic under aerobic condition, this bacterium has the denitrifying characteristics of high-efficiency aerobic, can utilize organic carbon source, and growth and denitrogenation fast do not produce simultaneously NO in sewage
2And N
2O, and growing under different physical environment temperature has very strong actual application value, the efficient denitrification performance of this bacterium the report that still belongs to the first time at home, and theoretical significance is huge.
The present invention is achieved through the following technical solutions:
The applicant separates from Jingzhou City, Chinese Hubei Province moat bed mud in July, 2006 and obtains a strain nitrite nitrogen is had the bacterial strain of high degradation capability, and it all has good degradation capability to ammonia nitrogen and nitrite nitrogen under aerobic condition.Through morphologic observation, physiological and biochemical test and 16S rDNA identify that this CY003 bacterial strain belongs to Pseudomonas stutzeri (Pseudomonas stutzeri) on taxonomy.The applicant delivers this bacterial strain to Chinese typical culture collection center (CCTCC) preservation in the Wuhan University of Wuhan City, Hubei Province on September 10th, 2009, and its deposit number (CCTCC NO): M 209196.
Meanwhile, the applicant discloses the method for screening Pseudomonas stutzeri (Pseudomonas stutzeri) CY003 that can remove nitrite nitrogen, nitric nitrogen and ammonia nitrogen in the water body, comprises the following steps:
1. the microorganism selective enrichment is cultivated:
Water sample is left standstill abandoning supernatant, get pond sludge seeding (denitrification substratum) in the test tube that adds the Du Shi tubule, inoculum size is 1～5%, cultivates 2～3d in 20～35 ℃ of incubators, and whether it is observed has gas to produce in the Du Shi tubule; For the test tube of aerogenesis, can determine not have substantially required bacterial strain substantially.
2. get the test tube of aerogenesis, therefrom draw 0.1ml, gradient dilution is coated in the BTB solid medium flat board to suitable concn, cultivates 2～3d in 30 ℃ of incubators, the dull and stereotyped inversion;
3. the blue single bacterium colony of picking from BTB substratum solid medium, be inoculated in the nitrated medium liquid substratum of denitrification, inoculum size 5～10%, 2～3d is cultivated in the constant temperature vibration under 30～35 ℃, the condition of 120～180rpm, measure nitrate, the content of nitrite.The nutrient solution that nitric nitrogen consumes substantially is the purpose bacterial strain.
4. the bacterial strain of getting institute's sieve mesh is 10 generations of switching continuously, measure its denitrification, determine whether inheritance stability, legacy is stable be required.
Whether this screening method can contain denitrifying bacterium in the high flux screening sample, verify by aerogenesis intuitively, and reach the effect of orienting enriching, reduce the follow-up work amount, reach the purpose of rapid screening, be fit to compartment analysis denitrifying microorganism from a large amount of samples.The aerobic denitrifying bacteria that is screened can utilize nitrite as normal growth breeding in the substratum of only nitrogen source, and environment is had stronger adaptive faculty, and inheritance stability has bigger development potentiality in practical engineering application.
Below the invention will be further described:
The characteristic of the bacterial strain that used strains separation screening method, degradability measuring method and the institute's separation screening of the present invention obtains is as follows:
One, strains separation screening, degradability are measured the preparation that reaches relevant substratum:
Denitrification substratum: saltpetre 2.0g, bitter salt 0.2g, dipotassium hydrogen phosphate 0.5g, Seignette salt 20g, distilled water 1L, pH 7.2 (add 20g agar and be solid medium), 121 ℃ of moist heat sterilizations 30 minutes.
BTB screening culture medium: altheine acid 1g, KNO
3(perhaps NaNO2) 1g, KH
2PO
41g, sodium succinate 8.5g, FeCl
2.6H
2O 0.05g, CaCl
2.2H
2O 0.2g, MgSO
4.7H
2O 0.07g, 1ml/L BTB[pH indicator Bromthylmol] (1%, be dissolved in ethanol), agar 20g, pH 7.0～7.2.
Two, the separation of Pseudomonas stutzeri CY003 strain and screening:
Water sample is left standstill abandoning supernatant, get active sludge and be inoculated in (denitrification substratum) in the test tube that adds the Du Shi tubule, inoculum size is 2%, cultivates 2d in 20～35 ℃ of incubators, and whether observe has gas to produce in the Du Shi tubule; The test tube that does not have aerogenesis can determine not have substantially required bacterial strain substantially.Get the test tube of aerogenesis, therefrom draw 0.1ml, gradient dilution is coated in the BTB solid medium flat board to suitable concn, cultivates 2～3d in 30 ℃ of incubators, the dull and stereotyped inversion.The blue single bacterium colony of picking from BTB substratum solid medium, be inoculated in the nitrated medium liquid substratum of denitrification, inoculum size 1%, 2～3d is cultivated in the constant temperature vibration under 25～30 ℃, the condition of 120～180rpm, measures nitrite nitrogen, nitric nitrogen and ammonia nitrogen content.The nutrient solution that nitrite nitrogen consumes substantially is the purpose bacterial strain.The bacterial strain of getting institute's sieve mesh is 10 generations of switching continuously, measure its denitrification, determine whether inheritance stability, legacy is stable be required.
The 16SrDNA sequence of the bacterial strain CY003 of the present invention screening is detected (Alfreider etc. with bacterial 16 S rDNA sequence measurement commonly used, 2002), this CY003 16SrDNA sequence sees Table 1, the CY00316SrDNA sequence is carried out the BLAST sequence alignment to the NCBI website, the sequence homology of finding itself and many strains Pseudomonas stutzeri reaches 99%, through identifying, determine that strain separated of the present invention is a strain Pseudomonas stutzeri, its classification called after Pseudomonas stutzeri.
Table 1 CY00316SrDNA sequence
| CY003 16S rDNA sequence | ??TCGGAGATCGATCCTCCGTCTTACCGTCCCCCCGAAGGTTAGACTAGCTACTTCT??GGAGCAACCCACTCCCATGGTGTGACGGGCGGTGTGTACAAGGCCCGGGAACGTA??TTCACCGTGACATTCTGATTCACGATTACTAGCGATTCCGACTTCACGCAGTCGA??GTTGCAGACTGCGATCCGGACTACGATCGGTTTTATGGGATTAGCTCCACCTCGC??GGCTTGGCAACCCTTTGTACCGACCATTGTAGCACGTGTGTAGCCCAGGCCGTAA??GGGCCATGATGACTTGACGTCATCCCCACCTTCCTCCGGTTTGTCACCGGCAGTC??TCCTTAGAGTGCCCACCTTAACGTGCTGGTAACTAAGGACAAGGGTTGCGCTCGT??TACGGGACTTAACCCAACATCTCACGACACGAGCTGACGACAGCCATGCAGCACC??TGTGTCAGAGTTCCCGAAGGCACCAATCCATCTCTGGAAAGTTCTCTGCATGTCA??AGGCCTGGTAAGGTTCTTCGCGTTGCTTCGAATTAAACCACATGCTCCACCGCTT??GTGCGGGCCCCCGTCAATTCATTTGAGTTTTAACCTTGCGGCCGTACTCCCCAGG??CGGTCGACTTAATGCGTTAGCTGCGCCACTAAGATCTCAAGGATCCCAACGGCTA??GTCGACATCGTTTACGGCGTGGACTACCAGGGTATCTAATCCTGTTTGCTCCCCA??CGCTTTCGCACCTCAGTGTCAGTATTAGCCCACGTGGTCGCCTTCGCCACTGGTG??TTCCTTCCTATATCTACGCATTTCACCGCTACACAGGAAATTCCACCACCCTCTG??CCATACTCTAGCTCGCCAGTTTTGGATGCAGTTCCCAGTTGAGCCCGGGGCTTTC??ACATCCACTTAACGACCCACCTACGCGCGCTTTACGCCCAGTAATTCCGATTAAC??GCTTGCACCCTTTCGTATACCGCGCTGCTGGCACGAAGTAAGCGGTGCTATTCTG??TTGTAACGTCAAAACACAAGGTATTACTTACTGCCTTTCCTCCCACTTAAGTGCT??TACAATCGAAGACTTCTTCACACACGCGGCATGCTGATCAGCTCGCCCATTGTCA??GTTCCCCACTGCTGCCTCGTAGGACTGAACGGTTCAGTCCCGTTACAGATACTCT??TTGACGTTACGGACTCGCTCTTGGGAGCTATCCAGAATGTCTAATCCGCAAGTCA??CTGAATCTGAGCTCGAGTTCGCGATTCCTGGGCCATCGTTGCCTTACGCT |
Three, the mycology feature of pseudomonas stutzeri strain CY003:
The CY003 strain is Gram-negative bacteria, and is shaft-like, and bacterium colony is faint yellow; Oxydase, product ammonia, starch hydrolysis and nitrate reduction test are positive, methyl red, V.P, hydrogen sulfide, gelatine liquefication, arginine decarboxylation, Methionin decarboxylation, hydrolysis of urea and Citrate trianion experiment are positive, can be at 6.5% NaCl solid culture basal growth, glucose, sucrose, wood sugar, lactose, maltose and mannose ferment produce not aerogenesis of acid, 4～55 ℃ of growth temperature ranges, 22～35 ℃ of optimum growth temperature scopes.
Four, bacterial strain CY003 is to the degradation property and the testing method of nitrite nitrogen, nitric nitrogen and ammonia nitrogen
1, to the degraded and the utilization of ammonia nitrogen:
(1) degradability is measured: bacterial strain CY003 is inoculated in the denitrifying bacterium liquid nutrient medium, and after the 24h activation, centrifugal 5 minutes of 6000r/min, outwelling supernatant liquor, with physiological saline experimental strain to be made bacteria concentration be 1 * 10
6The cfu/mL bacteria suspension, inoculating 1% bacteria suspension is the water sample of 100mg/L to ammonia nitrogen concentration, 25 ± 1 ℃ of water temperatures, pH7.2 ± 0.5, timing sampling is centrifugal, measures the degradation rate of ammonia nitrogen.
(2) under the differing temps to the degraded situation of ammonia nitrogen: will be 1 * 10 through 24 hours activation back concentration
6The CY003 bacteria suspension of cfu/mL inserts the allotrophic nitrobacteria nutrient solution by 1% inoculum size, is placed on 15 ℃ respectively, and 28 ℃, cultivate under 40 ℃ of conditions, get supernatant liquor after timing sampling is centrifugal, measure under the differing temps, bacterial strain CY003 is to the degradation capability of ammonia nitrogen.
To the degraded of ammonia nitrogen with utilize situation: bacterial strain CY003 is 15～40 ℃ to the temperature range of ammonia nitrogen degradation, under 15 ℃ of conditions, cultivation through 2 days, the ammonia nitrogen degradation rate reaches more than 87%, at optimum temperature range (25～40 ℃), bacterial strain CY003 can be in 24 hours will about 100mg/L ammonia nitrogen degradation 98%.Measure nitric nitrogen and nitrite nitrogen in the nutrient solution supernatant liquor simultaneously, do not have these two kinds of intermediate products and produce.
2, to the degraded and the utilization of nitrite nitrogen, nitric nitrogen:
With the ammonium sulfate in Sodium Nitrite or the SODIUMNITRATE replacement denitrification nutrient solution, making the C/N ratio is 8, and after the sterilization, inserting 1%24 hours activation back concentration is 1 * 10
6The CY003 bacteria suspension of cfu/mL, supernatant liquor is measured nitric nitrogen in 220nm and 275nm place content is got in sampling at regular intervals after centrifugal, and in the content of 540nm place mensuration nitrite nitrogen, the reference standard curve is determined the degradation rate of nitric nitrogen and nitrite nitrogen.
The degraded situation: under the aerobic culture condition, bacterial strain CY003 can grow in the substratum that with Sodium Nitrite or SODIUMNITRATE is only nitrogen source.Cultivating through 24h, can be that the nitric nitrogen of 100mg/L and the nitrite nitrogen of 100mg/L are degraded to 0.61mg/L and 0mg/L respectively with starting point concentration, and degradation rate is respectively 99.4% and 100%.Find that by measuring total nitrogen 24% nitrogen is converted into endogenous nitrogen by the cell assimilation, all the other nitrogen all are converted into nitrogen, no N
2O produces.In the two degradation process, all do not detect the accumulation of nitrite nitrogen and ammonia-state nitrogen.When being nitrogenous source with the nitric nitrogen, the accumulation volume of nitrite nitrogen has only 0.09mg/L at most.
Five, the animal safety of bacterial strain CY003 is measured
Bacterial strain CY003 is transferred in the meat soup liquid nutrient medium, the centrifugal supernatant liquor that removes behind the cultivation 24h, making bacterial content is 2 * 10
11The liquid aqua of cfu/mL is added to each aquaculture water respectively, observes its security to hydrocoles.Each concentration group test fish is 30 tails, 30 of each concentration groups of shrimp, as test chamber, each glass jar dress is through the abundant tap water 200L of aeration with the glass jar of long 74cm * wide 78cm * high 47cm, with heating rod heating and control water temperature is 25 ± 1 ℃, pH7.2～7.8.
A, to the acute toxicity test of hybridized prussian carp
After 10ppm, 100ppm, 1000ppm, 5000ppm CY003 bacterium were used, the equal feeding activity of test fish was normal in 9 days, surviving rate 100%, 2 * 10
11The CY003 bacterium of cfu/mL concentration is to 96 hours LD50＞5000ppm of hybridized prussian carp.
B, to the acute toxicity test of silver carp
After 10ppm, 100ppm, 1000ppm, 5000ppm CY003 bacterium were used, the equal feeding activity of test fish was normal in 9 days, surviving rate 100%, 2 * 10
11The CY003 bacterium of cfu/mL concentration is to 96 hours LD50＞5000ppm of silver carp.
C, to the acute toxicity test of fresh water freshwater shrimp
After the CY003 bacterium of 10ppm, 100ppm, 1000ppm was used, the equal feeding activity of test shrimp was normal in 9 days, surviving rate 100%, and after the CY003 bacterium of 5000ppm was used 48h, it is muddy that water body becomes, and shrimp has the anoxic symptom, and surviving rate 80% after 9 days.2 * 10
11The CY003 bacterium of cfu/mL concentration is to 96 hours LD50＞1000ppm of fresh water freshwater shrimp.
D, to the acute toxicity test of channel catfish
After the CY003 bacterium of 10ppm, 100ppm, 1000ppm, 5000ppm was used, the equal feeding activity of test fish was normal in 9 days, surviving rate 100%, 2 * 10
11The CY003 bacterium of cfu/mL concentration is to 96 hours LD50＞5000ppm of channel catfish.
E, to the small white mouse toxicity test
The small white mouse random packet, 20 every group, each 10 of male and female are weighed before the administration.Two groups of mouthful filling administration of test branch, spice administrations, each group are according to high, normal, basic three dosed administrations, and wherein mouthful filling of high dose group difference and spice are given bacterium 2 * 10
11Cfu/, be administered once every day.Establish the blank group of experiment contrast combination simultaneously.
After each is organized mouse and throws something and feeds with the CY003 bacterium, observe the situation such as appearance, behavior, diet of small white mouse every day.Off-test in 30 days 24h after the last administration, red corpuscle sum, oxyphorase, white corpuscle, lymphocyte, glutamic-oxal(o)acetic transaminase, gpt, AST, ALK index are measured in blood sampling, and each result is done variance test.Each is dissected after organizing the small white mouse blood sampling immediately, observes the pathological change of the heart, liver, spleen, lung, kidney, stomach, brain, uterus, testis.
After the off-test in 30 days, the activity of each test combinations control group small white mouse, color gloss, respiration apocatharsis are all normal, feed intake and the equal indifference of small white mouse body weight gain (P＞0.1), the heart, liver, spleen, lung, kidney, stomach, brain, uterus, testis are all normal, hematology of test group and control group small white mouse and blood parameters are through the t assay, behind the CY003 bacteria preparation that shows that the small white mouse mouth is irritated and spice is thrown something and fed, the t value all＞0.1, both indifferences show that the CY003 bacterium is to the small white mouse nontoxicity.
The pathogenic test that F, crucian mouth are irritated the CY003 bacterium
The CY003 inoculum is centrifugal, be diluted to 2 * 10 with physiological saline
11Cfu/mL, pour into the CY003 dilution of bacteria of 2mL, 0.6mL, 0.2mL respectively, per 2 days once, connects to irritate three times, establishes a mouthful control group of irritating 2mL sterilization LB substratum simultaneously, mouth is irritated and is finished the continuous observation in back 15 days, each test group and control group crucian mouth death all occurs after irritating the CY003 bacterium, and surviving rate is 100%, and comparing body weight with control group does not have considerable change, ANOMALOUS VARIATIONS does not appear in body surface and internal organ, and test shows that mouthful filling CY003 bacterium is to the crucian no pathogenicity.
The pathogenic test of G, injection CY003 bacterium.
The CY003 inoculum is centrifugal, be diluted to 2 * 10 with physiological saline
11Cfu/mL, abdominal injection crucian 20 tails are every group respectively, are divided into 2 groups, and each injects the CY003 dilution of bacteria of 1mL, 0.5mL, control group injection sterile saline 0.5mL; Mitten crab is injected 0.1mL for every in 20 of tripodia base portion injections, control group injection sterile saline 0.1mL; Penaeus vannamei is injected 20 tails, every tail 0.1mL, control group injection sterile saline 0.1mL; 20 every group of small white mouse is divided into 2 groups, and each injects the CY003 dilution of bacteria of 1mL, 0.5mL, control group injection sterile saline 0.5mL; Each animal groups was observed 15 days, the duration of test water temperature is 25 ± 1 ℃, the water body dissolved oxygen is more than 4.0mg/L, pH7.35, room temperature is 28 ± 2 ℃, and each test group is and death occurs, and comparing body weight with control group does not have considerable change, ANOMALOUS VARIATIONS does not appear in body surface and internal organ, and test shows that injection CY003 bacterium is to the aquatic animal no pathogenicity.
Six, the environmental safety of bacterial strain CY003 is measured
The flea class is the important monoid of limnobios, is the important step in material cycle and the flow of energy in the Freshwater ecosystems.They have very strong sensitivity to various poisonous substances.Extensively estimate the toxicity of various products both at home and abroad, carry out water pollution monitoring and formulate various water quality standard with it.This test and Selection the naked abdomen flea of the common biological thorniness of fresh water (Moina macrocopaStraus) as test organism, research bacterial strain CY003 is to its toxicity, for confirming that foundation provides necessary parameter to bacterial strain CY003 to ecological environment security.
The experimental animal kind: the naked abdomen flea of thorniness (Moina macrocopa Straus) is the gynecogenic single young flea of indoor long-term cultivation, and female flea is picked up from the adult fish testing ground in the Changjiang river aquatic products institute Yao Wan base.Select for use and cultivate young flea more than 3 generations, birth 6～24h under the laboratory condition for testing flea, the test flea should be the offspring of same parent, and it is active individual to choose health during test.Put into 10 of the test fleas cleaned with dilution water in every small beaker.
The raising condition: with high activity dried yeast (Angel Yeast Co.,Ltd's production) is bait.In the Circular glass cylinder or beaker of 1～2L, add 500～1000mL and cultivate water (tap water filter that natural aeration 3d is above), add the naked abdomen flea of 10～20 thorniness of people, with throwing something and feeding behind about 40 ℃ the water-solubleization dry yeast, every day is sooner or later respectively once.(cultivate 22 ± 1 ℃ of culture temperature, pH7.2 ± 0.5, dissolved oxygen 3mg/m under the distance 2～3m) at indoor fluorescent lamp
3More than, change nutrient solution weekly entirely 1～2 time.Select 10～20 of the high water fleas of fish brood amount, the competent bait of throwing something and feeding, before experiment 24h with the aperture be the sieve of 1mm with young flea elimination, 6～12h carries out sieving second time before experiment, the young water flea of resultant new birth is the young flea of 6～24h size of being born.
Test method:, be diluted to 2 * 10 with physiological saline with CY003 bacterial liquid medium centrifugal
11Cfu/mL selects 5 experimental concentration, and control group is set.The experimental concentration of bacterial strain CY003 in the test water is divided into 100ppm, 1000ppm, 2500ppm, 5000ppm, 10000ppm totally 5 test group, does not add bacterium liquid in the blank group.Inject the 800mL experimental liquid with the 1000mL beaker during test, put into 10 of the test fleas cleaned with dilution water in every glass.Then small beaker is put into water-bath and cultivate, controlled temperature is at 22 ± 1 ℃, and every day, (distance 2～3m) was shone 12h with fluorescent lamp.Not bait throwing in of duration of test, test period 24h.The determination methods of flea death generally adopts visual inspection, checks that the flea class stops action, and need rotate test chamber repeatedly, and the length that is no more than itself with flea class operating range in the 15s is sign, though the feeler and the gill can also activity or intestines still also stop action at wriggling.
Test-results: in the high density group, it is muddy that water quality becomes, but flea class mobility strengthens on the contrary, do not have the cluster behavior, and distribution is all arranged up and down in the beaker.The CY003 bacterium the results are shown in Table 2 to the acute experiment of the naked abdomen flea of thorniness.As can be seen from Table 2, concentration is 2 * 10
11The CY003 bacterium liquid of cfu/mL is to the 24h-LC of the naked abdomen flea of thorniness
50Greater than 10000ppm, be far longer than the denitrogenation working concentration of bacterial strain CY003, normal use can not cause environmental pollution and to the influence of aquatic attitude.
Table 2 bacterial strain CY003 is to the naked abdomen flea of thorniness The acute toxicity tests (21～23 ℃ of water temperatures)
| ??No | ??C??K | ??1 | ??2 | ??3 | ??4 | ??5 |
| Concentration (mg/L) | ??0 | ??1??0??0 | ??1??0??0??0 | ??2??5??0??0 | ??5??0??0??0 | ??100??00 |
| Mortality ratio (%) | ??0 | ??0 | ??0 | ??0 | ??0 | ??0 |
Seven, bacterial strain CY003 measures the processing power of actual sewage
The CY003 inoculum is centrifugal, be diluted to 1 * 10 with physiological saline
6Cfu/mL, the inoculum size by 1% inserts in the sanitary sewage, the amount of ammonia nitrogen and nitrous acid nitrogen in room temperature (22～25 ℃) the cultivation 72h mensuration water body, the result shows that bacterial strain CY003 is respectively 93.35% and 87.43% to the clearance of two kinds of nitrogenous sources.Inoculum size by 1 ‰ inserts in the breeding wastewater, the amount of ammonia nitrogen and nitrous acid nitrogen in room temperature (22～25 ℃) the cultivation 72h mensuration water body, and the result shows that bacterial strain CY003 is respectively 98.40% and 92.75% to the clearance of two kinds of nitrogenous sources.Experimental result shows that under the normally fermentation situation, the CY003 bacterial concentration reaches 1 * 10
10When cfu/mL was above, every liter of CY003 bacteria preparation can be used for 1000m
3Water body is used for fast denitrogenation.
Eight, bacterial strain CY003 liquid bacterial agent preparation
This bacteria fermentation flow process is: slant strains → activation → triangular flask liquid spawn → ferment tank → check → packing → finished product microbial inoculum.
The concrete processing step of described microbial inoculum is as follows
1) with above-described, be deposited in CCTCC, deposit number is: NO.M209196, preservation date: on September 10th, 2009, insert the denitrification substratum: extractum carnis 5.0g, saltpetre 3.0g, bitter salt 0.2g, dipotassium hydrogen phosphate 0.5g, Seignette salt 20g, distilled water 1L, pH 7.2 (add 20g agar and be solid medium), 121 ℃ of moist heat sterilizations 30 minutes.Cultivated 24 hours under 37 ℃ of conditions;
2) make the triangular flask liquid spawn: the test tube inoculation is cultivated on the denitrification liquid nutrient medium, this substratum is made by the following method: saltpetre 3.0g, glucose 5.0g, extractum carnis 5.0g, bitter salt 0.2g, dipotassium hydrogen phosphate 0.5g, Seignette salt 20g, 7.2,121 ℃ of moist heat sterilizations of distilled water 1L, pH 30 minutes.
2) ferment tank: in fermentor tank, mix up material: saltpetre 2.0kg, glucose 5.0kg, yeast extract paste 8.0kg, potassium nitrite sodium 2.0kg, bitter salt 0.2kg, dipotassium hydrogen phosphate 0.5kg, sodium-acetate 5kg, filter 1 ton of sterilized water by following prescription.Keep 32 ± 2 ℃, inoculate 5% triangular flask liquid spawn, fully aeration-agitation fermentation, fermentation in 72 hours is finished, check, packing.
The bacterium liquid living bacteria count of present method preparation is greater than 1 * 10
10Cfu/mL.
Description of drawings
Fig. 1: be the degradation curve of the pseudomonas CY003 that screens of the present invention to ammonia nitrogen.Ordinate zou is an ammonia nitrogen among the figure, nitric nitrogen, and the value of nitrite nitrogen concentration and OD600, X-coordinate is that different time is observed.
Fig. 2: be pseudomonas CY003 of the present invention degradation curve to ammonia nitrogen under 15,28 and 40 ℃ of conditions.Ordinate zou is the concentration of ammonia nitrogen under the differing temps among the figure, and ordinate zou is a different time.
Fig. 3: be the degradation curve of pseudomonas CY003 of the present invention to nitric nitrogen.Ordinate zou is an ammonia nitrogen among the figure, nitric nitrogen, and nitrite nitrogen concentration, X-coordinate is the different time.
Fig. 4: be the degradation curve of pseudomonas CY003 of the present invention to nitrite nitrogen.Ordinate zou is an ammonia nitrogen among the figure, nitric nitrogen, and nitrite nitrogen concentration, X-coordinate is the different time.
Embodiment
The separation of embodiment 1, bacterial strain, screening and evaluation
One, the separation screening of bacterial strain
1, be formulated as follows substratum:
Denitrification substratum: saltpetre 2.0g, bitter salt 0.2g, dipotassium hydrogen phosphate 0.5g, Seignette salt 20g, distilled water 1L, pH 7.2 (add 20g agar and be solid medium)
BTB screening culture medium: altheine acid 1g, KNO
3(perhaps NaNO
2) 1g, KH
2PO
41g, sodium succinate 8.5g, FeCl
2.6H
2O 0.05g, CaCl
2.2H
2O 0.2g, MgSO
4.7H
2O 0.07g, 1ml/L BTB[pH indicator Bromthylmol] (1%, be dissolved in ethanol), agar 20g, pH 7.2.
2, aerobic denitrifying bacteria screening
1., the microorganism selective enrichment is cultivated: water sample is left standstill abandoning supernatant, get active sludge and be inoculated in (denitrification substratum) in the test tube that adds the Du Shi tubule, inoculum size is 5%, cultivates 48h in 28 ℃ of incubators, and whether observe has gas to produce in the Du Shi tubule.
2., get the test tube of aerogenesis, therefrom draw 0.1ml, gradient dilution is coated in the BTB solid medium flat board to suitable concn, cultivates 48h in 28 ℃ of incubators, the dull and stereotyped inversion.
3., from BTB substratum solid medium the blue single bacterium colony of picking, be inoculated in the nitrated medium liquid substratum of denitrification, inoculum size 5%, 48h is cultivated in the constant temperature vibration under 28 ℃, the condition of 160rpm, measures nitrate, the content of nitrite.The nutrient solution that nitric nitrogen consumes substantially is the purpose bacterial strain.
4., the bacterial strain of getting institute's sieve mesh continuous 10 generations of switching, measure its denitrification, determine whether inheritance stability, legacy is stable be required.
Embodiment 2, Pseudomonas stutzeri CY003 measure the removal ability of sanitary sewage and breeding wastewater ammonia nitrogen
Test the Jing Zhou east gate moat sewage draining exit that used sanitary sewage is got, measuring its ammonia nitrogen concentration is 62.15mg/L, and nitrite concentration is 5.2mg/L, pH6.0-6.8; Changjiang University's culture of swamp eel base aquaculture wastewater that breeding wastewater is got, ammonia nitrogen concentration is 5.72mg/L, nitrite concentration is 0.7mg/L, pH7.5-8.5.With concentration is 1 * 10
7Cfu/mL CY003 microbial inoculum, the inoculum size by 1 ‰ inserts in the sewage.Incubated at room temperature 72 hours is measured ammonia nitrogen content in two kinds of sewage, determines the sewage water denitrification ability, and concrete experimental result sees Table 1.
Table 3 bacterial strain CY003 is to the ammonia nitrogen removal effect
Table 4 bacterial strain CY003 is to the nitrite nitrogen removal effect
Claims (4)
1. Pseudomonas stutzeri (Pseudomonasstutzeri) CY003 that can remove ammonia-state nitrogen, nitric nitrogen and nitrite nitrogen in the water body is deposited in Chinese typical culture collection center, and its deposit number is (CCTCC NO): M 209196.
2. the method for an efficient screening denitrifying bacteria comprises the following steps:
(1), the microorganism selective enrichment is cultivated:
Water sample is left standstill abandoning supernatant, get pond sludge seeding (denitrification substratum) in the test tube that adds the Du Shi tubule, inoculum size is 1～5%, cultivates 2～3d in 20～35 ℃ of incubators, and whether it is observed has gas to produce in the Du Shi tubule; For the test tube of aerogenesis, can determine not have substantially required bacterial strain substantially.
(2), get the test tube of aerogenesis, therefrom draw 0.1ml, gradient dilution is coated in the BTB solid medium flat board to suitable concn, cultivates 2～3d in 30 ℃ of incubators, the dull and stereotyped inversion;
(3), the blue single bacterium colony of picking from BTB substratum solid medium, be inoculated in the nitrated medium liquid substratum of denitrification, inoculum size 5～10%, 2～3d is cultivated in the constant temperature vibration under 30～35 ℃, the condition of 120～180rpm, measure nitrate, the content of nitrite.The nutrient solution that nitric nitrogen consumes substantially is the purpose bacterial strain.
(4), the bacterial strain of getting institute's sieve mesh continuous 10 generations of switching, measure its denitrification, determine whether inheritance stability, legacy is stable be required.
Denitrification substratum: saltpetre 2.0g, bitter salt 0.2g, dipotassium hydrogen phosphate 0.5g, Seignette salt 20g, distilled water 1L, pH7.2 (add 20g agar and be solid medium), 121 ℃ of moist heat sterilizations 30 minutes.
BTB screening culture medium: altheine acid 1g, KNO
3(perhaps NaNO
2) 1g, KH
2PO
41g, sodium succinate 8.5g, FeCl
2.6H
2O 0.05g, CaCl
2.2H
2O 0.2g, MgSO
4.7H
2O 0.07g, 1ml/L BTB[pH indicator Bromthylmol] (1%, be dissolved in ethanol), agar 20g, pH 7.0～7.2.
3. the application of the described bacterial strain of claim 1 in aquaculture system, landscape water body and industrial purifying domestic sewage.
4. the described application of claim 2 is comprising the application of removing in the water body in ammonia-state nitrogen, nitric nitrogen and the nitrite nitrogen.
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| CN112251387A (en) * | 2020-11-05 | 2021-01-22 | 广东省微生物研究所（广东省微生物分析检测中心） | Denitrifying bacterium and application thereof |
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