CN101712943B - Biological denitrogenation agent and application thereof - Google Patents
Biological denitrogenation agent and application thereof Download PDFInfo
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- CN101712943B CN101712943B CN2009102339902A CN200910233990A CN101712943B CN 101712943 B CN101712943 B CN 101712943B CN 2009102339902 A CN2009102339902 A CN 2009102339902A CN 200910233990 A CN200910233990 A CN 200910233990A CN 101712943 B CN101712943 B CN 101712943B
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Abstract
The invention relates to the microbe field and relates to a biological denitrogenation agent and application thereof. More specifically, the invention relates to a bacterium with denitrogenation biological activity, a method for culturing the bacterium, preparation of the biological denitrogenation agent with the bacterium as the main composition and application of the biological denitrogenation agent in degrading water nitrogen. The bacterium of the invention is named as pseudomonas sp. DN-2, the collection number of the bacterium is CGMCC No. 2917, the collection date is February 27, 2009 and the collection unit is China General Microbiological Culture Collection Center (CGMCC).
Description
Technical field
The present invention relates to microorganism field, be specifically related to a kind of the have bacterium of denitrogenation biologic activity, the method for cultivating this bacterium, is the preparation of the biological denitrifier of main moiety with this bacterium, and the application in degraded water body nitrogen.
Background technology
Nitrogen Cycling is one of composition of the of paramount importance ecosystem material cycle of occurring in nature.Yet the use of nitrogenous fertilizer and the development of high-density aquiculture have also caused serious environmental problem when bringing great economic benefit.In aquaculture, the eutrophication situation that causes because of the nitrogen phosphorus element content is too high frequently occurs, except that making water quality deterioration and destruction biotic balance wherein, ammonia nitrogen wherein and nitrite nitrogen have strong toxicity to cultivated animals, particularly nitrite nitrogen conversion rate under cultivating condition is low, make that nitrite accumulation is very outstanding in the water body of high-density breeding, especially after the heavy rain, nitrite content is severe overweight extremely easily, by its aquatic animal disease that causes even [the Ni JY that appear in the newspapers of the phenomena of mortality more, Gu B, ShaoCS.A case of detoxication of nitrite poisoning Litopenaeus vannamei.Scienti Fish Farming, 2004,4:49.Xu PF, Zeng XK.Aalysis of nitrite harm for farming Litopenaeus vannamei in freshwater andcountermeasure.Fishery Modernization, 2006,2:38-39.].As in prawn culturing, nitrite content surpasses 0.1ppm and reaches 0.3ppm when above, is easy to cause prawn to poison, and is difficult for rescue.Adopt active sludge directly to drop into traditionally, after adopt the immobilization photosynthetic bacterium to carry out denitrogenation processing again.People once utilized nitrifying bacteria community to denitrogenate, but the end product of nitrification is a nitrate, and balance and this product that can destroy the biosphere nitrogen can bring out human methemoglobinemia.Because must oxygenation in the raising fish and shrimp process guarantee the dissolved oxygen content in the water, the denitrogenation of anaerobic denitrifying bacteria can not be not fully exerted so.Therefore, people to aerobic denitrifying bacteria, change into diversion gaseous nitrogen because it can carry out denitrification under aerobic conditions with nitrate and nitrite, and ammonia directly can be changed into gaseous product under aerobic condition.
Compare with traditional biological denitrification process, the appearance of aerobic denitrifying bacteria can make biological denitrificaion finish in same reactor, realizes synchronous nitration and denitrification truly.The report that successful Application has been arranged about the biological denitrificaion that utilizes aerobic denitrifying bacteria to realize at present.[Gupta AB such as Gupta, Gupta SK.Simultaneous Carbon and NitrogenRemoval in a Mixed Culture Aerobic RBC Biofilm[J] .Wat Res, 1999,33 (2): 555-561.] use the blodisc that contains Thiosphaera pantotropha (now renaming Paracoccus denitrificans as) to handle the sanitary sewage of different concns; [Kshirsagar M such as Kshirsagar, Gupta AB, Gupta SK.Aerobic Denitrification Studies onActivated Sludge Mixed with Thiosphaera pantotropha[J] .Environ Technol, 1994,16 (1): 35-43.] utilize two identical oxidation ditches of operational condition to come treatment of simulated fertilizer industry waste water, wherein added Paracoccus denitrificans in the oxidation ditch, another oxidation ditch that does not add is a contradistinction system; [Pai SL such as Pai, Chong NM, ChenCH.Potential Applications of Aerobic Denitrifying Bacteria as Bioagents in Wastewater Treatment[J] .Bioresource Technology, 1999,68 (2): 179-185.] also once aerobic denitrifying bacteria T6 and nitrifying sludge were added in the same aerobic reactor.Utilize aerobic denitrifying bacteria to develop aerobic denitride technology and have following advantage [Huang HK, Tseng SK.Nitrate reduction by Citrobacter diversus under aerobic environment[J] .Appl MicrobiolBiotechnol, 2001,55:90-94.]: the first, nitrated-anti-nitration reaction is carried out in same reactor, can significantly reduce floor space and construction fund; The second, use aerobic denitrifying bacteria can reduce the chemical substance that adds regulation system pH in the treating processes, reduce cost; Three, the easier control of aerobic denitrifying bacteria in treating processes.
The key of aerobic denitrifying bacteria denitride technology is, and under certain conditions (as temperature, pH, the C/N ratio), this denitrifying bacterium itself has the ability that removes of nitre attitude, nitrite nitrogen efficiently, has external environment tolerance preferably simultaneously, as to salinity, the tolerance of heavy metal ion etc.Therefore, screening the aerobic denitrifying bacteria that has this two aspects ability simultaneously has very important significance for this The Application of Technology tool.Problems such as the aerobic denitrifying bacteria of having reported at present exists removing of nitre attitude, nitrite nitrogen indifferent mostly, lacks the tolerance to high concentration nitrate, nitrite, and the treatment time is long, and efficient is not high.[Wang Hongyu such as Wang Hongyu, horse is put, Su Junfeng, Deng the influence [J] of the different carbon sources of .2007. and carbon-nitrogen ratio to a strain aerobic denitrifying bacteria nitrogen removal performance. ACTA Scientiae Circumstantiae, 27 (6): 968-972] reported that a strain aerobic denitrifying bacteria can be at 8-10h, with the nitrate degraded of starting point concentration 150mg/L more than 90%.As seen, the tolerance to initial nitrate, nitrite also is one of important factor that limits its application.
The process of " nitrated-denitrification " denitrification process is organonitrogen → NH
4 +-N → NO
2 --N → NO
3 --N → NO
2 --N → nitrogen is overflowed, in this process, and NO
3 -The generation of-N has not only prolonged reaction mechanism, and has caused the waste of energy and additional carbon, therefore more takes " nitrosification-denitrification " denitrification process now.This just requires flora can utilize NO fast
2 --N carries out reaction fast, thereby improves the efficient of this denitrification process.
Summary of the invention
An object of the present invention is to provide a kind of new bacterium with denitrogenation biologic activity, this bacterium has the ability that removes the water body nitrogen.
Another object of the present invention provides a kind of combination culture condition, makes this bacterium can embody stronger nitrogen and removes ability.
A further object of the present invention has provided a kind of fermentor cultivation condition, makes this bacterium can realize the cultivation of large scale and high density.
A further object of the present invention provides a kind of formulation of biological denitrifier and forms mode, makes it have more practicality and operability when handling the water body nitrate pollution.
A further object of the present invention provides the application of bacterium of the present invention in the water body biological denitrificaion.This biological denitrifier of use gets final product the nitrogen in the effective elimination water body separately.
The invention discloses a strain and have the bioactive bacterium of denitrogenation, this bacterium has higher denitrification activity and individual plant nitric nitrogen/nitrite nitrogen removes ability.This class bacterial strain is an aerobic denitrifying bacteria, can be effectively with NO in biological denitrificaion
3 --N or NO
2 --N directly changes into self component of thalline, also can be with NO
3 --N or NO
2 --N is converted into gas mode such as NO, the N of various nitrogen elements by denitrification
2O and N
2Gas evolution.More specifically, this bacterioid is the bacterioid of Rhodopseudomonas.Disclosed bacterium called after pseudomonas DN-2 of the present invention, Pseudomonas sp.DN-2, preserving number are CGMCCNo.2917, the preservation time: on February 23rd, 2009, depositary institution: China Committee for Culture Collection of Microorganisms common micro-organisms center.
The present invention is with mire and 4 parts of (soil under the one place vegetation of the Purple Mountain, Nanjing of the more rich soil of vegetation; Mud in the one place's mire of the Purple Mountain, Nanjing; Soil in China Medicine University's campus flower bed; The XuanWu Lake, Nanjing lakeside mud) for for the examination soil sample, through enrichment culture, separation and purification, active check have obtained a large amount of bacterial strains with denitrification activity, confirm that tentatively they have nitric nitrogen/nitrite nitrogen and remove ability.These bacterial strains belong to Rhodopseudomonas, bacillus etc.
Particularly, the bacterium of this that from soil, screens ten strain different generas, this 9 strain bacterium all has similar biological function, the denitrification activity that it is higher and individual plant nitric nitrogen/nitrite nitrogen remove ability and have obtained concentrated expression by enrichment culture and separation screening, but 9 strain bacterium are variant on morphology.The morphological specificity of each strain bacterium on separation and Culture reaches sees Table 1.
Bacterium colony thalli morphology and the source thereof of table 1 bacterial strain DN-1 to DN-9
Annotate: source 1 is a soil under the one place vegetation of the Purple Mountain, Nanjing;
2 is mud in the one place's mire of the Purple Mountain, Nanjing;
3 is soil in China Medicine University's campus flower bed;
The XuanWu Lake, 4 Nanjing lakeside mud.
+ expression is dynamic;-expression is unpowered
The contriver removes ability from 9 strain bacteriums to its nitrite nitrogen and detects comparison repeatedly, the bacterium that found that separation numbering DN-2 of soil under the one place vegetation of the Purple Mountain, Nanjing shows higher denitrification activity and individual plant nitric nitrogen/nitrite nitrogen removes ability, belong to Rhodopseudomonas through evaluation, Pseudomonas sp.DN-2.This bacterial strain (hereinafter to be referred as pseudomonas DN-2) has following feature:
1. colony morphology characteristic: on the agar plate of denitrification substratum, 30 ℃ of aerobic cultivations of constant temperature form irregular polygon sample bacterium colony after 3 days, be faint yellow, surface folding, and quality is stiff, is difficult for provoking, and matrix does not see that tangible soluble pigment produces.
2. morphological features: shaft-like, about 0.3～0.5 * 1.5～2.0 μ m of thalline size, Gram-negative, no statospore.
3. the major physiological biochemical test sees Table 2: the suitableeest culture temperature is 30 ℃, and pH8.5 is aerobic.
The physio-biochemical characteristics of table 2DN-2 bacterial strain
Annotate :+expression positive reaction maybe can utilize;-expression negative reaction maybe can not be utilized.
4. phylogenetic analysis: the 16S rDNA gene to pseudomonas DN-2 increases and checks order, the row that check order carry out similarity analysis with BLAST software and GenBank database, and in Clustal X (1.8) software, carry out the multiple sequence compare of analysis with similar sequences, use Neighbor-Joining method constructing system evolutionary tree in MEGA (3.1) software at last.
5. denitrification activity: respectively with 0.616g/L NaNO
3Or 0.5g/L NaNO
2During for only nitrogen source, or being mixed nitrogen with the concentration of half-and-half, is carbon source with the 5.0g/L sodium acetate, cultivates about 12h, and the decreasing ratio of nitric nitrogen and nitrite nitrogen all can reach 100%; Respectively with 5.0g/L Zulkovsky starch, glucose, when N.F,USP MANNITOL is carbon source, when cultivating 24h, the decreasing ratio of nitric nitrogen or nitrite nitrogen can reach 100%.Work as NaNO
2When concentration reaches 4g/L, can in 54h, thoroughly reduce; When concentration reaches 5g/L, still can slowly degrade.
Classification with reference to uncle Jie Shi Bacteria Identification handbook the 9th edition is identified, this bacterial strain belongs to Rhodopseudomonas, its 16SrRNA is carried out phylogenetic analysis, with CLUSTAL_1.8 and Mega software building evolutionary tree, find that the sibship of Pseudomonas alcaligenes (Pseudomonas alcaligenes) in itself and the Rhodopseudomonas and pseudomonas mendocina (Pseudomonas mendocina) is nearest.Pseudomonas DN-2 is preserved in China Committee for Culture Collection of Microorganisms common micro-organisms center (CGMCC) on February 27th, 2009, and preserving number is CGMCC No.2917.
The present invention separates the pseudomonas DN-2 that obtains from soil, in containing the water body of nitrate/nitrite, nitrate/nitrite nitrogen can not only be converted into fast the component of thalline self, and have higher denitrification activity.The environment that this effect takes place is under aerobic condition, and under higher C/N mol ratio condition, biologic activity is stronger.
Pseudomonas DN-2 of the present invention can cultivate in order to following method: cultivate under following condition: temperature: 20～40 ℃; Dissolved oxygen: shake bottle rotating speed a 60～180rpm; PH5.0～10.0; Carbon source is selected from: sodium acetate, glucose, N.F,USP MANNITOL, maltose, sucrose or Zulkovsky starch; The C/N mol ratio is 1～20; Initial NaNO
2Concentration 0.5～5.0g/L.。
Preferred culture condition is: temperature: 25～35 ℃; Dissolved oxygen: shake bottle rotating speed a 90～150rpm; PH7.0～10.0; Carbon source is selected from: sodium acetate, glucose, N.F,USP MANNITOL or Zulkovsky starch; The C/N mol ratio is 12～20; Initial NaNO
2Concentration 0.5～4.0g/L.
Preferred culture condition is: temperature: 30 ℃; Dissolved oxygen: shake a bottle rotating speed 120rpm; PH 8.5; The C/N mol ratio is 12; Carbon source is a sodium acetate; Initial NaNO
2Concentration 2.0g/L.
A kind of large scale and high density is cultivated the method for pseudomonas DN-2 of the present invention, comprising: at 20～40 ℃, and mixing speed 80～120r/min, air flow 0.04～0.06vvm connects bacterium amount 3～6%, the condition bottom fermentation jar culturing bacterium of initial pH 8.5.Best method combination is included in 30 ℃, mixing speed 100r/min, air flow 0.05vvm, connects bacterium amount 5%, and the condition bottom fermentation jar of initial pH 8.5 is cultivated.
A kind of water body is carried out the method for biological denitrificaion, comprising: after pseudomonas DN-2 of the present invention is cultivated, be suspended in skimmed milk and freeze-drying, lyophilized powder is directly dropped into water body, the input amount of thalline is 1.0～100cfu/m
3More preferably, pseudomonas DN-2 of the present invention is carried out after large scale and high density cultivates, by 12,000g, 4 ℃ of centrifugal 15min results thalline are resuspended in 5～30% skimmed milk, and freeze-drying is handled.Thalline lyophilized powder (1.0 * 10
7Cfu/g) directly drop into the freshwater aquiculture water body, making its final concentration is 2.0～50cfu/m
3Best method combination comprises to be carried out Zymomonas mobilis DN-2 after large scale and high density cultivates, by 12, and 000g, 4 ℃ of centrifugal 15min results thalline are resuspended in 15% skimmed milk, and freeze-drying is handled.Thalline lyophilized powder (1.0 * 10
7Cfu/g) directly drop into the freshwater aquiculture water body, making its final concentration is 3cfu/m
3
Pseudomonas DN-2 of the present invention not only has higher denitrification activity but also have the bacterium that individual plant nitric nitrogen/nitrite nitrogen removes ability.Under the growth conditions of routine, just can show denitrification activity, and embodiment nitric nitrogen/nitrite nitrogen removes ability when Individual existence.At suitable pH value, temperature, sole carbon source, C/N ratio, wait under the combination culture condition, pseudomonas DN-2 can embody stronger denitrification activity and nitric nitrogen/nitrite nitrogen removes ability.
The pseudomonas DN-2 of the present invention's separation screening from soil can be in containing the nitrate of higher concentration/nitrite water body growth and breeding, by the intravital bioprocesses of bacterium, can apace a part of nitric nitrogen/nitrite nitrogen be changed into thalline self material, another part is converted into N
2Discharge, make that the remaining nitric nitrogen/nitrite nitrogen in the water body maintains lower level, the aquaculture water of carrying out a biological disposal upon through pseudomonas DN-2 can reach the comparatively safe level of fresh-water fishes shrimps.The invention provides the application of the pseudomonas DN-2 of separation screening from soil at the water body biological denitrificaion.In specific embodiments of the invention, pseudomonas DN-2, CGMCC No.2917 are applied to removing of nitrate pollution thing in the water body.
Pseudomonas DN-2 of the present invention is the aerobic heterotrophic microorganism of a strain, it can be to eutrophication water, the water body that particularly contains nitric nitrogen/nitrite carries out the nitrogen harmless biological to be handled, and the freshwater aquiculture water body of carrying out a biological disposal upon through pseudomonas DN-2 reaches the comparatively safe level of fresh-water fishes shrimps.The water body harmless treatment of pseudomonas DN-2 is carried out under aerobic conditions; do not need special equipment and extra treatment condition; nitric nitrogen/nitrite nitrogen is converted into the component of thalline self and the nitrogen of environmental sound in a large number; thereby bacterium can be used as the food circulation of fish and enters food chain; thereby when realizing environment protection, really accomplished harmless, the reasonable recirculation of the energy and material.
Denitrification is meant that microorganism is with NO in the present invention
3 -Be reduced to NO successively
2 -, N
2O, NO and N
2Process.
Description of drawings
The aspect graph of accompanying drawing 1 pseudomonas DN-2: A thalline transmission electron microscope photo (20000 *); B thalline Photomicrograph (1000 *); C plate bacterium colony figure
The 16S rDNA systematic evolution tree of accompanying drawing 2 pseudomonas DN-2
Accompanying drawing 3 strain growth curve and NaNO
2/ NaNO
3Virgin curve also
Accompanying drawing 4 bacterial strains are to NaNO
2And NaNO
3Go back virgin curve under existing simultaneously
Accompanying drawing 5 initial pH are to the influence of bacterial strain nitrite reducing activity
Accompanying drawing 6 initial NaNO
2Concentration is to the influence of bacterial strain nitrite reducing activity
NaNO in accompanying drawing 7 fermenting processs
2Concentration and pH change
Bacteria concentration and pO in accompanying drawing 8 fermenting processs
2Change
Accompanying drawing 9NaCl is to the influence of bacterial strain nitrite reducing activity
Accompanying drawing 10 peptones are to the influence of bacterial strain nitrite reducing activity
Accompanying drawing 11 metal ion Co
2+/ Cu
2+To the active influence of bacterial strain nitrite/nitrate reduction
Embodiment
The preparation of substratum and reagent
1.1 denitrifying bacterium enrichment medium: 4.0gNaNO
2, 5.0gNaAc, 0.03g MgSO
47H
2O, 0.01gMnSO
44H
2O, 0.75gK
2HPO
4, 0.25g NaH
2PO
4, 1,000ml dH
2O uses H
3PO
4Regulate pH to 8.0,0.7kg/cm
2High pressure steam sterilization 20min.
1.2 denitrifying bacterium isolation medium: 2.0gNaNO
2, 5.0gNaAc, 0.03g MgSO
47H
2O, 0.01g MnSO
44H
2O, 0.75gK
2HPO
4, 0.25g NaH
2PO
4, 1,000ml dH
2O, 1.5% agar powder is used H
3PO
4Regulate pH 8.0,0.7kg/cm
2High pressure steam sterilization 20min.
1.3 Griess reagent:
Solution I: take by weighing sulfanilic acid 0.5g and be dissolved in 150ml acetum (30%), be stored in brown bottle;
Solution II: take by weighing alpha-naphthylamine 0.5g and add in the 50ml distilled water, after boiling, slowly add in the 150ml acetum (30%), be stored in brown bottle.
NO
2 -Can generate the red-purple compound by sharp this reagent react of dative, this reaction is not subjected to NO
3 -The interference of-N.Get test liquid 50 μ l and drip on white plaque, drip Griess reagent I 50 μ l, mixing leaves standstill 10min, drips each 50 μ l of 2% acetum and Griess reagent II, observes color reaction, and displaing amaranth represents to contain in the test liquid NO
2 -, work as C
NO2-Show red-brown during＞1.0mg/L and generate precipitation.Make NO with this method
2 -Qualitative test.
1.4 denitrification substratum: NaNO
20.5g/L (or NaNO
30.616g/L), CH
3COONa 5.0g/L, MgSO
40.03g/L, MnSO
40.01g/L, FeSO
40.01g/L, K
2HPO
40.75g/L, NaH
2PO
40.25g/L, agar 15g/L, 8.0,121 ℃ of sterilizations of pH 20min.
1.5 naphthodiamide method NO
2-N measures and uses developer:
Take by weighing 20.0g 4-aminobenzene sulfonamide, 1.00g N-(1-naphthyl)-quadrol dihydrochloride is dissolved in 250ml H
2O and 50ml phosphoric acid are with ddH
2O is settled to 500ml, is stored in brown bottle, stablizes one month for 2～5 ℃.
1.6 pentanoic reagent: take by weighing the 0.5g pentanoic and be dissolved in the 100ml vitriol oil, pour in the 20ml distilled water, keep in Dark Place.
Get nutrient solution 50 μ l and drip on white plaque, other drips pentanoic reagent and each 50 μ l of the vitriol oil, if become blue, then explanation has nitrate to generate.
Isolation identification has the heterotrophic organism bacterial strain of denitrification activity
2.1 soil source: soil under the one place vegetation of the Purple Mountain, Nanjing; Mud in the fish pond, one place, the Purple Mountain, Nanjing; Soil in China Medicine University's campus flower bed; The XuanWu Lake, Nanjing lakeside mud.
2.2 enrichment culture
Take by weighing 10g respectively and put into the 250ml triangular flask, add denitrifying bacterium enrichment medium 100ml respectively, in 28 ℃, 120r/min cultivation, every 12h behind the cultivation 2d detects NO in the nutrient solution with lattice Lissajous method and pentanoic method respectively
2 -And NO
3 -Concentration.Select NO
2 -Concentration significantly reduces and does not produce NO
3 -Nutrient solution (be that redness shoals behind sharp this reagent react of dative, and do not produce blueness, get rid of allotrophic nitrobacteria) with the pentanoic reagent react, discard liquid, keep bed mud, adding fresh culture continues to cultivate, enrichment purpose bacterium.The all colours reaction does not all connect the blank substratum contrast of bacterium.
2.3 separation screening
The soil sample enrichment culture use agar plate with enrichment culture liquid separate application in separating after 6 days, and 28 ℃ leave standstill cultivation 2～3d and generate to bacterium colony, choose the flat board of colony growth quantity between 30～100, and each single bacterium colony of picking is in containing NaNO respectively
2Cultivate in the liquid enrichment medium of 500mg/L, and detect NO
2 -Concentration reduces situation and has or not NO
3 -Generate (be that redness shoals behind sharp this reagent react of dative, and do not produce blueness, get rid of allotrophic nitrobacteria) with the pentanoic reagent react.The strain cultured solution that selective action is strong, absorption 0.5ml moves in the fresh enrichment culture liquid and cultivates.Repeat above operation for several times, until obtaining the purpose bacterial strain.The all colours reaction does not all connect the blank substratum contrast of bacterium.
Form and cultural characteristic thereof according to each bacterial strain, judge that DN-1 has sliding phenomenon at the bacterium colony of solid culture primary surface, cell aggregation is agglomerating, may be embedded in the slime layer, but do not see sporocyst and suspensor, may belong to certain genus that produces the spore Myxobacteriaceae under the fruiting myxobacteria.The DN-2 thalline is a rod-short, and Gram-negative obtains energy by nitrite, and the fricton-tight phenomenon of the bacterium colony that generates on flat board is judged as denitrifying bacterium.DN-3 ... each strain cell of DN-9 is non-thread, has flagellum, and Gram-negative is non-Campylobacter, and wherein DN-3 is a genus bacillus.
2.4 the physiology of active bacterial strain is identified
With reference to uncle Jie Shi Bacteria Identification handbook the 9th edition.Characteristic of bacteria sees Table 3.
The physiological and biochemical property of table 3 bacterial strain DN-1 to DN-9
Judge that according to above test-results DN-2 may belong to Rhodopseudomonas, DN-3 is subordinated to Bacillaceae, fusobacterium, flaggellation, the urease test positive, generate hydrogen sulfide in broth culture, no milk solidification phenomenon may be class clostridium septicum or clostridium tetani.Each bacterial strain of DN-4～DN-9 all is a facultative anaerobic bacteria, the catalase positive, in broth culture, generate hydrogen sulfide, no milk solidification phenomenon, sugar fermentating test and IMVIC test-results are not quite identical, and wherein DN-4, DN-5, DN-7, DN-8 may belong to enterobacteriaceae Escherichieae citric acid Pseudomonas or salmonella; DN-6, DN-9 may belong to enterobacteriaceae Klebsiella family or Proteae.
Embodiment 3
The evaluation of pseudomonas DN-2
1. morphological features (seeing accompanying drawing 1-A/B): thalline is shaft-like, about 0.3～0.5 * 1.5～2.0 μ m of thalline size, and Gram-negative, no gemma can move.
2. colony morphology characteristic (seeing accompanying drawing 1-C): on the agar plate of denitrification substratum, 30 ℃ of aerobic cultivations of constant temperature form irregular polytropy sample bacterium colony after 3 days, be faint yellow, surface folding, quality is stiff, is difficult for provoking, and matrix does not see that tangible soluble pigment produces.
3. the major physiological biochemical characteristic sees Table 4, control strain is the type strain Pseudomonas aeruginosa (Pseudomonasaeruginosa ATCC27853) of pseudomonas, and Pseudomonas alcaligenes (Pseudomonas alcaligenes ATCC14909) and pseudomonas mendocina (Pseudomonas mendocina ATCC25411).
The comparison of relevant bacterial strain physio-biochemical characteristics in table 4 DN-2 and the Rhodopseudomonas
+: the positive;-: feminine gender; ND: do not detect; PHB: poly-beta-hydroxy-butanoic acid ester
4. phylogenetic analysis: the 16S rDNA gene to pseudomonas DN-2 increases and checks order, the row that check order carry out similarity analysis with BLAST software and GenBank database, and in Clustal X (1.8) software, carry out the multiple sequence compare of analysis with similar sequences, use Neighbor-Joining method constructing system evolutionary tree (seeing accompanying drawing 2) in MEGA (3.1) software at last.As seen, DN-2 belongs to Rhodopseudomonas from evolutionary tree, and Pseudomonas alcaligenes (Pseudomonasalcaligenes) and branch of pseudomonas mendocina (Pseudomonas mendocina) formation in belonging to this.
5. denitrification activity: with 0.5g/L NaNO
2During for only nitrogen source, be carbon source, cultivate about 12h that the decreasing ratio of nitrite nitrogen can reach 100% with the 5.0g/L sodium acetate; With 0.616g/L NaNO
3During for only nitrogen source, be carbon source, cultivate about 20h that the extrusion rate of nitric nitrogen can reach 100% (seeing accompanying drawing 3) with the 5.0g/L sodium acetate; Cultivate respectively with 5.0g/L Zulkovsky starch, glucose, when N.F,USP MANNITOL is carbon source, after cultivating 24h, the decreasing ratio of nitrite nitrogen can reach 100%.
Classification with reference to uncle Jie Shi Bacteria Identification handbook the 9th edition is identified, biochemical reactions and phylogenetic analysis in conjunction with bacterial strain, bacterial strain DN-2 belongs to Rhodopseudomonas, and it is nearest with the evolutionary distance of Pseudomonas alcaligenes (Pseudomonas alcaligenes) and pseudomonas mendocina (Pseudomonas mendocina), but features such as the starch hydrolysis of bacterial strain DN-2 and colonial morphology are consistent with pseudomonas stanieri (Pseudomonas stutzeri) again, are different from above with ramose two strain bacterium.So pseudomonas DN-2 may be novel species in the Rhodopseudomonas or new subspecies, temporarily with its called after pseudomonas DN-2.Pseudomonas DN-2 is preserved in China Committee for Culture Collection of Microorganisms common micro-organisms center (CGMCC) on February 27th, 2009, and preserving number is CGMCC No.2917.The cultural characteristic of pseudomonas DN-2 bacterial strain and individual morphology feature are seen A, B and the C among Fig. 1.
The cultivation of pseudomonas DN-2 and nitric nitrogen/nitrite nitrogen reducing activity
4.1 aerobic denitrifying bacteria is a heterotrophic bacterium, must add organic carbon source ability normal growth and carry out denitrification.In the denitrification substratum, respectively with 5.0g/L 1. sodium acetate, 2. glucose, 3. N.F,USP MANNITOL, 4. maltose, 5. sucrose and 6. Zulkovsky starch be sole carbon source, 0.5g/LNaNO
2During for only nitrogen source, 30 ℃, after the 120rpm shaking table was cultivated 12h, the naphthodiamide standard measure was measured remaining NO
2 -The concentration of-N is examined or check its influence to reducing activity (seeing Table 5).
NO when the different carbon sources of table 5 are cultivated
2 --N reduction ratio (12h)
Be incorporated with the 250ml Erlenmeyer flask of 100ml denitrification substratum 4.2 connect two ring lawns from the denitrification agar slant, at 28～30 ℃, 120～130rpm shaking table is cultivated 12h.By the inoculation of 5% inoculum size, the 5ml seed culture fluid is transferred in the 250ml Erlenmeyer flask that 100ml denitrification substratum is housed.Culture temperature is respectively 20,25,30,35,40 ℃; Dissolved oxygen, promptly shaking speed be respectively 60,90,120,150,180rpm; The initial pH of substratum is respectively 6,7,7.5,8,8.5,9,10; Change C/N (mol ratio) by the change sodium acetate concentration and be respectively 1,4,8,12,16,20; Initial NaNO
2Concentration is respectively 0.5,1,2,3,4,5g/L (with the sodium acetate is sole carbon source, and keeping C/N simultaneously is 12).If do not inoculate control group, every group of experiment is three repetitions.For initial pH group of difference and different initial NaNO
2The all every 2h sampling of concentration group is taken a sample behind all the other equal 12h, and 12, the centrifugal 1min of 000g, the naphthodiamide standard measure is measured remaining NO
2 -The concentration of-N, and calculate NO
2 -The reduction ratio of-N.The results are shown in Table 5.1, table 5.2, accompanying drawing 5, table 5.3, accompanying drawing 6.
NO when table 5.1 differing temps is cultivated
2 --N reduction ratio (12h)
Reduction ratio is the ratio of the concentration of nitrite in the concentration of the nitrite that is removed and the initial medium.
NO when table 5.2 different rotating speeds is cultivated
2 --N reduction ratio (12h)
NO when the different C/N of table 5.3 cultivate
2 --N reduction ratio (12h)
Aerobic denitrifying bacteria is a heterotrophic bacterium, must add organic carbon source ability normal growth and carry out denitrification.Experiment shows, when bacterial strain is carbon source with the sodium acetate, cultivates NO behind the 12h
2 --N all is reduced, and promptly the suitableeest carbon source is a sodium acetate.And factors such as culture temperature, initial pH all have very big influence to the effect that thalline removes nitrite nitrogen.When culture temperature was 30 ℃, strain growth was higher with the enzyme classes activity of carrying out denitrification, and bacterial strain suits to grow under the condition of meta-alkalescence and carry out denitrification.C/N was not less than 12 o'clock, NO behind the cultivation 12h
2 --N all is reduced, and thalli growth and denitrification no longer are subjected to carbon source restriction.It is consistent that the C source matrix of the surplus that this shows with present institute helps highly active aerobic denitrification.
In addition, bacterial strain is at initial NaNO
2When concentration is not higher than 4g/L, little to the reducing activity influence, initial NaNO
2When concentration is higher than 5g/L, can suppresses the growth of bacterial strain and influence its reducing activity.Initial NaNO
2When concentration is 2g/L, NO
2 -The average rate of reduction of-N is the highest, can reach 20.3mgL
-1H
-1
Pseudomonas DN-2 bacterial strain reaches best growth conditions and the condition of the highest nitrite nitrogen rate of reduction is: 30 ℃ of temperature, initial pH8.5 is a sole carbon source with the sodium acetate, and to keep C/N be 12 than (mol/mol), initially NaNO
2Concentration 2.0g/L; Dissolved oxygen: shake culturing bacterium under bottle rotating speed 120rpm condition.Because initial NaNO
2The required time is 20h during concentration 2.0g/L, and initial NaNO
2Then only need about 10h during concentration 0.5g/L, and can satisfy the growth of thalline fully, so initial NaNO in the subsequent experimental
2Concentration is decided to be 0.5g/L.With the initial NaNO of 0.5g/L
2Culture condition under, pseudomonas DN-2 bacterial strain when 24h, reach the growth stationary phase, weight in wet base is roughly equal to 25g/L (seeing accompanying drawing 3).
Same, with 0.616g/LNaNO
3Be only nitrogen source, 30 ℃ of temperature, initial pH8.5 is a sole carbon source with the sodium acetate, and to keep C/N be 12 than (mol/mol), dissolved oxygen: shake when cultivating this bacterium under bottle rotating speed 120rpm condition, denitrification percent reaches 100% (seeing accompanying drawing 3) in the time of about 20 hours.
When respectively with the NaNO of 0.25g/L and 0.308g/L (after the conversion, each 3.6mM of nitrite nitrogen and nitric nitrogen)
2, NaNO
3During as mixed nitrogen, pseudomonas DN-2 all reaches 100% (seeing accompanying drawing 4) when the 11h to the extrusion rate of these two kinds of nitrogens.Quick degraded appears in nitric nitrogen when 5h, degraded fast then appears in nitrite nitrogen when 7h.Illustrate that pseudomonas DN-2 preferentially utilizes nitrate when two kinds of nitrogens exist simultaneously.
The fermentor tank large scale culturing of pseudomonas DN-2
Connect two ring lawns from the denitrification agar slant and be incorporated with the 250ml Erlenmeyer flask of 100ml denitrification substratum, at 30 ℃, the 120rpm shaking table is cultivated 12h.By the inoculation of 5% inoculum size, the 500ml seed culture fluid is transferred in the 10L fermentor tank that the 9.5L fermention medium is housed.According to the result of study of shake flat experiment, determine the culture condition of best fermentor tank: 30 ℃, mixing speed 100r/min, air flow 0.05vvm, connect bacterium amount 5%, fermention medium is formed (g/L): NaNO
20.5, CH
3COONa 5, MgSO
47H
2O 0.03, MnSO
44H
2O 0.01, FeSO
47H
2O 0.01, K
2HPO
40.75, NaH
2PO
40.25, initial pH regulator to 8.5.NO
2 --N all is reduced in 10h, and pH rises in 0h～10h (seeing accompanying drawing 7), produces alkali with denitrification and coincide.Because bacterial strain is with NaNO
2Be only nitrogen source, behind the 10h with 5mgL
-1H
-1Speed add NaNO
2Add NaNO
2After still detect less than NO
2 --N, NaNO
2Transformed or utilize the synthetic cell material after the adding immediately by denitrification.After cultivating 24h, it is dense to measure bacterium with the viable bacteria technical process, and its concentration reaches maximum value 1.2 * 10
11Cfu/ml, and pO
2Bottom out behind 24h (seeing accompanying drawing 8), strain growth enters plateau.Cultivate the thalline that 24h can gather in the crops higher concentration, help the extensive use in the practical application.
The denitrogenation of pseudomonas DN-2 in water body
6.1 under the NaCl of different concns, the denitrification effect of pseudomonas DN-2 in water body
Connect two ring lawns from the denitrification agar slant and be incorporated with the 250ml Erlenmeyer flask of 100ml denitrification substratum, at 30 ℃, the 120rpm shaking table is cultivated 12h.By the inoculation of 5% inoculum size, the 5ml seed culture fluid is transferred in the 250ml Erlenmeyer flask that 100ml denitrification substratum is housed.Denitrification NaCl in medium concentration is respectively 0,0.5,1,1.5,2.5,3.5%, and the denitrification effect of pseudomonas DN-2 in water body seen accompanying drawing 9.
The result shows, when NaCl concentration is not higher than 15g/L, to the not obviously influence of reducing activity of bacterial strain; During the NaCl excessive concentration, then can influence thalli growth and suppress its denitrification.Have certain salt concn in the water body of aquaculture, the fresh water salt concn generally is no more than 0.5g/L, and seawater average salt concentration is 35g/L.Bacterial strain is obtained by screening in the soil, and the high salt concentration of incompatibility seawater.But with regard to the water body of common freshwater aquiculture, do not influence its reducing activity, can remove the nitrite in the water body fast.
6.2 under the peptone of different concns, the denitrification effect of pseudomonas DN-2 in water body
Connect two ring lawns from the denitrification agar slant and be incorporated with the 250ml Erlenmeyer flask of 100ml denitrification substratum, at 30 ℃, the 120rpm shaking table is cultivated 12h.By the inoculation of 5% inoculum size, the 5ml seed culture fluid is transferred in the 250ml Erlenmeyer flask that 100ml denitrification substratum is housed.Peptone concentration is respectively 0,0.05,0.1,0.2,0.5,1.0% in the denitrification substratum, and the denitrification effect of pseudomonas DN-2 in water body seen accompanying drawing 10.
The result shows that peptone concentration reducing activity to bacterial strain in the scope of 0g/L～10g/L does not obviously influence.When having certain density itrogenous organic substance in the water body, do not influence the reduction of bacterial strain to nitrite.
6.3 under the certain density heavy metal ion, the denitrification effect of pseudomonas DN-2 in water body
Connect two ring lawns from the denitrification agar slant and be incorporated with the 250ml Erlenmeyer flask of 100ml denitrification substratum, at 30 ℃, the 120rpm shaking table is cultivated 12h.By the inoculation of 5% inoculum size, the 5ml seed culture fluid is transferred in the 250ml Erlenmeyer flask that 100ml denitrification substratum is housed.Be under the condition of only nitrogen source with nitrite nitrogen and nitric nitrogen respectively, when cultivating 8h, add Co respectively through the 2.5mM of membrane filtration degerming
2+And Cu
2+Solution, the denitrification effect of pseudomonas DN-2 in water body seen accompanying drawing 11 (A/B).
The result shows, the Co of 2.5mM
2+And Cu
2+Can obviously suppress the reduction of bacterial strain to nitrogen.Therefore, when having the heavy metal ion of high density in the water body, should handle water body earlier and remove too high heavy metal ion, re-use pseudomonas DN-2 denitrogenation.
The denitrogenation of pseudomonas DN-2 in actual freshwater aquiculture water body
According to embodiment 5, (B.Braun Biotech International 10L) cultivates 24 hours pseudomonas DN-2 thalline by 12, and 000g 4 ℃ of down centrifugal 15min collections, is suspended in 15% skimmed milk and freeze-drying with fermentor tank.The lyophilized powder (1.0 * 10 that obtains
7Cfu/g) directly drop into the freshwater aquiculture pond, the input amount of thalline is 10.0cfu/m
3Measure the concentration (seeing Table 6) of pH, nitric nitrogen and the nitrite nitrogen of pond waters every day.
The result shows, under the pond water environment of 25 ℃ of water temperatures, pH8.6, and NO in the water
2 -3d after-N throws in from thalline begins to descend, dropped to 0.49mg/L by 1.47mg/L during to 18d, the effect of pseudomonas DN-2 degraded nitrite nitrogen is very obvious, can be under the situation of not taking additive method, quickly with water nitrite attitude nitrogen degradation to the comparatively safe level of fishes and shrimps.In whole observation process, pH fluctuates between the scope of 8.4-8.9, and this scope is fit to the growth of pseudomonas DN-2 and other denitrifying bacteriums.
Table 6 pseudomonas DN-2 removes nitrite nitrogen in the freshwater aquiculture water body
Claims (5)
1. one kind has the bioactive bacterium of denitrogenation, and preserving number is: CGMCC No.2917.
2. the method for bacterium described in the large scale culturing claim 1 is included in 20～40 ℃, mixing speed 80～120r/min, and air flow 0.04～0.06vvm connects bacterium amount 3～6%, the condition bottom fermentation jar culturing bacterium of initial pH 8.5.
3. the method for claim 2 is included in 30 ℃, mixing speed 100r/min, air flow 0.05vvm, connects bacterium amount 5%, the condition bottom fermentation jar culturing bacterium of initial pH 8.5.
4. the application in the nitrite nitrogen of bacterium in removing the fresh water water body described in the claim 1.
5. method that removes the nitrite nitrogen in the fresh water water body: after comprising the microbial culture with claim 1, be suspended in skimmed milk and freeze-drying, lyophilized powder is directly dropped into water body, the input amount of thalline is 1.0～100cfu/m
3
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