CN1590532A - Denitrogen bacteria composition and its application - Google Patents

Denitrogen bacteria composition and its application Download PDF

Info

Publication number
CN1590532A
CN1590532A CN 03118599 CN03118599A CN1590532A CN 1590532 A CN1590532 A CN 1590532A CN 03118599 CN03118599 CN 03118599 CN 03118599 A CN03118599 A CN 03118599A CN 1590532 A CN1590532 A CN 1590532A
Authority
CN
China
Prior art keywords
bacteria
nitrogen
bacterium
denitrification
bacteria composition
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Granted
Application number
CN 03118599
Other languages
Chinese (zh)
Other versions
CN100500831C (en
Inventor
王一明
彭光浩
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Institute of Soil Science of CAS
Original Assignee
Institute of Soil Science of CAS
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Institute of Soil Science of CAS filed Critical Institute of Soil Science of CAS
Priority to CNB031185991A priority Critical patent/CN100500831C/en
Publication of CN1590532A publication Critical patent/CN1590532A/en
Application granted granted Critical
Publication of CN100500831C publication Critical patent/CN100500831C/en
Expired - Fee Related legal-status Critical Current
Anticipated expiration legal-status Critical

Links

Abstract

A denitrifying bacterial composition is composed of heterotrophic nitrobacteria and heterotrophic aerobic denitrobacteria, and can be used to convert the nitrogen elements in water to non-pollution N2.

Description

Denitrifying bacteria composition and application thereof
Field that the present invention belongs to:
The invention belongs to microorganism field, specifically, the present invention relates to have the bacteria composition and the application of this bacteria composition in the water body biological denitrificaion of denitrogenation biologic activity
Background technology:
Nitrogen is the necessary nutritive elements of all life, also is the important component part of Global Ecological system material cycle.Use nitrogenous fertilizer to become the particularly main means of food crop high yield and volume increase of farm crop.But the use of nitrogenous fertilizer has also brought serious environmental problem when increasing substantially crop yield: as the continuous growth of tap water Nitrate Accumulation, and the eutrophication in the semiclosed waters of sealing such as lake, NH 4 +-NH 3Enter aquatic resources such as water body harm fish shellfish; And greenhouse gases N 2O constantly discharges and has aggravated the atmosphere Greenhouse effect and to destruction of ozonosphere etc.Improve nitrogen utilization efficiency, reduce loss of nitrogen fertilizer and the detrimentally affect of environment has been become the huge challenge that present agriculture production and environment protection face.Simultaneously, nitrogen form transforms again the improvement with sewage sludge nitric efficiency, denitrification process, and the solution of body eutrophication is closely related.The nitrogen eutrophication that solves water body at present has physical method, chemical process and biological method.Along with the continuous aggravation of nitrate pollution and people to the pay attention to day by day of environmental problem, denitrogenate technology, particularly bio-denitrification technology has become the important directions and the means of control water pollution.
Ammonia nitrogen is one of main nitrate pollution thing that causes body eutrophication.Generally believe that at present in biological denitrification process, the ammonia nitrogen in the waste water at first is oxidized to NO by the autotrophy nitrifier under aerobic condition x -, NO then x -Under anoxia condition, be reduced to gaseous nitrogen such as N by denitrifying bacterium 2Deng from water, overflowing.Nitrification and denitrification both can carry out in activated sludge reactor, can in biofilm reactor, carry out again, and the maximum still activated sludge process of practical application, nitrifier and denitrifying bacteria are in the same active sludge.Owing to find obviously different with the anoxic and the heterotrophism characteristic of denitrifying bacteria at present with the aerobic and autotrophy characteristic of the nitrifier of using, denitrification process needs independently to carry out in two reactors usually, as Bardenpho technology, UCT (University ofCapetwon) technology, two channel type oxidation channel technologies etc., or in a reactor, carry out in turn as SBR (Sequencing Batch Reactor, sequencing batch reactor).Situation is then opposite when mixing sludge enters Aerobic Pond or has been in oxygen condition, nitrifier work, and denitrifying bacteria is in holddown; When mixing sludge enters anoxic pond or is in anoxic condition, denitrifying bacteria work, nitrifier is in holddown.
But no matter traditional microorganic adhesion type sewage treatment structure, as biological filter, blodisc and submerged biological filter, or some high-performance bio film processing systems newly developed, as two-phase fluidization bed, three-phase fluidized bed, anaerobic fluidized bed, electrode-biomembrance process, the nitrifying bacteria community that these methods adopted mostly is autotrophic bacteria, rate of propagation is slow and be difficult to keep higher biological concentration, need to handle to reduce organic concentration through aeration earlier, biologic activity be grown and be showed to bacterial strain could, a little less than the impact resistance; Ammonia nitrogen in high density and nitrite can suppress the growth of nitrifier again, make nitrification incomplete, cause nitrogen removal rate very low.
At present, there is the investigator that the biological denitrification process under the aerobic condition is studied abroad, utilizes the certain micro-organisms population under aerobic condition, to have denitrifying characteristic and realize synchronous nitration and denitrification.Result of study shows that general foster sulphur coccus (Thiosphera pantotroph), Alcaligenes faecalis (Alcaligenea faecalis), pseudomonas (Pseadonmonas sp), comamonas microorganisms such as (Comamonos sp.) can utilize NO under aerobic condition x-N carries out denitrification.Nitrifier and denitrifying bacteria are placed mixed culture in same reactor such as the aeration tank, can reach the synchronous nitration and denitrification of single reactor.But the denitrification result is unsatisfactory, and a large amount of nitrogen are converted to oxidation state nitrogen, and this oxidation state nitrogen is again the arch-criminal that atmosphere greenhouse gases effect and ozone cavity form, and causes secondary pollution easily.
Domesticly also carried out some relevant research work, (Geng Jinju, Liu Dengru etc. use and the environmental organism journal 2002,8 (1): 78-82) to utilize aerobic denitrifying bacteria group and autotrophy nitrifying bacteria community combined denitrification.Though bacteria composition has ammonia nitrogen removal ability preferably, impact resistance a little less than, the ammonia nitrogen that ammonia nitrogen concentration is higher than the high density of 0.3 grams per liter can suppress the growth of thalline, and ammonia nitrogen concentration is when being higher than 0.2 grams per liter, the ammonia nitrogen residual volume is more after the denitrogenation; Not anti-high organic carbon concentration of while, and the organic carbon concentration of 0.5 grams per liter can suppress thalli growth and reduce denitrification effect.And all kinds of microbial culture and growth conditions in this combination flora are inconsistent, the opposing party but is in holddown during one side performance function, cause inharmonious each other, the biological denitrification process time lengthening, cost increases, the more important thing is that nitric efficiency is general, the water body after the denitrogenation also has distance from environmental requirement.
The invention technology contents:
An object of the present invention is to provide a kind of bacteria composition, this bacteria composition can remove the nitrogen in the water body effectively by acting synergistically, to be harmful to nitrogen and change into, and the denitrogenation time shortens dramatically to the free of contamination biomass of water body with to the free of contamination nitrogen of atmospheric environment.
Another object of the present invention provides the application of bacteria composition in the water body biological denitrificaion.
A kind of bacteria composition with denitrogenation biologic activity is characterized in that it contains:
(A) allotrophic nitrobacteria,
(B) heterotrophism aerobic denitrifying bacteria,
Wherein component (A) can mix the purpose that just can realize the water body combined denitrification by arbitrary proportion with component (B).The component (B) of trace is arranged in component (A) or micro-component (A) is arranged in component (B), this bacteria composition can remove the nitrogen in the water body effectively by acting synergistically, will be harmful to nitrogen and change into to the free of contamination biomass of water body with to the free of contamination nitrogen of atmospheric environment.
In a preferred version of the present invention, component provided by the invention (A) allotrophic nitrobacteria is Arthrobacter globiformis WR-2, (Arthrobacter globiformis WR-2), and its preservation registration number is CCTCCM202043.In another preferred version of the present invention, component disclosed by the invention (B) heterotrophic denitrification bacterium is plant bacillus pumilis WO-8, (Curtoacterium plantarum WO-8), and its preservation registration number is CCTCC M202044.
A kind of bacteria composition with denitrogenation biologic activity is characterized in that it contains following component (volume ratio):
(A) allotrophic nitrobacteria 1-99%,
(B) heterotrophism aerobic denitrifying bacteria 99-1%,
Wherein allotrophic nitrobacteria is a bacterioid of Arthrobacter globiformis, and the heterotrophic denitrification bacterium is a bacterioid of plant bacillus pumilis.In the present invention, investigator of the present invention serves as for the examination soil sample with this caustic lime soil of the North China moisture soil of Fengqiu, Henan Province, and through PM plate isolation, purifying, active check, Griess reagent is identified, obtained the heterotrophism bacterial strain that the Ge Lisi reaction is positive, confirmed that tentatively they have ammoxidation capability.Further screening is selected the strong bacterial strain of stable performance and nitrification activity and individual plant ammonia nitrogen removal ability as the purpose bacterial strain in these bacterial strains, and we find that Arthrobacter globiformis WR-2 has this performance.Through being accredited as Arthrobacter globiformis WR-2, Arthrobacter globiformis WR-2.This bacterial strain has following feature:
1. colony morphology characteristic: go up at nutrient agar plate (PM flat board) and to form very little bacterium colony after 28 ℃ of good air cultures of constant temperature are supported 7 days, circle, flat, full edge; Rough have wrinkle, lawn surface to be khaki color, and matrix does not see that tangible soluble pigment produces.
2. morphological features: Gram-positive; Cultivate in early days, it is irregular shaft-like that cell is, and prolongs incubation time and sphere then occurs, and the aged cell almost is spherical entirely; How to arrange with the chain form; Do not move, do not form statospore.
3. the major physiological biochemical characteristic sees Table 1: the suitableeest culture temperature is 28-32 ℃, and pH7.0-7.2 is aerobic.
4. ammoxidation activity: the nitrite concentration of cultivating accumulation in 14 days at the 90-100r/min shaking table all is higher than 60.0mg NO 2-NL -1, indivedual individual plants can be up to 95mg NO 2-NL -1More than.
5. denitrification activity: with concentration is 0.075mol L -1When sodium acetate was carbon source, C/N was 5: 1, cultivated 28
The physio-biochemical characteristics of table 1 WR-2 bacterial strain
Test item is the test item result as a result
Growth need vitamin H+liquefy gelatin+
VitB1-hydrolyzed starch+
NO 3→ NO 2+ D-wood sugar-/+
Uric acid+melizitose+
M-hydroxy-benzoic acid+D-glucuronate-/+
α-ketone group-pentanedioic acid+nucite-
Raffinose-D-seminose-
D-glycine+sucrose-/+
Lactose-
Annotate :+expression positive reaction maybe can utilize :-expression negative reaction maybe can not be utilized.My god, ammonia-nitrogen removal rate is 65%, full nitrogen removal efficiency is 62%; When pyruvic acid was carbon source, concentration was 0.050molL -1The time, C/N is 5: 1, cultivates 28 days, and ammonia-nitrogen removal rate is 90%, and full nitrogen removal efficiency is 68%.Remaining full nitrogen be almost somatic cells.
Classification with reference to uncle Jie Shi Bacteria Identification handbook the 9th edition identifies that this bacterial strain is Arthrobacter globiformis (Arthrobacter globiformis).Therefore with its called after Arthrobacter globiformis WR-2.Arthrobacter globiformis WR-2 on November 5th, 2002 at specified depositary institution of Patent Office of the People's Republic of China-Chinese typical culture collection center (CCTCC) preservation, preservation registration number CCTCC M202043.
In the present invention, the strain plant Curtobacterium bacterium that the inventor also is separated to from the North China moisture soil of Fengqiu, Henan Province, further repeated screening detects relatively, this strain bacterium has the heterotrophism aerobic denitrification capability as a result, through being accredited as plant bacillus pumilis WO-8, Curtoacterium plantarum WO-8.This bacterial strain has following feature:
1. colony morphology characteristic: after 28 ℃ of good air cultures of constant temperature on the PM nutrient agar plate were supported 2 days, the bacterium colony of formation was circular, flat, full edge, smooth surface; The lawn surface is orange, and matrix does not see that tangible soluble pigment produces.
2. morphological features: the positive reaction of gramstaining; How rod-short, thick shape, the blunt circle in thalline two ends occur with single spread pattern; Do not see statospore formation; Motion.
3. the major physiological biochemical characteristic sees Table 2, and the suitableeest culture temperature is 28-32 ℃, and pH7.0-7.2 is aerobic.
4. the denitrification activity of bacterial strain: have strong denitrification activity, have nitrate, nitrite reducing power, experiment records in 7 days 200mg N L -1NO 2 -Remove 99%; In 10 days with 280mg N L -1
The physio-biochemical characteristics of table 2 WO-8 bacterial strain
Test item is the test item result as a result
Cyclohexaamylose-soil temperature 40+
Dextrin+soil temperature 80+
Glycogen+N-acetyl-D-glycosamine-
Amygdaloside-N-acetyl-D-mannosamine-
L-arabinose-arbutin-
D-pectinose+cellobiose-
Fructose+fucose-
D-semi-lactosi-gentiobiose-
Alpha-D-glucose+α-D-lactose-
M-inositol-lactose-
Maltose-/+D-N.F,USP MANNITOL+
The D-seminose-/+the D-melibiose-
Alpha-Methyl-D-galactoside-Alpha-Methyl-D-glucoside-
Beta-methyl-D-galactoside-Beta-methyl-D-glucoside-
Beta-methyl glucose-D-psicose-
D-raffinose-L-rhamnosyl-
D-ribose+sucrose-
D-tagatose-trehalose-
Turanose-Xylitol-
The D-wood sugar-/+acetic acid+
Alpha-hydroxy-butyric acid+beta-hydroxy-butyric acid+
α-ketone group-pentanedioic acid+methyl-prop ketone acid+
List-methylsuccinic acid+propionic acid+
Pyruvic acid+citric acid-
Succsinic acid+N-acetate-L-L-glutamic acid+
Propionic acid amide+D-L-Ala+
L-L-Ala+L-alanyl-glycine+
Altheine+L-L-glutamic acid+
Glycine-L-L-glutamic acid+L-Pyrrolidonecarboxylic acid-
L-Serine-2, the 3-butyleneglycol-
Glycerine+inosine-
Thymidine-uridine-/+
Cori's eater Cori-2-glucose 1-phosphate1--
D-L-α-Phosphoric acid glycerol esters-
Annotate: in the table+ecbatic is positive, the reaction that is negative of-expression ecbatic.NO 3 -Remove 97%, and lose with the form ease of gaseous nitrogen.When being nitrogenous source, there is not the generation of nitrite in the culturing process, no heterotrophism nitrification activity with the ammonium salt.
Classification with reference to uncle Jie Shi Bacteria Identification handbook the 9th edition identifies that this bacterial strain is plant bacillus pumilis (Curtobacterium plantarum).Therefore with its called after plant bacillus pumilis WO-8.Plant bacillus pumilis WO-8 on November 5th, 2002 at specified depositary institution of Patent Office of the People's Republic of China-Chinese typical culture collection center (CCTCC) preservation, preservation registration number CCTCC M202044.
In the present invention, cultivate Arthrobacter globiformis WR-2 and plant bacillus pumilis WO-8 respectively, and survey bacterium liquid OD respectively by the liquid shaking table 600Value, phase modulation is with thalline OD 600Value, by volume per-cent is measured the bacterium mixing, obtains bacteria composition provided by the present invention.In the present invention, the component (B) of trace is arranged in component (A) or micro-component (A) is arranged in component (B), the bacteria composition of being made up of this two strains bacterium can remove nitrogen in the water body effectively by synergy, to be harmful to nitrogen changes into to the free of contamination biomass of water body with to the free of contamination nitrogen of atmospheric environment, this biological denitrification process carries out under aerobic conditions and heterotrophism state, bacterium with organic carbon as the energy, ammonia-state nitrogen is changed into the gas of dinitrogen, and this biotransformation does not need the physics and the aeration of chemistry to handle, compare with traditional method, the time of biological respinse shortens dramatically.
Except that containing these two kinds of components of Arthrobacter globiformis WR-2 and plant bacillus pumilis WO-8, bacteria composition of the present invention also can contain (volume ratio) in the composition of the present invention:
(C) receivable carrier 0-10% in the water body denitrification technology;
Wherein receivable carrier is meant polyvinyl alcohol in the water body denitrification technology, the nitrification and denitrification bacterium.In the present invention, polyvinyl alcohol is meant that weight ratio is 20% polyvinyl alcohol, gets the conventional dissolving of 20 gram polyvinyl alcohol when 20% polyvinyl alcohol solution is prepared and gets, and measures an amount of volume then as required, the formation composition.In the present invention, the nitrification and denitrification bacterium is meant bacillus cereus NBB-135, (CGMCC No.0560); Bacillus pumilus NBB-112, (CGMCC No.0561); Bacillus circulans NBB-295, (CGMCCNo.0557); Bacillus licheniformis NBB-072, (CGMCC No.0562); Alcaligenes faecalis, (CGMCCNo.1.1799); Pseudomonas fluorescens, (CGMCC No1.1802) etc.These bacterial strains carry out the patented procedure preservation or can ask for to this culture presevation mechanism at China Committee for Culture Collection of Microorganisms common micro-organisms center.Nitrobacteria commonly used and denitrifying bacterium are being surveyed bacterium liquid OD 600After the value, phase modulation is with thalline OD 600Value, by volume per-cent mixes after measuring bacterium.
In a preferred version of the present invention, this bacteria composition contains following component (volume ratio):
(A) allotrophic nitrobacteria 30-70%,
(B) heterotrophic denitrification bacterium 70-30%.
Of the present invention one more preferably in the scheme this bacteria composition contain following component (volume ratio):
(A) allotrophic nitrobacteria 40%,
(B) the heterotrophic denitrification bacterium 60%.
Allotrophic nitrobacteria provided by the invention mainly is meant under aerobic state the NH in the water body 4+ change into NO by nitrification 2 -And NO 3 -, or oxidation state N and with the NH in the water body 4+ be converted into nitrogen or oxidation state nitrogen.Heterotrophic denitrification bacterium provided by the invention mainly be under aerobic state with in the water body with oxidation state nitrogen such as NO 2 -And NO 3 -Be converted into nitrogen and discharge, the two combined action water body of allotrophic nitrobacteria and heterotrophic denitrification bacterium can transform the nitrate pollution in the water body effectively the nitrogen of atmosphere also non-secondary pollution is overflowed and the shortening sewage disposal time.Usually adopt bacteria combination of the present invention to shorten 7 days than the individual plant water body denitrification time, preferred bacterium combination of the present invention is shortened 17 days than WR-2 individual plant in the water body denitrification time.Nitrification and denitrification technological process denitrogenation speed than traditional autotrophy improves 19-30%.
Ammonium nitrogen in the present invention or ammonia-state nitrogen are meant the ammonium radical ion NH of solubility 4 +-NH 3Oxynitride is meant NO and N 2O.
Nitrification is meant that microorganism is with NH in the present invention 4 +Be oxidized to NO 2 -, NO then 2 -Reoxidize and be NO 3 -Process, perhaps because the microbial process process that causes oxidation state nitrogen to increase.It is the important step of nature Nitrogen Cycling, also is one of critical path of farmland nitrogen loss.The biological nitration effect can be divided into the three major types type: chemosynthetic autotroph nitrification, heterotroph nitrification and methane nutritional type nitrification.
Heterotrophic nitrification is meant that broadly organic and inorganic nitrogen compound is the process of multiple oxidation state from going back ortho states through bio-oxidation in the present invention, the sense stricto process that is meant that heterotrophic microorganism is oxidized to azanol, nitrite and nitrate with the ammonium nitrogen or the organic nitrogen of oxidation state-3 under aerobic condition.
Denitrification is meant that microorganism is with NO in the present invention 3 -Be reduced to NO 2 -, NO then 2 -Restore and be N 2O, NO or N 2Process.
Carbon/nitrogen ratio claims C/N than the ratio of the volumetric molar concentration that is meant carbon with the volumetric molar concentration of nitrogen element again in the present invention, is equivalent to ammonium sulfate 0.015mol L as C/N=1 -1: sodium acetate 0.015mol L -1
Denitrogenation in the present invention is meant the removal of soluble nitrogen in the water body, and soluble nitrogen is meant NH 4 +, NO 2 -And NO 3 -
Receivable carrier comprises sorbent material in the water body denitrification technology in the present invention, nitrification and denitrification bacterium etc.There is the same employing national standard of national standard in various units of Shi Yonging in the present invention, the employing industry standard of no national standard, and nitrite concentration is 60.0mg NO 2-N L -1, represent to contain in every liter of solution 60 milligrams of nitrite nitrogens, nitrate content is 0.18mg N L -1Represent to contain in every liter of solution 0.18 milligram of nitric nitrogen,
Description of drawings
The thalline Photomicrograph of accompanying drawing 1 Arthrobacter globiformis WR-2
The thalline Photomicrograph of accompanying drawing 2 plant bacillus pumilis WO-8
The denitrification effect figure of the different time of accompanying drawing 3 bacteria compositions.NH wherein 4, TN, NO 2Ammonia nitrogen residual concentration in the expression system respectively, the accumulated concentrations of full nitrogen residual concentration and nitrite.
Below in conjunction with specific embodiment.Further set forth the present invention, be to be understood that, these embodiment only are used to the present invention is described and are not used in restriction the scope of protection of present invention, unreceipted concrete experiment condition and method in the following example, usually compile as: soil microorganisms research association " day " according to normal condition, Ye Weiqing etc. translate, Science Press, 1983, the soil microorganisms laboratory method; Permitted volumes such as radiance, Beijing agricultural press, 1986, soil microorganisms analytical procedure handbook; And microbial room of Nanjing Soil Inst., Chinese Academy of Sciences volume, Science Press, 1985, the condition described in the books such as soil microorganisms organon, or connect the condition of advising according to manufacturer.
Embodiment
The preparation of embodiment 1. substratum and reagent
1.1PM liquid nutrient medium (beef-protein medium) preparation
Claim 3 gram beef extracts, 5 gram peptones are dissolved in and form the PM liquid nutrient medium in 1000 ml distilled waters, add 20 gram agar in above-mentioned unpasteurized liquid PM substratum, form the PM solid medium.
Transfer medium pH to 7.1 with 1N sodium hydroxide.Be divided in the triangular flask, sterilization is fallen dull and stereotyped when substratum is cooled to 50 ℃.The PM liquid nutrient medium is through high pressure liquid chromatographic analysis, and the result is that nitrite and nitrate content are the mark amount: nitrite does not detect, and nitrate content is 0.18mg N L -1
1.2 the preparation of Griess reagent
1.2.1 Sulphanilic Acid reagent (A liquid): 0.5 gram Sulphanilic Acid (Sulfanilic acid) is dissolved in 150 milliliter 20% the dilute acetic acid solution, stores in brown bottle, refrigerates standby.
1.2.2 alpha-naphthylamine reagent (B liquid): (α-naphthylamine) is added in the dilute acetic acid solution of 20 ml distilled waters and 150 milliliter 20% 0.5 gram alpha-naphthylamine, stores in brown bottle, refrigerates standby.
1.2.3 the use liquid of Griess reagent: the A liquid of getting equal proportion mixes and can use with B liquid.
1.3 the preparation of NB liquid nutrient medium
1.3.1 the preparation of inorganic salt solution
Take by weighing (NH 4) 2SO 42.1 gram, NaH 2PO 40.25 gram, K 2HPO 40.75 gram,
MgSO 47H 2O 0.03 gram, MnSO 4H 2O 0.01 gram,
FeSO 47H 2O 0.01 gram is dissolved in 1000 ml distilled waters,
Transferring the pH value of substratum with 1M NaOH is 7.0, is divided in the triangular flask sterilization.
1.3.2 the preparation of organic carbon solution
1.3.2.1 the preparation of sodium acetate solution: take by weighing 27.2 gram sodium acetate trihydrate and be dissolved in the sodium acetate solution that forms 0.20M in 1000 ml distilled waters, sterilization.
1.3.2.2 the preparation of sodium formate solution: take by weighing 20.8 grams, two water sodium formiates and be dissolved in the sodium formate solution that forms 0.20M in 1000 ml distilled waters, sterilization.
1.3.2.3 the preparation of pyruvic acid solution: draw 14.0 milliliters of pyruvic acid and be dissolved in the pyruvic acid solution that forms 0.20M in 1000 ml distilled waters, sterilization.
Get inorganic salt solution 90 parts (volume ratios) during use and mix for 10 parts, form the NB substratum with organic carbon solution.
1.4 the Nitrate bacteria culture medium preparation takes by weighing NaNO 21.0 gram, MgSO 47H 2O 0.03 gram, K 2HPO 40.75 gram, NaH 2PO 40.25 gram, MnSO 4H 2O 0.01 gram, NaCO 31.0 gram is dissolved in 1000 ml distilled waters, transferring the pH value of substratum with 1M NaOH is 7.2, is divided in the triangular flask sterilization.
1.5 the preparation of pentanoic reagent
Pentanoic 0.5 gram, 100 milliliters of the vitriol oils, 20 milliliters of distilled water; Earlier pentanoic 0.5 gram is dissolved in 20 milliliters of the distilled water, adds 100 milliliters of the vitriol oils more slowly, be stored in the brown bottle standby
1.6 denitrification culture medium preparation
1.6.1 the preparation of inorganic salt solution
Take by weighing KNO 32.0 gram, KH 2PO 41.0 gram, K 2HPO 41.0 gram, MgSO 47H 2O 0.2 gram is dissolved in 1000 ml distilled waters, and transferring the pH value of substratum with 1M NaOH is 7.2, is divided in the triangular flask sterilization.
1.6.2 the preparation of organic carbon solution
Carbon source is Trisodium Citrate, sodium acetate or glucose
1.6.2.1 the preparation of glucose solution: take by weighing the glucose solution that 20 gram glucose are dissolved in formation 2% in 1000 ml distilled waters, sterilization.
1.6.2.2 the preparation of sodium acetate solution: take by weighing the sodium acetate solution that 20 gram sodium acetate trihydrate are dissolved in formation 2% in 1000 ml distilled waters, sterilization.
Get 90 parts of inorganic salt solutions during use by volume and mix for 10 parts, form the denitrifying bacterium substratum with organic carbon solution.
Embodiment: 2, isolation identification has the heterotrophic organism bacterial strain of nitrification activity
2.1 pedotheque
Sampling position: Zhao Gang township, Fengqiu County, Henan Province; Specific name: the yellow moisture soil of middle loamy texture;
The source: topsoil soils, soil becomes silted up; Soil sample is handled: air-dry, ground 20 mesh sieves;
2.2 take by weighing 1.0 gram wind desiceted soils in 250 milliliters of triangular flasks that contain 50 milliliters of sterile distilled waters, 90r/min shaking table vibration 4 hours.
2.3 soil suspension is coated the PM flat board, three repetitions of each extent of dilution after diluting by 10 times of methods.Cultivate after 7 days for 28 ℃, picking list bacterium colony is to the PM flat board, the purifying of ruling.Microscopy proves purity.
2.4 primary dcreening operation (discriminating of active bacterium)
The heterotrophism bacterial strain after separation and purification that obtains is inoculated in the PM flat board, cultivated 10 days for 28 ℃, Griess reagent directly point drips to flat board, carries out nitrification activity and confirms, and make blank with the flat board that does not connect bacterium.In 1 minute, the Griess reagent colour developing takes on a red color, and showing has nitrite to generate.Repeated authentication, color reaction still is positive, and tentatively confirms as the nitrification activity bacterium.(seeing Table 3).The PM substratum is through high pressure liquid chromatographic analysis, and show that wherein nitrite and nitrate content are the mark amount: wherein nitrite does not detect, and nitrate content is lower than 0.2mg N L -1, can not constitute and just disturb the result.
2.5 multiple sieve
The representative bacterial strain that activity is stronger when choosing primary dcreening operation carries out.Scrape and get the pure bacterium lawn that grows in the PM flat board and go into 30 ml sterile waters, it fully is uniformly dispersed makes bacteria suspension.Inoculate 1 milliliter of bacteria suspension respectively and go into to contain in 250 milliliters of Erlenmeyer flasks of 50 milliliters of NB substratum that different organism are carbon source every group of three repetitions.And make blank with the substratum of not inoculating.28 ℃ of static cultivations are after 42 days, 4 ℃ of centrifugal 15min of following 5000g of nutrient solution, the nitrite concentration in the Griess reagent method colorimetric estimation supernatant liquor.Bacterial classification sample cultivation supernatant colourimetric number subtracts the difference of blank supernatant colourimetric number greater than 0.3mg N L -1The time be judged to the active bacterium (table 4) of heterotrophic nitrification.WR-2 bacterial strain nitrification activity is the highest, is asserted type strain.
2.6 the physiology of active bacterial strain is identified
With reference to uncle Jie Shi Bacteria Identification handbook the 9th edition.Form and the physiological characteristic of bacterium see Table 5, table 6.
2.7 Arthrobacter globiformis WR-2 bacterial strain has following feature:
2.7.1 colony morphology characteristic: after supporting 7 days, 28 ℃ of good air cultures of constant temperature on the nutrient agar medium PM flat board form very little bacterium colony, circle, flat, full edge; Rough have wrinkle, lawn surface to be khaki color, and matrix does not see that tangible soluble pigment produces, and sees Fig. 1.
2.7.2 morphological features: Gram-positive; Cultivate in early days, it is irregular shaft-like that cell is, and prolongs incubation time and sphere then occurs, and the aged cell almost is spherical entirely; How to arrange with the chain form; Do not move, do not form statospore.
The classification of table 3 part active bacterial strain
Plant (genus) name representative strain
Genus arthrobacter (Arthrobacter) WY-1, WR-2
Arthrobacter globiformis (Arthrobacter globiformis) WR-2
Erwinia (Erwinia) WT-1, XY-3
Corynebacterium (Corynebacterium) WY-19
Micromonospora (Micromonospora) WH-1
Bacillus (Bacillus)
Bacillus pumilus (Bacillus brevis) WY-2, WY-21
Bacillus licheniformis (Bacillus licheniformis) WX-2
Bacillus circulans (Bacillus circulans) WR-8
Bacillus firmus (Bacillus firmus) WR-9
The colourimetric number of each bacterial strain of table 4 (is represented NO with nitrite 2 --N mg L -1)
Bacterial strain carbon source Trisodium Citrate sodium acetate
XY-3 0.4 1.0
WY-2 0.0 1.7
WY-19 0.7 0.3
WY-20 0.0 0.4
WY-21 0.7 0.8
WR-2 0.6 8.2
WT-1 0.3 1.9
WH-1 0.0 0.4
CK 0.0 0.1
The physio-biochemical characteristics of table 5 arthrobacter bacteria
Bacterial strain anaerobic growth catalase starch hydrolyzation of glucose fermentation decomposition of cellulose indoles experiment nitrate reduction
B173 + + - + - - +
B256 + + + + - - +
B459 - + + + - - +
B464 - + + + - - +
B602 - + + + - - +
B605 - + + + - - +
WY-1 - + + + - - +
WR-2 - + + + - - +
Annotate: in the table+ecbatic is positive, the reaction that is negative of-expression ecbatic.
The individual morphology feature of table 6 arthrobacter bacteria
Bacterial strain number
Bacterium colony circle on the B173 PM flat board, light yellow, microprotrusion; Gram-positive, no endogenous spore,
12 hours is bacillus different in size, is globoferous cell in the time of 7 days, and particular arrangement is arranged
Bacterium colony circle on the B256 PM flat board, yellow, microprotrusion; Gram-positive, no endogenous spore, 12 hours
Be straight or crooked dialister bacterium, what have has a branch, is club shape cell in the time of 7 days
Bacterium colony circle on the B459 PM flat board, light yellow, microprotrusion; Gram-positive, no endogenous spore, 12 is little
The time be bacillus, two branches are arranged, out-of-shape is coccus, coccobacillus in the time of 7 days
Bacterium colony circle on the B464 PM flat board, light yellow, microprotrusion; Gram-positive, no endogenous spore, 12 is little
The time be bacillus, branch is arranged, be coccus, coccobacillus in the time of 7 days
Bacterium colony circle on the B602 PM flat board, yolk yellow, opaque, projection, smooth; Gram-positive is in the nothing
The spore of sprouting, 12 hours is bacillus, is coccus, sphere or elliposoidal in the time of 7 days
Bacterium colony circle on the B605 PM flat board, yolk yellow, projection, smooth; Gram-positive, no endogenous spore, 12
Hour be bacillus, elongate curved is coccus, coccobacillus in the time of 7 days
Bacterium colony circle on the WY-1 PM flat board, yellow, projection, smooth; Gram-positive, no endogenous spore,
12 hours is bacillus, and what have has a branch, is coccus, coccobacillus in the time of 7 days
WR-2 can form very little bacterium colony on the PM flat board, circle is flat, full edge; Rough have wrinkle,
The lawn surface is khaki color; Gram-positive; Cultivate in early days, it is irregular shaft-like that cell is, and prolongs when cultivating
Between then occur spherically, the aged cell almost is spherical entirely; How to arrange with the chain form; Do not move, do not form
Statospore
2.7.3 the major physiological biochemical characteristic sees Table 1: the suitableeest culture temperature is 28-32 ℃, and pH7.0-7.2 is aerobic.
2.7.4 ammoxidation activity: the nitrite concentration of cultivating accumulation in 14 days at the 90-100r/min shaking table all is higher than 60.0mg NO 2-N L -1, indivedual individual plants can be up to 95mg NO 2-N L -1More than.
2.7.5 denitrification activity: when being carbon source with the sodium acetate, concentration is 0.075mol L -1The time, C/N is 5: 1, cultivates 28 days, and ammonia-nitrogen removal rate is 65%, and full nitrogen removal efficiency is 62%; When pyruvic acid was carbon source, concentration was 0.050mol L -1The time, C/N is 5: 1, cultivates 28 days, and ammonia-nitrogen removal rate is 90%, and full nitrogen removal efficiency is 68%.Remaining full nitrogen be almost somatic cells.
Classification with reference to uncle Jie Shi Bacteria Identification handbook the 9th edition identifies that this bacterial strain is Arthrobacter globiformis (Arthrobacter globiformis).Therefore with its called after Arthrobacter globiformis WR-2.Arthrobacter globiformis WR-2 on November 5th, 2002 in specified depositary institution of Patent Office of the People's Republic of China---Chinese typical culture collection center (CCTCC) preservation, deposit number are CCTCC M202043.
Separation and the evaluation of embodiment 3 WO-8
3.1 take by weighing 1.0 the gram wind desiceted soils in 50 milliliters sterilized, contain in 250 milliliters of triangular flasks of Nitrate bacteria enriched medium, 28 ℃, static cultivation.Cultivate after 7 days and took a sample every three days, the Griess reagent drop is determined the disappearance of nitrite, etc. color reaction pinkiness or redfree, after the apparent feminine gender or the weak positive, with the generation of pentanoic reagent drop affirmation nitrate, if color reaction is dark blue or black-and-blue; Be light blue or colourless etc. color reaction, promptly get 2 milliliters of nutrient solutions after the apparent feminine gender or the weak positive and be inoculated in the fresh culture.After the reaction of Griess reagent drop color reaction, pentanoic reagent colour development all was negative once more, strains separation was carried out in sampling.After diluting by 10 times of methods, rich nutrient solution coats the PM flat board, three repetitions of each extent of dilution 3.2 add.Cultivate after 7 days for 28 ℃, picking list bacterium colony is to the PM flat board, the purifying of ruling.Microscopy proves purity.
3.3 the screening of denitrification activity bacterium is confirmed:
Scrape and get the pure bacterium lawn that grows in the PM inclined-plane and go into 9 ml sterile waters, it fully is uniformly dispersed makes bacteria suspension.Inoculating 1 milliliter of bacteria suspension respectively, to go into to contain 50 milliliters be 250 milliliters of Erlenmeyer flasks of carbon source Nitrate bacteria substratum and 250 milliliters of Erlenmeyer flasks that contain 50 milliliters of denitrification substratum with glucose.And make blank with the substratum of not inoculating.28 ℃ of static cultivations, every sampling in 3 days, the Griess reagent drop was confirmed the existence and the disappearance of nitrite, by nitrate reduction and nitrite-oxidizing, proves its denitrification activity, the results are shown in Table 7.
By each active bacterial strain is further compared, find that the WO-8 bacterial strain can be with 200mg NL in 7 days -1NO 2 -Remove 99%; In 10 days with 280mg N L -1NO 3 -Remove 97%.Confirm that WO-8 bacterial strain nitrite reducing power and nitrate reduction ability are the strongest.
3.4 the WO-8 bacterial strain is through being accredited as plant bacillus pumilis WO-8, (Curtoacterium plantarum WO-8).This bacterial strain has following feature:
3.4.1 colony morphology characteristic: after 28 ℃ of good air cultures of constant temperature on the PM nutrient agar plate were supported 2 days, the bacterium colony of formation was circular, flat, full edge, smooth surface; The lawn surface is orange, and matrix does not see that tangible soluble pigment produces.
3.4.2 morphological features: the positive reaction of gramstaining; How rod-short, thick shape, the blunt circle in thalline two ends occur with single spread pattern; Do not see statospore formation; Fig. 2 is seen in motion.
3.4.3 the major physiological biochemical characteristic sees Table 2: the suitableeest culture temperature is 28-32 ℃, and pH7.0-7.2 is aerobic.
3.4.4 the denitrification activity of bacterial strain has strong denitrification activity, has nitrate, nitrite reducing power, experiment records can be with 200mg N L in 7 days -1NO 2 -Remove 99%; In 10 days with 280mg N L -1NO 3 -Remove 97%, and lose with the form ease of gaseous nitrogen.When being nitrogenous source, there is not the generation of nitrite in the culturing process, no heterotrophism nitrification activity with the ammonium salt.
The denitrification activity performance (28 ℃, static cultivation) of each bacterial strain of table 7
Nitrite reduction nitrate reduction
Bacterial strain glucose Trisodium Citrate glucose Trisodium Citrate
WO-1 + + + +
WO-2 - - - -
WO-3 - - - -
WO-4 - - - -
WO-5 - - + +
WO-4 - - - -
WO-7 - - + +
WO-8 + + + +
WO-9 + + + +
WO-10 + + - -
WO-11 - - + +
WO-12 - - + +
WO-12 - - + +
Annotate :-expression is negative, and+expression is positive
Classification with reference to uncle Jie Shi Bacteria Identification handbook the 9th edition identifies that this bacterial strain is plant bacillus pumilis (Curtobacterium plantarum).Therefore with its called after plant bacillus pumilis WO-8.Plant bacillus pumilis WO-8 on November 5th, 2002 at specified depositary institution of Patent Office of the People's Republic of China-Chinese typical culture collection center (CCTCC) preservation, preservation registration number CCTCC M202044.
The denitrification effect of embodiment 4