CN1210395C - Heterotrophic nitrobacteri, culturing method and application thereof - Google Patents

Heterotrophic nitrobacteri, culturing method and application thereof Download PDF

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CN1210395C
CN1210395C CN 03118597 CN03118597A CN1210395C CN 1210395 C CN1210395 C CN 1210395C CN 03118597 CN03118597 CN 03118597 CN 03118597 A CN03118597 A CN 03118597A CN 1210395 C CN1210395 C CN 1210395C
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bacterium
nitrogen
nitrification
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concentration
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CN1483813A (en
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彭光浩
王一明
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Institute of Soil Science of CAS
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Abstract

The present invention discloses a new bacterium having nitration activity and individual amino and nitrogen removal capability and a composition containing the bacteria. The bacterium can effectively remove ammoniacal nitrogen and protect environment. The present invention also discloses a method for culturing the bacterium and the application of the bacterium to biological denitrogenation and nitration inhibitor screening.

Description

Allotrophic nitrobacteria, cultural method and application thereof
Technical field:
The invention belongs to microorganism field, specifically, the present invention relates to new bacterium, the method for cultivating this bacterioid with denitrogenation biologic activity, the composition that contains this bacterioid that can denitrogenation in water body, and the application of this bacterioid in biological denitrificaion and screening nitrification inhibitor.
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, volume increase of farm crop.But the use of nitrogenous fertilizer has also brought serious environmental problem when increasing substantially crop yield: as eutrophication, the NH in the semiclosed waters of sealing such as the continuous growth of tap water Nitrate Accumulation, lake 4 +-NH 3Enter aquatic resources such as water body harm fish shellfish, and the N of one of greenhouse gases 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 reduce nitrogen the detrimentally affect of environment has been become the huge challenge that present industrial and agricultural production and environment protection face.Simultaneously, the improvement that nitrogen form transforms again with sewage sludge nitric efficiency, denitrification process improves, 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 a few days ago 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).When mixing sludge enters anoxic pond or is in anoxic condition, denitrifying bacteria work, nitrifier is in holddown; Situation is then opposite when mixing sludge enters Aerobic Pond or has been in oxygen condition, nitrifier work, and denitrifying bacteria 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 etc., 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 through aeration earlier to reduce organic concentration, 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, 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, though can reach the synchronous nitration and denitrification of single reactor.But the denitrification result is unsatisfactory, no actual application value.
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 have 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, the organic carbon concentration of 0.5 grams per liter suppress thalli growth and also reduce denitrification effect.
Nitrification inhibitor is the obligate chemicals that selectivity suppresses ammonia oxidizing bacteria, and developing various nitrification inhibitors is one of effective measure of control nitrification, raising utilization rate of nitrogen fertilizer.Though the numerous nitrification inhibitor of kind class has been arranged at present, but practical application both at home and abroad and wide coverage be limited to 2-chloro-6 (trichloromethyl) pyridine (N-serve that filters out at the beginning of the 60-70 age more, also claim nitrapyrin or western pyrrole CP), 4-amino-1,2,4-triazolium salt hydrochlorate (ATC), Dyhard RU 100 (DCD), thiocarbamide (TU), amidinothiourea (ASU), 2-amino-4-chloro-6-methylpyrimidine (AM) wait a few compound; New research is also also not fully up to expectations.Cause the one of the main reasons of this situation to be, nitrated microorganism is formed complexity, nitrification inhibitor is very not clear and definite to target action of microorganisms mechanism.
Existing result of study shows that nitrification inhibitor differs greatly to the inhibition concentration of different soils, and as N-Serve, concentration can be at 0.5-20g N-Serve g -1The soil variation (Norton J.M., Handbookof soil science, 2000,160-175).Except the soil texture, pH, water content, organic content, temperature etc. have (Keeney D.R. the substantial connection, Ntrification inhibitors potentialsnandlimitations, 1980,33-46), wherein as the composition of the nitrated microorganism of target, difference on the nitrated microorganism of different soils is formed, the especially existence of heterotrophic nitrification microorganism and their activity have significant effects to the application effect of nitrification inhibitor.There is experiment to show, some nitrification inhibitors commonly used at present, as acetylene, N-Serve, 1-allyl group-2-thiocarbamide under the concentration that can suppress the autotrophy nitrobacteria, can not suppress the nitrification of part for the pure culture of examination allotrophic nitrobacteria, they need much higher inhibition concentration (Verstraete W.and Alexander M., J.Bacterial., 1972,110:955-961; Hynes R.K.and Knowles R., Can.J.Microbiol., 1982,28:334-340).The experiment of carrying out with the ammonia monooxygenase (AMO) of purifying in the allotrophic nitrobacteria has obtained similar result, the acetylene of 1mM (being equivalent to 2.5%) can not suppress the enzyme of heterotrophic nitrification microorganism-Paracoccus denitrificans AMO and live, pseudomonas putida, 5% concentration acetylene could suppress its oxidation to ammonia, and Nitrosomonas europaea, but 0.25 the acetylene of μ M just strongly inhibited it to the oxidation of ammonia, 3.8 the acetylene of μ M can suppress its enzyme (HynesR.K.and Knowles R. alive fully, Can.J.Microbiol., 1982,28:334-340; Moir J.W.B.et al, FEBS Lett., 387:71-74; Daum M.et al, Curr.Microbiol., 1998,37:281-288).
And mostly the screening study of present most of nitrification inhibitors and effect comparison are to carry out under soil is cultivated; Up to now, the autotrophy nitrobacteria still is considered to the especially main executive of agricultural soil nitrification of soil, and the exploitation screening of nitrification inhibitor is that target carries out with the autotrophy nitrobacteria mainly also.In addition, methodology from inhibitor screening, except that ATC, sodiumazide or potassium, Dyhard RU 100, a few inhibitor of thiocarbamide, a large amount of test compounds mostly is the organic compound that is insoluble in water, must add organic solvent or tensio-active agent during use, and the investigator these organism have often been ignored to the composition of nitrated microorganism, the influence of active performance.
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 nitrification activity and ammonia nitrogen removal ability.
A further object of the present invention provides the application of new bacterium in the water body biological denitrificaion.Separately or unite and use new bacterium can remove nitrogen in the water body effectively.
A further object of the present invention provides the application of new bacterium in the screening nitrification inhibitor.New bacterial strain can be used as type strain screening nitrification inhibitor.
A further object of the present invention provides a kind of bacteria composition with denitrogenation biologic activity, and the application of this bacteria composition in the water body biological denitrificaion.
Can reach the above each purpose by the present invention.
A kind of have a bioactive bacterium of denitrogenation, and this bacterium has higher nitrification activity and individual plant ammonia nitrogen removal ability.This class bacterial strain is an allotrophic nitrobacteria, can be effectively with NH in biological denitrificaion 4 +Be converted into NO by nitrification 2 -Or NO 3 -, also can be with NH 4 +Directly change into gas mode such as NO, the N of various nitrogen elements 2O and N 2Gas evolution.More specifically, this bacterioid is a bacterioid of genus arthrobacter Arthrobacter globiformis kind.In an object lesson of the present invention, the bacterium that had not only had higher nitrification activity but also had an individual plant ammonia nitrogen removal ability is Arthrobacter globiformis (Arthrobacter globiformis) WR-2, this strain bacterium 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, preservation registration number is CCTCC M202043.
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, through PM plate isolation, purifying, active check, Griess reagent is identified, has obtained the heterotrophism bacterial strain that a large amount of Ge Lisi reactions is positive, and confirms that tentatively they have ammoxidation capability.These bacterial strains belong to genus arthrobacter, erwinia, corynebacterium, micromonospora, bacillus etc.
Particularly, the 8 strain bacteriums that have that belong to genus arthrobacter Arthrobacter globiformis kind that from soil, screen, this 8 strain bacterium all has similar biological function, other kind bacterium denitrogenation biological activity height that its nitrification activity and individual plant ammonia nitrogen removal energy force rate screen from soil, but 8 strain bacterium are variant on morphology.Each strain characteristic of bacteria is as follows:
(1) bacterium colony circle on the B173:PM flat board is 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;
(2) bacterium colony circle on the B256:PM flat board, yellow, microprotrusion; Gram-positive, no endogenous spore, 12 hours is straight or crooked dialister bacterium, what have has a branch, is club shape cell in the time of 7 days;
(3) bacterium colony circle on the B459:PM flat board is light yellow, microprotrusion; Gram-positive, no endogenous spore, 12 hours is bacillus, and two branches are arranged, out-of-shape is coccus, coccobacillus in the time of 7 days;
(4) bacterium colony circle on the B464:PM flat board is light yellow, microprotrusion; Gram-positive, no endogenous spore, 12 hours is bacillus, and branch is arranged, and is coccus, coccobacillus in the time of 7 days;
(5) bacterium colony circle on the B602:PM flat board is yolk yellow, opaque, and projection is smooth; Gram-positive, no endogenous spore, 12 hours is bacillus, is coccus in the time of 7 days, sphere or elliposoidal;
(6) bacterium colony circle on the B605:PM flat board is yolk yellow, and projection is smooth; Gram-positive, no endogenous spore, 12 hours is bacillus, elongate curved is coccus, coccobacillus in the time of 7 days;
(7) bacterium colony circle on the WY-1:PM flat board, yellow, projection, smooth; Gram-positive, no endogenous spore, 12 hours is bacillus, what have has a branch, is coccus, coccobacillus in the time of 7 days;
(8) WR-2: can on the PM flat board, form very little bacterium colony, circle, flat, full edge; Rough have wrinkle, lawn surface to be khaki color; 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 inventor further detects relatively repeatedly from 8 strain bacteriums, the result is separated to the bacterium that a strain has higher nitrification activity and individual plant ammonia nitrogen removal ability from the North China moisture soil of Fengqiu, Henan Province, through being accredited as Arthrobacter globiformis (Arthrobacter globiformis) WR-2.This Arthrobacter globiformis WR-2 bacterial strain has following feature:
1. colony morphology characteristic: support on the agar plate in that the PM of battalion is dull and stereotyped, 28 ℃ of good air cultures of constant temperature are supported and are formed very little bacterium colony after 7 days, and circle is 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.
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.
4. ammoxidation activity: with the pyruvic acid is carbon source, and ammonium sulfate is nitrogenous source, and bacterial strain is at initial pH5.0-9.0, all can well grow in the culture temperature 15-35 ℃ of scope and nitrification takes place; 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.Initial pH is lower than at 5 o'clock, and thalline does not have the no nitrification activity of growth.
5. denitrification activity: with concentration is 0.075mol L -1Sodium acetate is a carbon source, and C/N is 5: 1 o'clock, cultivates 28 days, and ammonia-nitrogen removal rate is 65%, and full nitrogen removal efficiency is 62%; Concentration is 0.050mol L -1Pyruvic acid be carbon source, C/N is 5: 1 o'clock, cultivates 28 days, ammonia-nitrogen removal rate is 90%, 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 is CCTCC M202043.
The Arthrobacter globiformis WR-2 that we are separated to from soil in the water body that has nitrogen such as ammonia-state nitrogen and/or nitrate and/or nitrite, not only has higher nitrification activity, and ammonium nitrogen can be transformed dinitrogen gas N 2Ability, the environment that this effect takes place is under aerobic condition, and at higher carbon/nitrogen than under the condition, biologic activity is stronger, like this, to the water body of eutrophication, need not carry out chemical aeration treating processes.
A kind of stronger nitrification activity of Arthrobacter globiformis embodiment and method of ammonia nitrogen removal ability of cultivating, this method are included in a), and pH is 5.0-9.0; B) temperature is 15-35 ℃; C) iron concentration is with FeSO 47H 2O counts the 0.005-0.02 grams per liter; D) carbon/nitrogen ratio is 1: 10-20: culturing bacterium under 1 condition.The preferable methods combination comprises that a) pH is 6.0-8.0; B) temperature is 18-30 ℃; C) iron concentration is with FeSO 47H 2O counts the 0.01-0.02 grams per liter; D) carbon/nitrogen ratio is 1: 1-10: culturing bacterium under 1 condition, and best method combination comprises that a) pH is 7.0; B) temperature is 30 ℃; C) iron concentration is with FeSO 47H 2O counts 0.01 grams per liter; D) carbon/nitrogen ratio is a culturing bacterium under 5: 1 conditions.
Arthrobacter globiformis WR-2 is the bacterium that not only has higher nitrification activity but also have individual plant ammonia nitrogen removal ability.Under the growth conditions of routine, just can show nitrification activity, and when Individual existence, embody the ammonia nitrogen removal ability.In suitable pH value, temperature, under the combination culture condition such as carbon/nitrogen ratio and iron concentration, Arthrobacter globiformis WR-2 can embody stronger nitrification activity and ammonia nitrogen removal ability.
The Arthrobacter globiformis WR-2 of the present invention's separation screening from soil can be in containing the water body of nitrate pollution growth and breeding, by the intravital bioprocesses of bacterium, a part of nitrogen transformation becomes dinitrogen gas to discharge, another partly changes into bacterium thalline material, remaining nitrogen is in common water body reasonable content scope, and the sewage of carrying out a biological disposal upon through Arthrobacter globiformis WR-2 reaches environmental emission standard.The invention provides the application of Arthrobacter globiformis WR-2 in the water body biological denitrificaion of separation screening from soil.In specific embodiments of the invention, Arthrobacter globiformis WR-2, CCTCC M202043 are applied to removing of nitrate pollution thing in the water body.
Arthrobacter globiformis WR-2 is the aerobic microorganism of a strain heterotrophism, water body particularly eutrophication water comprise that can carry out the nitrogen harmless biological in the water body that has ammonium salt, nitrite, organonitrogen handles, the qualified water body standard that reaches discharging or drink through the water body of Arthrobacter globiformis WR-2 biological treatment.It is to carry out under aerobic conditions that the water body harmless biological of Arthrobacter globiformis WR-2 is handled, and does not need special equipment and extra experiment condition, and the harmless biological of water body nitrogen is handled the process of carrying out nitrification and denitrification in a reaction tank simultaneously.
The invention also discloses the application of Arthrobacter globiformis WR-2 in the screening nitrification inhibitor.This pattern bacterium of Arthrobacter globiformis WR-2 can make the inhibitor of experimental bacteria screening nitrification, in a specific embodiment of the present invention, with Arthrobacter globiformis (Arthrobacter globiformis) WR-2, preservation registration number CCTCCM202043 is for supplying the examination bacterium, find that vitamins C can suppress the generation of nitrification in the soil effectively, this helps reasonably applied nitrogen.
A kind of bacteria composition of denitrogenation in water body contains receivable carrier in the Arthrobacter globiformis WR-2 of significant quantity and the water body denitrification technology.Receivable carrier comprises sorbent material in the water body denitrification technology, nitrification and denitrification bacterium etc.The sorbent material that adopts among the present invention is as 20% polyvinyl alcohol; Nitrobacteria such as bacillus cereus NBB-135 (CGMCC No.0560); Denitrifying bacterium such as plant bacillus pumilis WO-8 (CCTCCM202044) etc.Bacillus cereus NBB-135, CGMCC No.0560 carries out the patented procedure preservation at China Committee for Culture Collection of Microorganisms common micro-organisms center; Plant bacillus pumilis WO-8 (CCTCC M202044) carries out the patented procedure preservation at China typical culture collection center.
The invention provides the application of bacteria composition in the water body biological denitrificaion.Use bacteria composition provided by the invention can remove ammonia-state nitrogen in the water body effectively.
Arthrobacter globiformis WR-2 provided by the invention is the aerobic microorganism of a strain heterotrophism, not only have higher nitrification activity and have individual plant ammonia nitrogen removal ability, and nitrification inhibitor such as 4-amino-1,2 to generally using, 4-triazolium salt hydrochlorate (ATC) insensitivity; The inventor is the pattern bacterium with Arthrobacter globiformis WR-2, carries out the screening operation of nitrification inhibitor.In a specific embodiment of the present invention, we are the pattern bacterium with Arthrobacter globiformis WR-2, find that vitamins C can obviously suppress its growth and breeding, and influence its biologic activity.
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 are the same employing national standard of national standard, the employing industry standard of no national standard in various units of Shi Yonging in the present invention.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.
In the present invention, Arthrobacter globiformis WR-2 separation screening from soil obtains, and has done the preservation of patented procedure, and preserving number is CCTCC M202043, and in the present invention, Arthrobacter globiformis WR-2 is equal to CCTCC M202043.
Description of drawings
The aspect graph 1:A plate bacterium colony figure of accompanying drawing 1 Arthrobacter globiformis WR-2; B thalline Photomicrograph
The thalli growth curve of accompanying drawing 2 Arthrobacter globiformis WR-2 when the different concns sodium acetate is cultivated, C1 wherein, C3, C5, C10 represent that respectively sodium acetate concentration is 0.015mol L -1, 0.045mol L -1, 0.075mol L -1, 0.15mol L -1
The nitrite accumulation of accompanying drawing 3 Arthrobacter globiformis WR-2 when the different concns sodium acetate is cultivated, C1 wherein, C3, C5, C10 represent that respectively sodium acetate concentration is 0.015mol L -1, 0.045mol L -1, 0.075mol L -1, 0.15mol L -1
The upgrowth situation of accompanying drawing 4 Arthrobacter globiformis WR-2 in different concns ATC, wherein carbon source is a pyruvic acid, culture temperature is 28 ℃ static cultivation
The nitrification activity performance of accompanying drawing 5 Arthrobacter globiformis WR-2 in different concns ATC, wherein carbon source is a pyruvic acid, culture temperature is 28 ℃ static cultivation
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
1.1 the PM liquid nutrient medium is also named the preparation of beef-protein medium
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 1 N 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, and 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 90 parts of inorganic salt solutions during use by volume and mix for 10 parts, form the NB substratum with organic carbon solution.
1.4 4-amino-1,2, the preparation of 4-triazolium salt hydrochlorate (ATC): take by weighing 18 gram ATC and be dissolved in the ATC solution that forms 1800 mg/litre in 1000 ml distilled waters, sterilization.
1.5 ascorbic preparation: take by weighing the vitamin c solution that 2.60 gram vitamins Cs are dissolved in formation 2.60% in 100 ml distilled waters, sterilization.
1.6 the preparation of polyvinyl alcohol (PVA2000) solution: take by weighing the PVA solution that 20.0 gram PVC2000 are dissolved in formation 20% in 100 ml distilled waters.
1.7 calcium chloride (CaCl 2) preparation of solution: take by weighing 11.1 gram CaCl 2Be dissolved in and form 1.0M CaCl in 100 ml distilled waters 2Solution.
1.8 the preparation of polyvinyl alcohol and calcium chloride mixing solutions: get 80 parts of 20% PVA solution by volume, 1.0MCaCl 220 parts of solution mix, and are polyvinyl alcohol and calcium chloride mixing solutions.
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 coat the PM flat board behind 10 times of gradient dilutions of soil suspension, three repetitions of each extent of dilution.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, the 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.Inoculated bacteria arrives flat board once more, repeated authentication, and color reaction still is positive, and tentatively confirms as the nitrification activity bacterium.(seeing Table 2).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 the establishment of multiple sieve, type strain
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 (seeing Table 3) of heterotrophic nitrification.
The classification of table 2 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 3 (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
2.6 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 4, table 5.
Embodiment: the evaluation of 3 Arthrobacter globiformis WR-2
1. colony morphology characteristic: after 28 ℃ of good air cultures of constant temperature on the nutrient agar medium PM flat board are supported 7 days, form very little
The physio-biochemical characteristics of table 4 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 :+expression positive reaction maybe can utilize;-expression negative reaction maybe can not be utilized.
The individual morphology feature of table 5 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
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.
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, and the suitableeest culture temperature is 28-32 ℃, and pH7.0-7.2 is aerobic.
4. ammoxidation activity: with ammonium sulfate is nitrogenous source, and bacterial strain is at initial pH5.0-9.0, all can well grow in the culture temperature 15-35 ℃ of scope and nitrification takes place; 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-NL -1More than.Initial pH is lower than at 5 o'clock, and thalline does not have the no nitrification activity of growth.
5. denitrification activity: when being carbon source with the sodium acetate, concentration is 0.075mol L -1The time, C/N=5: 1, to cultivate 28 days, ammonia-nitrogen removal rate is: 65%, full nitrogen removal efficiency is 62%; When pyruvic acid was carbon source, concentration was 0.050mol L -1The time, C/N=5: 1, to cultivate 28 days, ammonia-nitrogen removal rate is: 90%, 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 is CCTCC M202043.The cultural characteristic of WR-2 bacterial strain and individual morphology feature are seen A and the B among Fig. 1.
Embodiment: the cultivation of 4 Arthrobacter globiformis WR-2 and biologic activity performance
4.1 scrape and get the pure bacterium lawn that grows in the PM flat board and go into 20 ml sterile waters, it fully is uniformly dispersed makes bacteria suspension.Inoculate 1 milliliter of bacteria suspension respectively and go into to contain 1. sodium acetate, 2. oxysuccinic acid, 3. pyruvic acid, 4. sodium formiate, 5. Trisodium Citrate is in 250 milliliters of Erlenmeyer flasks of 50 milliliters of NB substratum of 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, and the nitrite concentration in the Griess reagent method colorimetric estimation supernatant liquor the results are shown in Table 6.
The growth and the nitrification activity of the pure culture of WR-2 bacterium during the different carbon source of table 6
Carbon?source Cell(Log?N) Net?NO 2 -(mg?N?L -1)
1. sodium acetate 8.8 8.2
2. oxysuccinic acid 8.7 8.0
3. pyruvic acid 9.1 26.1
4. sodium formiate 7.9 9.2
5. Trisodium Citrate 6.0 0.3
CK 6.1 0.3
Annotate: CK represents not add the cultivation that any organism is a carbon source
Go into to be equipped with 250 milliliters of Erlenmeyer flasks of 50 milliliters of NB substratum 4.2 connect a ring lawn from the PM inclined-plane, at 28-30 ℃, 120-130r/min shaking table shaking culture 24 hours.By 2% amount inoculation, 1 milliliter of bacterium liquid is connected in 250 milliliters of Erlenmeyer flasks that 50 milliliters of substratum are housed.Initial pH value of medium is set at respectively: 5.0,6.0,7.0,8.0,9.0; Culture temperature is made as respectively: 15 ℃, 25 ℃, 30 ℃, 30 ℃, 35 ℃, 37 ℃; Iron concentration is with FeSO 47H 2The O score is not made as 0 grams per liter, 0.005 grams per liter, 0.01 grams per liter, 0.02 grams per liter, 0.03 grams per liter; Carbon/nitrogen ratio was respectively 1: 10,1: 5,2: 1,5: 1,10: 1,20: 1.If do not inoculate control group, 3 repetitions.Cultivated 7 days, and 14 days, after 21 days, got 4 ℃ of centrifugal 15min of following 5000g of nutrient solution, the nitrite concentration in the Griess reagent method colorimetric estimation supernatant liquor.The results are shown in Table 7.1,7.2,7.3,7.4,7.5,7.6.
The growth of bacterial strain and nitrite accumulation (shaking table was cultivated 14 days) under the different initial pH conditions of table 7.1
Initial pH 5.0 6.0 7.0 8.0 9.0
OD 600 1.12 1.18 1.17 1.14 1.12
NO 2 - 59.51 70.53 80.16 83.98 84.54
Temperature is 30 ℃ during experiment, iron concentration 0.01g L -, carbon/nitrogen was than 2: 1.NO in the table 2 -With mg N L -1Expression
The growth of bacterial strain and nitrite accumulation under table 7.2 differing temps (static cultivation 21 days)
15 ℃ 25 ℃ 32 ℃ 35 ℃ 37 ℃ of culture temperature
OD 600 0.52 1.10 1.00 0.92 0.83
NO 2 - 4.45 17.43 29.91 10.0 0.031
PH 6.5 during experiment, iron concentration 0.01g L -, carbon/nitrogen was than 2: 1.NO in the table 2 -With mg N L -1Expression
The growth of bacterial strain and nitrite accumulation under table 7.3 differing temps (static cultivation 14 days)
28 ℃ 30 ℃ 35 ℃ of culture temperature
OD 600 0.98 0.98 0.87
NO 2 - 3.96 17.5 14.71
PH 6.5 during experiment, iron concentration 0.01g L -, carbon/nitrogen was than 2: 1.NO in the table 2 -With mg N L -1Expression
The growth of bacterial strain and nitrite accumulation under the different iron concentrations of table 7.4 (shaking table was cultivated 14 days)
FeSO 47H 2O counts (gL -1) 0 0.005 0.01 0.02 0.03
OD 600 0.50 0.87 1.11 0.94 0.90
NO 2 - 1.18 17.3 52.6 36.2 19.2
PH 6.5 during experiment, 30 ℃ of temperature, and carbon/nitrogen was than 2: 1.NO in the table 2 -With mg N L -1Expression
The cultivation situation of bacterial strain under the different carbon-nitrogen ratios of table 7.5 (30 ℃, static cultivation 28 days)
Carbon-nitrogen ratio 1: 10 1: 52: 14: 15: 1 10: 1 20: 1
OD 600 0.18 0.30 0.80 1.09 1.12 1.08 0.90
NO 2 - 0.55 0.80 10.55 26.10 5.57 2.03 1.56
NH 4 +％ 92.45％ 89.22％ 41.82％ 18.36％ 10.63％ 19.63％ 17.50％
T N％ 94.62％ 93.81％ 57.22％ 39.19％ 32.64％ 37.61％ 38.50％
Annotate: NO 2 -With mg N L -1Expression
Residual per-cent (the NH of ammonium nitrogen 4 +%)=connect the ammonium nitrogen concentration of bacterium culture/the do not connect ammonium nitrogen concentration of bacterium contrast
The complete residual per-cent (T of nitrogen N%)=connect the full nitrogen of bacterium culture/the do not connect full nitrogen of bacterium contrast
Each cultivation of organizing the different concns carbon source all has the minimizing of total nitrogen.Wherein concentration is C/N=4: 1, C/N=5: 1, C/N=10: denitrification effect was all fine in 1 o'clock, and with C/N=5: 1 the best: cultivated 28 days, ammonia-nitrogen removal rate is 90%, and full nitrogen removal efficiency is 68%, remaining full nitrogen be almost somatic cells, the nitrite of accumulation is at 5.57mg N L -1Below.C/N=4: 1 and C/N=10: it is close that 1 cultivation ammonia nitrogen and full nitrogen remove ability.
The optimum temps of Arthrobacter globiformis WR-2 bacterial strain generation nitrification is 30 ℃, best iron concentration 0.01g L -, best carbon/nitrogen is than 5: 1, best initial pH is pH9.0, when considering pH9.0 ammonia volatilization serious, the initial pH of subsequent experimental is decided to be pH7.0.
Embodiment: the denitrogenation in water body of 5 Arthrobacter globiformis WR-2
The denitrification effect when 5.1 the sodium acetate of different concns is carbon source
5.1.1 substratum is the NB substratum, is carbon source with the sodium acetate, concentration is 0.015mol L respectively -1, C/N=1: 1; 0.045mol L -1, C/N=3: 1; 0.075mol L -1, C/N=5: 1; 0.15mol L -1, C/N=10: 1.Compound method is with 1.3.
5.1.2 culturing process: cultivate in advance and connect a ring lawn from the PM inclined-plane and go into to be equipped with L with 0.015mol -1Sodium acetate is in 250 milliliters of Erlenmeyer flasks of 50 milliliters of NB substratum of carbon source, at 30 ℃, and 120-130r/min shaking table shaking culture 24 hours.By 2% amount inoculation, the bacterium liquid of cultivation in advance is connected to be equipped with the different concns sodium acetate be in 250 milliliters of Erlenmeyer flasks of 50 milliliters of NB substratum of carbon source for 1 milliliter.If do not inoculate contrast, 3 repetitions.30 ℃, static cultivation.Result such as table 8, table 9, Fig. 2, shown in Figure 3.
The growth and the nitrite accumulation of WR-2 bacterial strain when table 8 different concns sodium acetate is carbon source
(30 ℃, static cultivation 42 days)
Carbon-nitrogen ratio 1: 13: 15: 1 10: 1
OD 600 0.50±0.010 0.78±0.020 0.90±0.021 0.83±0.018
NO 2 - 7.26±0.050 0.41±0.098 0.12±0.020 0.09±0.016
NH 4 + 16.38±5.37 5.62±0.22 4.59±1.34
Full nitrogen 89.81 ± 1.99
Annotate: C 0With mol L -1Expression; Full nitrogen, NH 4 +, NO 2 -With mg N L -1Expression; Data representation: mean value ± standard error n=3.
Cultivation situation when table 9 different concns sodium acetate is carbon source (30 ℃, static cultivation)
Carbon-nitrogen ratio 1: 13: 15: 1
28 days 42 days 28 days 48 days 28 days 38 days
NO 2 - 1.19±0.29 4.15±1.33 0.074±0.021 0.41±0.19 0.094±0.010 0.090±0.016
NH 4 +％ - 42.08±6.16％ 37.4±1.90％ - 34.5±1.84％ 9.38±0.78％
T N％ 70.9±1.24％ 54.7±3.03％ 45.0±2.97％ 23.6±1.85％ 37.7±1.20％ 22.3±1.82％
Annotate: NO 2 -With mg N L -1Expression.
Residual per-cent (the NH of ammonium nitrogen 4 +%)=connect the ammonium nitrogen concentration of bacterium culture/the do not connect ammonium nitrogen concentration of bacterium contrast
The complete residual per-cent (T of nitrogen N%)=connect the full nitrogen of bacterium culture/the do not connect full nitrogen of bacterium contrast
Data representation: mean value ± standard error n=3.
Arthrobacter globiformis WR-2 has good ammonia nitrogen removal ability, and the measurement nitrite concentration only is 0.1mg N L in the biological denitrification process -1, the accumulation of no nitrite.With the sodium acetate concentration 0.075mol L wherein -1, C/N is 5: 1 and 0.15mol L -1, when C/N was 10: 1 cultivation, the ammonia nitrogen removal ability was strong, is being 0.045mol L with the sodium acetate concentration -1, C/N takes second place at 3: 1.
Embodiment: 6 WR-2 and nitrifier bacillus cereus NBB-135, the mixed culture of CGMCC No.0560
6.1 substratum is with 0.015mol L -1Sodium acetate is the NB substratum of carbon source, and compound method is with 1.3.
6.2 pre-the cultivation:
6.2.1 meet a ring bacillus cereus NBB-135 from the PM inclined-plane, the lawn of CGMCC No.0560 goes into to be equipped with the L with 0.015mol -1Sodium acetate is in 250 milliliters of Erlenmeyer flasks of 50 milliliters of NB substratum of carbon source, at 30 ℃, and 120-130r/min shaking table shaking culture 24 hours, transferring thalline OD value is OD 600=0.40.
Go into to be equipped with L 6.2.2 connect the lawn of a global shape Arthrobacter WR-2 from the PM inclined-plane with 0.015mol -1Sodium acetate is in 250 milliliters of Erlenmeyer flasks of 50 milliliters of NB substratum of carbon source, at 30 ℃, and 120-130r/min shaking table shaking culture 24 hours, transferring thalline OD value is OD 600=0.40.
6.3 cultivate: NBB-135 (culture of 6.2.1) and WR-2 bacterium (culture of 6.2.2) mix by 1: 1 (volume ratio), again with 2% 1 milliliter of access of inoculation mixed bacteria liquid of measuring with 0.075mol L -1Acetate be the NB substratum (prescription with 1.3) of carbon source at 30 ℃, static cultivation 28 days.Survey ammonia-state nitrogen with the indophenol blue colorimetry, Griess reagent colorimetric method for determining nitrite nitrogen, the Kelvin method is measured total nitrogen.
6.4 result: mixed culture has removed 62.3% of the full nitrogen of system after cultivating 28 days, the ammonium nitrogen removal efficiency is 78.1%, and nitrite concentration only is 0.52mg N L -1
Embodiment: 7 WR-2 and denitrifying bacteria plant bacillus pumilis WO-8, the mixed culture of CCTCC M202044
7.1 substratum is with 0.015mol L -1Sodium acetate is the NB substratum of carbon source, and compound method is with 1.3.
7.2 cultivate:
7.2.1 meet a ring plant bacillus pumilis WO-8 from the PM inclined-plane, the lawn of CCTCC M202044 goes into to be equipped with the L with 0.015mol -1Sodium acetate is in 250 milliliters of Erlenmeyer flasks of 50 milliliters of NB substratum of carbon source, at 30 ℃, and 120-130r/min shaking table shaking culture 24 hours, transferring thalline OD value is OD 600=0.40.
Go into to be equipped with L 7.2.2 connect the lawn of a global shape Arthrobacter WR-2 from the PM inclined-plane with 0.015mol -1Sodium acetate is in 250 milliliters of Erlenmeyer flasks of 50 milliliters of NB substratum of carbon source, at 30 ℃, and 120-130r/min shaking table shaking culture 24 hours, transferring thalline OD value is OD 600=0.40.
7.2.3 WO-8 (culture of 7.2.1) and WR-2 bacterium (culture of 7.2.2) mix by 1: 2 (volume ratio), the inoculum size with 2% with 1 milliliter of access of mixed bacteria liquid with 0.075mol L -1Acetate is the NB substratum (prescription is with 1.3) of carbon source, 30 ℃, and static cultivation 28 days.With indophenol blue colorimetry ammonia-state nitrogen, Griess reagent colorimetric method for determining nitrite nitrogen, the Kelvin method is measured total nitrogen.
7.3 result: mixed culture has removed 68.2% of the full nitrogen of system after cultivating 28 days, the ammonium nitrogen removal efficiency is 79.7%, and nitrite concentration only is 0.32mg N L -1
Embodiment: the denitrogenation of 8 immobilization Arthrobacter globiformis WR-2
8.1 the preparation of the fixed film of Arthrobacter globiformis WR-2
8.1.1 Arthrobacter globiformis WR-2 concentrates the preparation of thalline
Connect a ring lawn from the PM inclined-plane and go into to be equipped with L with 0.015mol -1Sodium acetate is in 250 milliliters of Erlenmeyer flasks of 50 milliliters of NB substratum of carbon source, at 30 ℃, and 120-130r/min shaking table shaking culture 24 hours.By 1% amount inoculation bacterium liquid is connected to 0.015mol L is housed -1Sodium acetate is in 500 milliliters of Erlenmeyer flasks of 150 milliliters of NB substratum of carbon source, at 30 ℃, and 120-130r/min shaking table shaking culture 72 hours.At 5000r/min, 4 ℃ centrifugal 15 minutes down, wash centrifugal twice with physiological saline after, be suspended in 15ml physiological saline.
8.1.2 Arthrobacter globiformis WR-2 fixes
Thalline be will concentrate and 20%PVA and 1.0mol L joined -1CaCl 2Mixing solutions in, be tiled on the poly (methyl methacrylate) plate after stirring evenly, place refrigerator ,-20 ℃ of freeze overnight are at room temperature thawed again.Repeated freezing thaws 3-4 time, has the distilled water thorough washing to show, promptly gets tabular fixation cell after birth.
8.1.3 fixed film reactor and denitrogenation experiment
With the blue fixing and assembling biological denitrification reactor of the fixation cell after birth usage of gained.It is 1600 milliliters that reactor is contained liquid measure, with 0.015mol L -1Acetate is the NB substratum of carbon source, and wherein ammonium nitrogen concentration is kept to 100mg N L -1, place 28 ℃ of constant incubators to activate, treat that cytoactive is stable after, carry out the denitrogenation experiment.In the experimentation, the control dissolved oxygen concentration is the 5-8 mg/litre.Sample analysis ammonium nitrogen, nitrite nitrogen and nitric nitrogen concentration wherein at regular intervals takes a morsel.The result is presented at and cultivates after 16 days, and the ammonium nitrogen removal efficiency is 92.7%, and nitrite concentration only is 0.22mg N L -1
Embodiment: 9 Arthrobacter globiformis WR-2 are as pattern bacterium screening nitrification inhibitor.
9.1 different concns ATC is to the influence of WR-2 bacteria growing and nitrification activity
Go into to be equipped with L 9.1.1 connect a ring lawn with 0.020mol from the PM inclined-plane -1Pyruvic acid is in 250 milliliters of Erlenmeyer flasks of 50 milliliters of NB substratum of carbon source, at 30 ℃, and 120-130r/min shaking table shaking culture 24 hours.By 2% amount inoculation bacterium liquid is connected in 250 milliliters of Erlenmeyer flasks that 50 milliliters of NB substratum are housed.If do not inoculate contrast, 3 repetitions.ATC establishes 4 concentration gradients and is respectively 0,15mg L -1, 75mg L -1, 150mg L -1ATC, carbon source are sterilized separately, mix before using.At 30 ℃, static cultivation.
9.1.2 result such as Fig. 4, Fig. 5.The result shows, at 0-75mg L -1Concentration range in, ATC is to growth and the nitrification activity unrestraint effect of heterotrophic nitrification active bacterial strain WR-2.At 150mg L -1During high density, ATC suppresses the growth and the nitrification activity of bacterial strain.
8.2 vitamins C is to the influence of WR-2 bacterial growth and nitrification activity
Go into to be equipped with L 8.2.1 connect a ring lawn with 0.015mol from the PM inclined-plane -1Sodium acetate is in 250 milliliters of Erlenmeyer flasks of 50 milliliters of NB substratum of carbon source, at 30 ℃, and 120-130r/min shaking table shaking culture 24 hours.By 2% amount inoculation, bacterium liquid is connected in 250 milliliters of Erlenmeyer flasks that 50 milliliters of NB substratum are housed.If do not inoculate contrast, 3 repetitions.Vitamins C concentration is 2.17 grams per liters; Vitamins C, carbon source are sterilized separately, mix before using.30 ℃, static cultivation.
9.2.2 the result is as shown in table 11:
Table 11. adds Vc and does not add Vc the growth and the nitrite accumulation of bacterial strain when cultivating
(30 ℃, static cultivation 21 days)
The mensuration project is added Vc contrast (not adding Vc)
OD 600 0.020 0.51
NO 2 - 0.12 7.10
Annotate: NO 2 -With mg N L -1Expression
Bacterial strain does not have growth, no nitrification activity, but show that Vc concentration is the growth and the nitrification activity of 2.17 grams per liter strongly inhibited bacterial strains.
Bacterium protects catalogue
Bacterial strain number
WR-2 CCTCC M202043 Arthrobacter globiformis WR-2 Arthrobacter globiformis
WO-8 CCTCC M202044 Curtoacterium plantarum WO-8 plant bacillus pumilis
The cured shape genus bacillus of NBB135 CGMCC NO.0560 Bacillus cereus NBB-135

Claims (8)

1. the bacterium with denitrogenation biologic activity is characterized in that this bacterium is Arthrobacter globiformis (Arthrobacter globiformis) WR-2, and its preservation registration number is CCTCC M202043.
2. method of cultivating bacterium described in the claim 1, this method are to be 5.0-9.0 at a) pH; B) temperature is 15-35 ℃; C) iron concentration is with FeSO 47H 2O counts the 0.005-0.02 grams per liter; D) carbon/nitrogen ratio is 1: 10-20: culturing bacterium under 1 condition.
3. method according to claim 2, this method are to be 6.0-8.0 at a) pH; B) temperature is 20-35 ℃; C) iron concentration is with FeSO 47H 2O counts the 0.01-0.02 grams per liter; D) carbon/nitrogen ratio is 1: 1-10: culturing bacterium under 1 condition.
4. method according to claim 3, this method are to be 7.0 at a) pH; B) temperature is 30 ℃; C) iron concentration is with FeSO 47H 2O counts 0.01 grams per liter; D) carbon/nitrogen ratio is a culturing bacterium under 5: 1 conditions.
5. the bacteria composition of a denitrogenation in water body, contain significant quantity a kind of described in claim 1 bacterium and the water body biological denitrificaion in receivable carrier.
6. the application of bacterium in the water body biological denitrificaion described in the claim 1.
7. the application of bacteria composition in the water body biological denitrificaion in the claim 5.
8. the application of bacterium described in the claim 1 in the screening nitrification inhibitor.
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