CN103497908B - Pseudomonas stutzeri and cultivation, immobilization and application - Google Patents
Pseudomonas stutzeri and cultivation, immobilization and application Download PDFInfo
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Abstract
The invention discloses a strain Pseudomonas stutzeri and cultivation, immobilization and application.This bacterial strain is Pseudomonas stutzeri (Pseudomonas stutzeri) WZUF25, is preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center, registers on the books and be numbered CGMCC NO.7685 in preservation center.This bacterial strain carries out the appropriate pH of aerobic denitrification and temperature range is wide, not only to NO
3 --N and NO
2 -the clearance of-N is high, and has very high assimilation NH
4 +-N ability, can be simultaneous nitrification and denitrification and provides provenance.The immobilization Pseudomonas stutzeri that the present invention provides employing gac-calcium alginate embedded legal system standby simultaneously, it has good removal NO equally
3 -the ability of-N and stability.
Description
Technical field
The invention belongs to field of environment microorganism, be specifically related to a strain Pseudomonas stutzeri and cultivation, immobilization and application.
Background technology
The pollution problem that nitrogen causes to environment becomes increasingly conspicuous in recent years, and its hazardness is also day by day familiar with by people and is paid attention to.As ammonia nitrogen, nitrate nitrogen and nitrite nitrogen are likely converted into the nitrosamine of carcinogenic, mutagenesis and teratogenesis; And for example nitrogen flows into water body and causes body eutrophication, causes water quality deterioration and even lake to degenerate.Biological denitrificaion has that treatment effect is good, treating processes is reliable and stable, the advantage of convenient operation and management etc. and being used widely.
Tradition denitrogenation theory thinks that wastewater biological denitrificaion must be that ammonium nitrogen experiences typical nitrification and denitrification process, namely first the ammonium nitrogen in waste water is oxidized to nitrite nitrogen, nitrate nitrogen by ammonia oxidizing bacteria and nitrite-oxidizing bacteria successively under aerobic condition, then under anaerobism or double oxygen condition, nitrate nitrogen is reduced to nitrite nitrogen, nitrogen or oxynitride successively by denitrifying bacterium.Denitrifying bacteria on traditional understanding preferentially utilizes oxygen as electron acceptor(EA) under aerobic conditions, only has nitrate under anoxic conditions that oxygen just can be replaced to carry out denitrification as final electron acceptor(EA).
The eighties in 20th century, Robertson and Kuene is separated to aerobic denitrifying bacteria first in sulphur removal and denitrification treatment system
thiosphaera pantotropha(now rename as
paracoccus pantotrophus),
pseudomonasspp. and
alcaligenes faecalisso far the existing separated acquisition of many aerobic denitrifying bacterias, the aerobic denitrifying bacteria now reported is distributed in soil, the common kinds of waste water such as Rhodopseudomonas (Pseudomonas), bacillus (Bacillus) and Alcaligenes (Alcaligenes) mostly, and finds that aerobic denitrifying bacteria shows the characteristic of Heterotrophic nitrification mostly.But the bacterial strain be separated from varying environment, its physiological property and the ability of denitrogenating differ from one another, and they can be practical application or further strain improvement provides abundant provenance.As Bruce Lee etc. be separated to from the water sample of fish pond 1 strain acinetobacter calcoaceticus (
acinetobacter sp.), with KNO
3, (NH
4)
2sO
4, NaNO
2for in the nutrient solution of only nitrogen source, can by NO in nutrient solution in 24 h
3 --N is from 161. 61 mg L
-1be down to 55. 69 mg L
-1, removing speed is 4.41 mgL
-1h
-1nO
3 --N; By NH in 15 h
4 +-N is by 220. 24 mgL
-1be down to 14.78 mg L
-1, removing speed is 13.70 mgL
-1h
-1nH
4 +– N; NO in 12 h
2 --N concentration is by 101. 27 mg L
-1be down to 21. 85 mgL
-1, removal speed is 6.62mgL
-1h
-1nO
2 --N; But the appropriate pH of its denitrogenation is meta-alkalescence; Do not grow when 20 DEG C, at NH when 40 DEG C
4 +-N measures poor growth in liquid, at NO
2 --N measures in liquid and does not grow, and optimum growth temp is 30 DEG C (microorganism journal, 2011,51(8): 1062-1070).
Traditional biological denitrificaion theory has been broken in the discovery of aerobic denitrification and heterotrophic nitrification, makes simultaneous nitrification and denitrification become possibility.Up to now, mechanism for heterotrophic nitrification, aerobic denitrification still needs to continue further investigation, also little to the application of nitrification bacteria and aerobic denitrifying bacteria, but because of its unique advantage in denitrogenation, will be widely used in the long term, therefore the bacterial strain of seed selection premium properties provides provenance to have important practical significance for practical application.
Immobilized cell technology is used for biological wastewater treatment compared with traditional suspended biological facture, and energy purifying and maintenance high-efficiency strain, microorganism concn is high, and sludge yield is few, good effect of separating solid from liquid.Therefore, this technology, in biological wastewater treatment, especially in extraordinary water treatment field, obtains and studies widely.Fixing
Change cell technology for the removal of BOD material, nitrification-denitrification, dephosphorization, the degraded of removing phenol, cyanogen, LAS degraded, the removal of heavy metal ion and the desolventing technology etc. of recovery and dyeing waste water.In recent years, immobilized nitrobacteria denitride technology enters large-scale industrial experimentation from laboratory and bench-scale testing stage
Stage.At present, immobilization aerobic denitrifying bacteria denitride technology is also in laboratory and bench-scale testing stage.
Summary of the invention
In order to solve the problems of the technologies described above, the invention provides a strain aerobic denitrifying bacteria, its feature is this bacterial strain is adopt conventional separation methods to be separated from the active sludge picking up from Wenzhou District of Zhejiang Province western movie sewage work to obtain, through 16SrDNA sequencing sequencing result compared by GeenBank Blast, with
pseudomanos stutzerihomology be 99.9%, compile and be
p. stutzeriwZUF25.
A strain Pseudomonas stutzeri provided by the invention, this bacterial strain be Pseudomonas stutzeri (
pseudomonas stutzeri) WZUF25, being preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center, registers on the books and is numbered CGMCC NO.7685 in preservation center.
The invention provides the cultural method of above-mentioned Pseudomonas stutzeri, comprise the steps:
1) preservation strain WZUF25 is inoculated in LB substratum, cultivates 12 more than h, centrifugal, obtains thalline, makes with after sterilized water washing
oD 680it is the bacteria suspension of 0.900 ~ 1.000;
2) step 1) gained bacterial suspension inoculation is cultivated in denitrification substratum;
Wherein, the formation of described denitrification substratum is: carbon source, nitrogenous source, K
2hPO
4, FeSO
4, MgSO
4, H
2o, described carbon source is Soduxin, sodium acetate, glycerine or glucose, and described nitrogenous source is the compound containing nitrate ion.
Preferably, step 2) described in the formula of denitrification substratum be: carbon source, the quality 1.228g of nitrate radical, K in nitrogenous source
2hPO
41g, FeSO
47H
2o 0.20g, MgSO
47H
2o 0.10 g, H
2o 1000 mL, pH are 5 ~ 9, the NO of described carbon source and nitrogenous source
3 -mass ratio be 5:1.228 ~ 15:1.228.
As optimal technical scheme, step 1) preservation strain WZUF25 is inoculated in LB substratum, in 20 ~ 40 DEG C, and dissolved oxygen 3.5 ~ 6.1 mgL
-1condition under cultivate; Step 2) in gained bacterial suspension inoculation in denitrification substratum, in 25 ~ 40 DEG C, dissolved oxygen 3.5 ~ 6.1 mgL
-1lower cultivation.
As optimal technical scheme, step 2) described in the pH 5 ~ 9 of denitrification substratum.
Preferably, described bacteria suspension with 5% volume ratio be inoculated in denitrification substratum.
The invention provides the application of above-mentioned Pseudomonas stutzeri, it is characterized in that, described Pseudomonas stutzeri WZUF25 is inoculated in the nitrogenous aqueous solution, carries out denitrogenation.
As optimal technical scheme, the described nitrogenous aqueous solution is for containing NH
4 +, NO
3 -and NO
2 -a kind of or its combination aqueous solution, described Pseudomonas stutzeri WZUF25 takes off NO
3 --N, NH
4 +-N and NO
2 -the carbon source of-N contains one of them or its combination of sodium acetate, Soduxin, glycerine or glucose.Nitrogenous source in the nitrogenous aqueous solution is only NH
4 +during-N, NH in carbon source and nitrogenous effluent
4 +mass ratio be preferably 5:0.0.34 ~ 15:0.34; Nitrogenous source in the nitrogenous aqueous solution is only NO
3 -time, NO in carbon source and nitrogenous effluent
3 -mass ratio be 5:1.228 ~ 15:1.228; Nitrogenous source in the nitrogenous aqueous solution is only NO
2 -during-N, NO in carbon source and nitrogenous effluent
2 -mass ratio be 5:0.67 ~ 15:0.67.
As optimal technical scheme, described nitrogenous aqueous solution pH is 4 ~ 10, and described Pseudomonas stutzeri WZUF25 is in temperature 20 DEG C ~ 45 DEG C, and dissolved oxygen is 1.3 ~ 7.3mgL
-1condition under denitrogenation is carried out to the nitrogenous aqueous solution.
The invention provides the method that microcapsule are prepared in above-mentioned Pseudomonas stutzeri immobilization, step is as follows:
1) bacterial strain WZUF25 is centrifugal after being inoculated in and cultivating in LB substratum obtains thalline, and thalline is with distilled water wash, and thalline mixes with the sodium alginate soln being dispersed with active carbon powder;
2) CaCl is prepared
2in the aqueous solution, after 37 DEG C of water-bath 10 ~ 30min, step 1) gained mixed solution is added dropwise to the CaCl of temperature 37 DEG C
2in the aqueous solution, obtain microcapsule.
The application of above-mentioned microcapsule: described microcapsule are inoculated in the nitrogenous aqueous solution, carry out denitrogenation.
Preferably, the described nitrogenous aqueous solution is for containing NO
3 -and NO
2 -a kind of or its combination aqueous solution, the microcapsule of described immobilization Pseudomonas stutzeri WZUF25 take off NO
3 --N, NH
4 +-N and NO
2 -the carbon source of-N contains one of them or its combination of sodium acetate, Soduxin, glycerine or glucose.Nitrogenous source in the nitrogenous aqueous solution is only NO
3 -time, NO in carbon source and nitrogenous effluent
3 -mass ratio be 5:1.228 ~ 15:1.228; Nitrogenous source in the nitrogenous aqueous solution is only NO
2 -time, NO in carbon source and nitrogenous effluent
2 -mass ratio be 5:0.67 ~ 15:0.67.
As optimal technical scheme, described nitrogenous aqueous solution pH is 4 ~ 10, and the microcapsule of described immobilization Pseudomonas stutzeri WZUF25 are in temperature 20 DEG C ~ 45 DEG C, and dissolved oxygen is 1.3 ~ 7.3mgL
-1condition under denitrogenation is carried out to the nitrogenous aqueous solution.
The present invention can reach following technique effect:
1, Pseudomonas stutzeri (
pseudomanos stutzeri) WZUF25 carries out the appropriate pH of aerobic denitrification and temperature range is wide, not only to NO
3 --N and NO
2 -the clearance of-N is high, and has higher assimilation NH
4 +-N ability, can be simultaneous nitrification and denitrification and provides provenance.The immobilization Pseudomonas stutzeri simultaneously providing employing gac-calcium alginate embedded legal system standby removes NO
3 -the ability of-N and stability.
2, the present invention provides denitrification substratum and the cultural method of applicable bacterial strain WZUF25 cultivation after deliberation, can cultivate and obtain a large amount of thalline.
3, under the best denitrogenation condition of bacterial strain WZUF25, NO
3 --N and NO
2 -the clearance of-N can reach more than 99%; NH
4 +-N clearance can reach about 60%.
4, utilize bacterial strain of the present invention effectively to process waste water, reduce waste water COD, and denitrification effect is good.
5, bacterial strain of the present invention has NO equally through the microcapsule that immobilization is obtained
3 --N and NO
2 -the feature that the clearance of-N is high.
Accompanying drawing explanation
Fig. 1 adopts MEGA4.1 software, ortho position connection method display bacterial strain WZUF25 grows to relevant 16S rDNA sequential system of planting and sets, carry out the similarity double counting of 1000 times, grow tree node in figure and only show Bootstrap value and be greater than 50% numerical value, upper target " T " intermediate scheme bacterial strain (
p.,
pseudomonas).
Fig. 2 is the NH that bacterial strain WZUF25 removes artificial preparation
4 +-N sewage process.
Fig. 3 is the NO that bacterial strain WZUF25 removes artificial preparation
3 --N sewage process.
Fig. 4 is the NO that bacterial strain WZUF25 removes artificial preparation
2 --N sewage process.
Fig. 5 is the NO that gac-calcium alginate embedded bacterial strain WZUF25 removes artificial preparation
3 --N sewage process.
Bacterial strain preservation
Pseudomonas stutzeri of the present invention (
pseudomanos stutzeri) WZUF25, be preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center, preservation address: No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City, register on the books and be numbered CGMCC NO.7685 in preservation center, preservation from date is on June 8th, 2013.
Embodiment
Below in conjunction with the drawings and specific embodiments, the invention will be further described, can better understand the present invention and can be implemented, but illustrated embodiment is not as a limitation of the invention to make those skilled in the art.
Embodiment one: the separation of aerobic denitrifying bacteria
Sample is the active sludge taking from Wenzhou District of Zhejiang Province western movie sewage work, adopts conventional separation technique, by one
Qualitative activity sludge seeding is in enrichment medium (KNO
31g, Soduxin 5g, KH
2pO
41g, FeSO
47H
2o 0.20g, MgSO
47H
2o 0.10g, H
2o 1000mL, pH 7.2-7.6) under 30 DEG C of 160 ~ 180 rpm, carry out enrichment culture 3 ~ 4 days, the enrichment medium of transferring new after growing bacterium continues enrichment culture, so repeats 4 ~ 5 times.Then the bacterium liquid through suitably dilution is coated isolation medium (agar 18gL
-1, the same enrichment medium of other compositions) and at 30 DEG C, cultivate 48 h, picking list bacterium colony new isolation medium of transferring carries out line purifying, until be pure growth through microscopy, storage medium of then transferring (yeast extract paste 5g, peptone 10g, NaCl 10g, agar 18g, H
2o 1000 mL, pH 7.0) at 30 DEG C, cultivate rear preservation.
Preservation strain is inoculated in denitrification substratum (Soduxin 10gL respectively
-1, the same enrichment medium of other compositions) and under 30 DEG C of 160 ~ 180 rpm, cultivate (the bottled substratum 100mL of 250mL taper), timing sampling detects NO with Griess reagent I and II and pentanoic respectively
2 --N and NO
3 --N, according to NO
2 -the generation of-N and degraded situation and NO
3 -the degraded situation of-N, preliminary screening is carried out to the aerobic denitrification capability of preservation strain, denitrification capability mensuration is carried out to the aerobic denitrifying bacteria that preliminary screening obtains, measuring method carries out (eastern elegant pearl by document, common bacteria system identification handbook, Beijing: Science Press, 2001), to determine aerobic denitrification capability further.
The bacterial strain with aerobic denitrification capability that primary dcreening operation obtains is carried out multiple sieve, and method is in denitrification substratum (Soduxin 10gL by inoculation
-1, the same enrichment medium of other compositions) and under 30 DEG C of 160 ~ 180 rpm, cultivate the bottled substratum 100mL of 24 h(250mL taper), under 8000rpm, then after centrifugal 10min, measure the NO of supernatant liquor
2 --N concentration and NO
3 --N concentration, calculates NO
3 -the clearance of-N and NO
2 -the accumulation of-N, screening NO
3 --N removal ability is strong, NO
2 --N accumulates the low strain excellent even do not accumulated.
Obtain the excellent aerobic denitrifying bacteria of 1 strain through aforesaid method, compile as WZUF25.
NO
3 --N measures and adopts disulfonic acid phenol spectrophotometry, NO
2 --N measures and adopts N-(1-naphthyl)-quadrol light-intensity method (State Bueau of Environmental Protection. water and waste water method for monitoring and analyzing (third edition). Beijing: China Environmental Science Press, 1989).
NO
3 --N clearance (%)=(supernatant liquor NO before cultivating
3 -supernatant liquor NO after-N concentration-cultivation
3 --N concentration)/cultivate front supernatant liquor NO
3 --N concentration × 100%
Embodiment two: the qualification of aerobic denitrifying bacteria
After isolation medium cultivates 48h, bacterium colony is rounded, translucent, rough, edge is irregular, bacterium colony is faint yellow, mycetocyte rod-short, size 0.7 ~ 0.8 × 1.5 ~ 3.2 μm for bacterial strain WZUF25, without gemma, and Gram-negative, one pole flagellum.
With bacterial genomes DNA for template amplification 16SrDNA, amplification employing a pair universal primer: upstream primer (P1): 5 '-AGAGTTTGATCCTGGTCAGAACGAACGCT-3 ', downstream primer (P6): 5 '-TACGGCTACCTTGTTACGACTTCACCCC-3 ', purifying and the order-checking of PCR primer are completed by Shanghai biotechnology company limited, and sequencing result is compared by GeenBank Blast.With in GeenBank Rhodopseudomonas (
pseudmonas sp.) 16SrDNA sequence there is very high homology, with
p. stutzerihomology be 99.9%.Adopt MEGA4.1 software, ortho position connection method display bacterial strain WZUF25 grows tree to relevant 16S rDNA sequential system of planting and sees Fig. 1.
Pseudomonas stutzeri (
pseudmonas stutzeri) WZUF25, being preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center, registers on the books and is numbered CGMCC NO. 7685 in its preservation center, and preservation from date is on June 8th, 2013.
Embodiment three: bacterial strain WZUF25 aerobic denitrification removes NO
3 -the characteristic of-N
Adopt one-factor experiment method, research bacterial strain WZUF25 carries out aerobic denitrification and removes NO
3 -the characteristic of-N.Experimentation is: be inoculated in and 200mL LB substratum (yeast extract paste 5g, peptone 10g, NaCl 10g, H are housed by preservation strain (the bacterium liquid that 2.0mL freeze pipe melts)
2o 1000 mL, pH 7.0) 500mL Erlenmeyer flask in, 30 DEG C, cultivate 24h under 160rpm, under 8000 rpm, centrifugal 10min obtains thalline, and with sterile distilled water washing thalline 2 times, with sterilized water make bacteria suspension (
oD 6800.900 ~ 1.000).
Then bacteria suspension is transferred in 100mL denitrification substratum (carbon source, KNO are housed by the inoculum size of 5% volume ratio
3, K
2hPO
41g, FeSO
47H
2o 0.20g, MgSO
47H
2o 0.10 g, H
2o 1000mL, pH 4 ~ 10) 250mL Erlenmeyer flask in, under the certain rotating speed (0 ~ 250rpm) of certain temperature (20 ~ 45 DEG C), cultivate 24 h, under 8000rpm, after centrifugal 10min, measure the NO of supernatant liquor
2 --N concentration and NO
3 --N concentration, calculates NO
3 -the clearance of-N and NO
2 -the accumulation of-N.
NO
2 --N and NO
3 -the mensuration of-N is with embodiment one, and the 250mL Erlenmeyer flask that being determined as of dissolved oxygen is equipped with 100mL substratum vibrates and measures the DO value of substratum after 24h under certain temperature, certain rotating speed.
NO
3 --N clearance calculates with embodiment one.
Main discussion carbon source, carbon source and KNO
3weight ratio, temperature, pH and dissolved oxygen NO is removed to bacterial strain WZUF25
3 -the impact of-N, result is as shown in table 1 ~ table 5.
1, carbon source removes NO to bacterial strain WZUF25
3 -the impact of-N
Bacteria suspension is transferred in 100mL denitrification substratum (carbon source 10g, KNO are housed by the inoculum size of 5% volume ratio
32.0g, K
2hPO
41g, FeSO
47H
2o 0.20g, MgSO
47H
2o 0.10 g, H
2o 1000mL, pH 7.0) 250mL Erlenmeyer flask in, in temperature 30 DEG C, rotating speed 150rpm(dissolved oxygen 4.9mgL
-1) under cultivate 24 h, measure the NO of supernatant liquor under 8000rpm after centrifugal 10min
2 --N concentration and NO
3 --N concentration, calculates NO
3 -the clearance of-N and NO
2 -the accumulation of-N.
Table 1 carbon source removes NO to bacterial strain WZUF25
3 -the impact of-N
As shown in Table 1, the optimum carbon source of bacterial strain WZUF25 aerobic denitrification is Soduxin and sodium acetate,
When they are carbon source, NO
3 --N clearance more than 90%, and without NO
2 -the accumulation of-N.Secondly be glycerine and glucose, when they are carbon source, NO
3 --N clearance more than 60%, almost without NO
2 -the accumulation of-N.
2, carbon source and KNO
3weight ratio to bacterial strain WZUF25 remove NO
3 -the impact of-N
Bacteria suspension is transferred in 100mL denitrification substratum (carbon source 2 ~ 15 g, KNO are housed by the inoculum size of 5% volume ratio
32.0g, K
2hPO
41g, FeSO
47H
2o 0.20g, MgSO
47H
2o 0.10g, H
2o 1000mL, pH 7.0) 250mL Erlenmeyer flask in, in temperature 30 DEG C, rotating speed 150rpm(dissolved oxygen 4.9mgL
-1) under cultivate 24 h, measure the NO of supernatant liquor under 8000rpm after centrifugal 10min
2 --N concentration and NO
3 --N concentration, calculates NO
3 -the clearance of-N and NO
2 -the accumulation of-N.
Table 2 carbon source and KNO
3weight ratio to bacterial strain WZUF25 remove NO
3 -the impact of-N
As shown in Table 2, along with carbon source and KNO
3weight ratio increase to NO
3 -the clearance of-N all increases, but declines again more than after 10:2.Soduxin and the optimum carbon source of glycerine and KNO
3weight ratio be 10:2, glucose and sodium acetate are 5:2 ~ 10:2.
2gL
-1kNO
3denitrification substratum be equivalent to containing 1.228 gL
-1nO
3 -, therefore succinic acid and glycerine and NO
3 -optimum mass ratio is 10:1.228, glucose and sodium acetate and NO
3 -optimum mass ratio is 5:1.228 ~ 10:1.228.
3, temperature removes NO to bacterial strain WZUF25
3 -the impact of-N
Bacteria suspension is transferred in 100mL denitrification substratum (Soduxin 10g, KNO are housed by the inoculum size of 5% volume ratio
32.0g, K
2hPO
41g, FeSO
47H
2o 0.20g, MgSO
47H
2o 0.10 g, H
2o 1000mL, pH 7.0) 250mL Erlenmeyer flask in, in temperature 20 ~ 45 DEG C, rotating speed 150rpm(dissolved oxygen 4.9mgL
-1) under cultivate 24 h, measure the NO of supernatant liquor under 8000rpm after centrifugal 10min
2 --N concentration and NO
3 --N concentration, calculates NO
3 -the clearance of-N and NO
2 -the accumulation of-N.
Table 3 temperature removes NO to bacterial strain WZUF25
3 -the impact of-N
As shown in Table 3, within the scope of 25 DEG C ~ 40 DEG C, bacterial strain WZUF25 is to NO
3 -the clearance of-N is similar, all more than 90%, 20 DEG C or 45 DEG C time to NO
3 -the clearance of-N declines, and within the scope of 20 DEG C ~ 45 DEG C, almost all without NO
2 -the accumulation of-N.
4, pH removes NO to bacterial strain WZUF25
3 -the impact of-N
Bacteria suspension is transferred in 100mL denitrification substratum (Soduxin 10g, KNO are housed by the inoculum size of 5% volume ratio
32.0g, K
2hPO
41g, FeSO
47H
2o 0.20g, MgSO
47H
2o 0.10 g, H
2o 1000mL, pH 4 ~ 10, pH NaOH or HCl regulate) 250mL Erlenmeyer flask in, in temperature 30 DEG C, rotating speed 150rpm(dissolved oxygen 4.9mgL
-1) under cultivate 24 h, measure the NO of supernatant liquor under 8000rpm after centrifugal 10min
2 --N concentration and NO
3 --N concentration, calculates NO
3 -the clearance of-N and NO
2 -the accumulation of-N.
Table 4 pH removes NO to bacterial strain WZUF25
3 -the impact of-N
As shown in Table 4, in the scope of pH5 ~ 9, bacterial strain WZUF25 is to NO
3 -the clearance of-N is close, and all without NO
2 -the accumulation of-N; To NO when pH 4 or 10
3 -the clearance of-N declines.
5, dissolved oxygen removes NO to bacterial strain WZUF25
3 -the impact of-N
Bacteria suspension is transferred in 100mL denitrification substratum (Soduxin 10g, KNO are housed by the inoculum size of 5% volume ratio
32.0g, K
2hPO
41g, FeSO
47H
2o 0.20g, MgSO
47H
2o 0.10g, H
2o 1000mL, pH 7.0) 250mL Erlenmeyer flask in, control dissolved oxygen in temperature 30 DEG C, rotating speed 0 ~ 250rpm(by rotating speed) under cultivate 24 h, measure the NO of supernatant liquor under 8000rpm after centrifugal 10min
2 --N concentration and NO
3 --N concentration, calculates NO
3 -the clearance of-N and NO
2 -the accumulation of-N.
Table 5 dissolved oxygen removes NO to bacterial strain WZUF25
3 -the impact of-N
As shown in Table 5, dissolved oxygen is 1.3mgL
-1time, bacterial strain WZUF25 is to NO
3 -the clearance of-N is very low; Along with dissolved oxygen increases, to NO
3 -the clearance of-N increases thereupon; Dissolved oxygen is 3.5 ~ 6.1 mgL
-1time, to NO
3 -the clearance of-N is close, and without NO
2 -the accumulation of-N; Dissolved oxygen is 7.3 mgL
-1time, NO
3 -the clearance of-N starts to decline.
Embodiment four: bacterial strain WZUF25 removes the NO of artificial preparation
3 --N, NO
2 --N and NH
4 +-N sewage process
Preservation strain (2.0 mL freeze pipes melt bacterium liquid) is inoculated in the 500mL Erlenmeyer flask that 200mL LB substratum (same embodiment three of filling a prescription) is housed, 30 DEG C, cultivate 24h under 160rpm, under 8000 rpm, centrifugal 10min obtains thalline, after sterile distilled water washing thalline 2 times, with sterilized water make bacteria suspension (
oD 6800.900 ~ 1.000).The NH of artificial preparation is inoculated in by the inoculum size of 5% volume ratio
4 +-N sewage (NH
4cl 1g, Soduxin 10g, K
2hPO
41g, FeSO
47H
2o 0.20g, MgSO
47H
2o 0.10 g, H
2o 1000 mL, pH 7.0), NO
3 --N sewage (Soduxin 10g, KNO
32.0g, K
2hPO
41g, FeSO
47H
2o 0.20g, MgSO
47H
2o 0.10 g, H
2o 1000 mL, pH 7.0) and NO
2 --N sewage (Soduxin 10g, NaNO
21.0g(nitrite anions content 0.67g), K
2hPO
41g, FeSO
47H
2o 0.20g, MgSO
47H
2o 0.10 g, H
2o 1000 mL, pH 7.0), in 30 DEG C of 150 rpm(DO value 4.3 mgL
-1) under cultivate, timing sampling measure biomass (
oD 680), under 8000rpm, then after centrifugal 10min, measure the NH of supernatant liquor
4 +-N concentration, NO
2 --N concentration and NO
3 --N concentration, calculates NH
4 +-N, NO
3 --N and NO
2 -the clearance of-N, result is as Fig. 2 ~ Fig. 4.
NO
2 --N and NO
3 -the mensuration of-N and NO
3 --N clearance calculates with embodiment one.
NH
4 +-N mensuration employing reagent colorimetric method (State Bueau of Environmental Protection. water and waste water method for monitoring and analyzing (third edition). Beijing: China Environmental Science Press, 1989).
NH
4 +-N clearance (%)=(supernatant liquor NH before cultivating
4 +supernatant liquor NH after-N concentration-cultivation
4 +-N concentration)/cultivate front supernatant liquor NH
4 +-N concentration × 100%
NO
2 --N clearance (%)=(supernatant liquor NO before cultivating
2 -supernatant liquor NO after-N concentration-cultivation
2 --N concentration)/cultivate front supernatant liquor NO
2 --N concentration × 100%
Found out by Fig. 2, bacterial strain WZUF25 can by 1gL in 19 h
-1nH
4nH contained by Cl
4 +-N(is after measured on average containing 0.260 mgmL
-1nH
4 +-N) remove 59.79%, removing speed is 8.18 mgL
-1h
-1nH
4 +-N, its biomass, also at this moment close to stable, grow and removes NH
4 +-N is synchronous.But in the process removed, do not form NO
2 --N or NO
3 --N, therefore bacterial strain WZUF25 does not have heterotrophic nitrification performance, only assimilates NH4
+-N, absorbs ammonia nitrogen.
As can be seen from Figure 3, bacterial strain WZUF25 can by 2gL in 12 h
-1kNO
3contained NO
3 --N(is after measured on average containing 0.205 mgmL
-1nO
3 --N) remove completely, removing speed is 17.08 mgL
-1h
-1nO
3 --N, now biomass also reaches stable, growth and NO
3 -it is synchronous that-N removes.NO
2 -the 9h that is accumulated in of-N peaks, to during 12h close to zero.
Found out by Fig. 4, bacterial strain WZUF25 can by 1gL in 12 h
-1naNO
2contained NO
2 --N(is after measured containing 0.200 mgmL
-1nO
2 --N) remove 98.58%, removing speed is 16.43 mgL
-1h
-1nO
2 --N, now biomass also reaches stable, growth and NO
2 -it is synchronous that-N removes.
Embodiment five: gac-calcium alginate embedding immobilized method bacterial strain WZUF25 and immobilized cell remove the NO of artificial preparation
3 -the process of-N sewage
Preservation strain WZUF25(2mL freeze pipe is melted bacterium liquid) be inoculated in the 500mL Erlenmeyer flask that 200mL LB substratum (same embodiment three of filling a prescription) is housed, 30 DEG C, cultivate 24h under 160rpm, under 8000 rpm, centrifugal 10min obtains thalline, and washs thalline 2 times with sterile distilled water.
2.5g wet thallus is suspended from 5mL sterile distilled water, adds 5mL mass percent concentration 4% sodium alginate aqueous solution (being dispersed with mass percent 1% Powdered Activated Carbon in the aqueous solution) and fully mix.200mL mass percent concentration 4% CaCl is added in triangular flask
2solution, stretches into the tampon of scalp intravenous needle by triangular flask bottleneck in triangular flask, and is connected with syringe, is immersed 10min in 37 DEG C of water-baths.Moved into by sodium alginate thalline suspension in syringe, appropriateness is afterburning, and sodium alginate thalline suspension is instilled 4% CaCl
2in solution.Remove place 1h hypsokinesis in triangular flask immigration 20 ~ 22 DEG C of water-baths to solution after dripping off, add after 200mL sterile distilled water washs 1 time and rejoin 200mL 4% CaCl
2solution, 4 DEG C of equilibrate overnight, obtained gac-calcium alginate microcapsule.
The NO that 50mL manually prepares about 50 microcapsule accesses is equipped with
3 --N sewage (Soduxin 10g, KNO
32.0g, K
2hPO
41g, FeSO
47H
2o 0.20g, MgSO
47H
2o 0.10 g, H
2o 1000 mL, pH 7.0) Erlenmeyer flask in, at 30 DEG C of 100 ~ 120rpm(DO value 4.5mgL
-1) under cultivate, timing sampling measure NO
3 -the clearance of-N and NO
2 -the accumulation of-N, and the stability of observing microcapsule, the results are shown in Figure 5.
Found out by Fig. 5, immobilized bacterium is NO when 12h
3 -the clearance of-N reaches 92.70%, and removing speed is 15.84 mgL
-1h
-1nO
3 --N, removes NO with without immobilized bacterial strain
3 -the speed of-N is close.NO
2 -the concentration of-N peaks when 9h, to being down to 11.585 μ gmL during 12h
-1, be less than 1 μ gmL to being down to during 16h
-1.At this moment, microcapsule are stable.
Embodiment six: bacterial strain WZUF25 is to the decontamination effect improving of the waste water of livestock poultry after anaerobic treatment
Preservation strain (2.0 mL freeze pipes melt bacterium liquid) is inoculated in the 500mL Erlenmeyer flask that 200mL LB substratum (same embodiment three of filling a prescription) is housed, 30 DEG C, cultivate 24h under 160rpm, under 8000 rpm, centrifugal 10min obtains thalline, after sterile distilled water washing thalline 2 times, with sterilized water make bacteria suspension (
oD 6800.900 ~ 1.000).In the waste water of livestock poultry of anaerobic treatment (the bottled 200mL of 500mL taper) is inoculated in, in 30 DEG C of 150 rpm(DO value 4.3 mgL by the inoculum size of 5% volume ratio
-1) under cultivate, every 24 h sampling and measuring biomasss (
oD 680), under 8000rpm, then after centrifugal 10min, measure the NH of supernatant liquor
4 +-N concentration, NO
2 --N concentration, NO
3 --N concentration and COD value, the results are shown in Table 6-1.
Table 6-1 bacterial strain WZUF25 is to the decontamination effect improving of the waste water of livestock poultry after anaerobic treatment
Can be made to decline 106.33 mgL through the COD of the waste water of livestock poultry of anaerobic treatment in 48 h from table 6-1, bacterial strain WZUF25
-1, NH
4 +-N declines 116.32 mgL
-1, NO
3 --N declines 42.21 mgL
-1, NO
2 --N declines 19.90 mgL
-1.
In the waste water of livestock poultry after anaerobic treatment, add carbon source Soduxin, make its concentration in waste water reach 10gL
-1, the method identical with the waste water of livestock poultry not adding sodium acetate, research bacterial strain WZUF25, to the decontamination effect improving of waste water of livestock poultry, the results are shown in Table 6-2.
From table 6-2, in the waste water of livestock poultry after anaerobic treatment, add 10 gL
-1after Soduxin, the COD value of waste water increases by 821.80 mgL
-1, decline after cultivating 24 h 379.61 mgL
-1, decline after cultivating 48 h 447.17 mgL
-1; Cultivate NH after 24 h
4 +-N declines 163.84 mgL
-1; Cultivate NH after 48 h
4 +-N declines 183.39 mgL
-1; After cultivating 24 h, NO
3 --N declines 43.87 mgL
-1, NO
2 --N declines 22.21 mgL
-1.Therefore, bacterial strain WZUF22 has good practical application potentiality.
Table 6-2 bacterial strain WZUF22 is to the decontamination effect improving of the waste water of livestock poultry after the anaerobic treatment that with the addition of nitrogenous source
Embodiment seven: gac-calcium-alginate-immobilized cell is to the decontamination effect improving of the waste water of livestock poultry after anaerobic treatment
100mL is housed in the 500mL Erlenmeyer flask of the waste water of livestock poultry of anaerobic treatment, at 30 DEG C of 100 ~ 120rpm(DO value 3.5mgL by 100 by gac-calcium alginate microcapsule access that embodiment five legal system is standby
-1) under cultivate, sample the NH measuring supernatant liquor under 8000rpm after centrifugal 10min every 24 h
4 +-N concentration, NO
2 --N concentration, NO
3 --N concentration and COD value, the results are shown in Table 7.
Table 7 gac-calcium-alginate-immobilized cell is to the decontamination effect improving of the waste water of livestock poultry after anaerobic treatment
From table 7-1, in 48 h, gac-calcium alginate microcapsule can make to decline 32.09 mgL through the COD of the waste water of livestock poultry of anaerobic treatment
-1, NH
4 +-N declines 22.68 mgL
-1, NO
3 --N decline 41.21mgL
-1, NO
2 --N declines 20.31 mgL
-1.Therefore, immobilized cell mainly can be used for removing the NO in waste water
3 --N and NO
2 --N.
The above embodiment is only that protection scope of the present invention is not limited thereto in order to absolutely prove the preferred embodiment that the present invention lifts.The equivalent alternative or conversion that those skilled in the art do on basis of the present invention, all within protection scope of the present invention.Protection scope of the present invention is as the criterion with claims.
Claims (8)
1. a strain Pseudomonas stutzeri, is characterized in that, this bacterial strain be Pseudomonas stutzeri (
pseudomonas stutzeri) WZUF25, being preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center, registers on the books and is numbered CGMCC NO.7685 in preservation center.
2. the cultural method of Pseudomonas stutzeri described in claim 1, is characterized in that, comprises the steps:
1) preservation strain WZUF25 is inoculated in LB substratum, cultivates 12 more than h, centrifugal, obtains thalline, makes with after sterilized water washing
oD 680it is the bacteria suspension of 0.900 ~ 1.000;
2) step 1) gained bacterial suspension inoculation is cultivated in denitrification substratum;
Wherein, the formation of described denitrification substratum is: carbon source, nitrogenous source, K
2hPO
4, FeSO
4, MgSO
4, H
2o, described carbon source is Soduxin, sodium acetate, glycerine or glucose, and described nitrogenous source is the compound containing nitrate ion.
3. cultural method according to claim 2, is characterized in that, step 1) preservation strain WZUF25 is inoculated in LB substratum, in 20 ~ 40 DEG C, and dissolved oxygen 3.5 ~ 6.1 mgL
-1condition under cultivate; Step 2) in gained bacterial suspension inoculation in denitrification substratum, in 25 ~ 40 DEG C, dissolved oxygen 3.5 ~ 6.1 mgL
-1lower cultivation.
4. cultural method according to claim 2, is characterized in that, step 2) described in the formula of denitrification substratum be: carbon source, the quality 1.228g of nitrate radical, K in nitrogenous source
2hPO
41g, FeSO
47H
2o 0.20g, MgSO
47H
2o 0.10 g, H
2o 1000 mL, pH are 5 ~ 9, the NO of described carbon source and nitrogenous source
3 -mass ratio be 5:1.228 ~ 15:1.228.
5. the application of Pseudomonas stutzeri according to claim 1, is characterized in that, is inoculated in the nitrogenous aqueous solution, carries out denitrogenation by described Pseudomonas stutzeri WZUF25.
6. the application of Pseudomonas stutzeri according to claim 5, is characterized in that, the described nitrogenous aqueous solution is for containing NH
4 +, NO
3 -and NO
2 -a kind of or its combination aqueous solution, described Pseudomonas stutzeri WZUF25 takes off NO
3 --N, NH
4 +-N and NO
2 -the carbon source of-N contains one of them or its combination of sodium acetate, Soduxin, glycerine or glucose.
7. application according to claim 6, is characterized in that, described nitrogenous aqueous solution pH is 4 ~ 10, and described Pseudomonas stutzeri WZUF25 is in temperature 20 DEG C ~ 45 DEG C, and dissolved oxygen is 1.3 ~ 7.3mgL
-1condition under denitrogenation is carried out to the nitrogenous aqueous solution.
8. microcapsule are prepared in Pseudomonas stutzeri immobilization according to claim 1, and it is characterized in that, step is as follows:
1) bacterial strain WZUF25 is centrifugal after being inoculated in and cultivating in LB substratum obtains thalline, and thalline is with distilled water wash, and the thalline after washing mixes with the sodium alginate soln being dispersed with active carbon powder;
2) CaCl is prepared
2the aqueous solution, after 37 DEG C of water-bath 10 ~ 30min, is added dropwise to the CaCl of temperature 37 DEG C by step 1) gained mixed solution
2in the aqueous solution, obtain microcapsule.
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| CN106754570B (en) * | 2017-02-28 | 2019-06-07 | 重庆大学 | One plant of Pseudomonas stutzeri and its application |
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| CN110468066B (en) * | 2019-07-29 | 2021-09-17 | 中成进出口股份有限公司 | Aerobic denitrifying strain and application thereof |
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| CN114395505B (en) * | 2021-12-21 | 2023-08-18 | 江苏南资环保科技有限公司 | Low-temperature denitrifying bacterium and application thereof |
| CN114703093B (en) * | 2022-03-18 | 2023-08-22 | 曲阜师范大学 | Facultative anaerobic complete denitrification gas producing bacterium Y23 and application thereof |
| CN115261259B (en) * | 2022-06-08 | 2023-04-25 | 安徽师范大学 | Aeromonas veronii, product and nitrogen removal application thereof |
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