CN102604870A - Recovery culturable viable but non culturable (VBNC) arthrobacterium DSC4 strain and recovering method and application thereof - Google Patents
Recovery culturable viable but non culturable (VBNC) arthrobacterium DSC4 strain and recovering method and application thereof Download PDFInfo
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- Purification Treatments By Anaerobic Or Anaerobic And Aerobic Bacteria Or Animals (AREA)
Abstract
The invention belongs to the fields of agricultural biotechnology and environmental protection and relates to a 'viable but non culturable (VBNC)' arthrobacterium strain which is obtained through separation by using recovery culturable technology, a recovering method of the strain and application of the strain. The preservation number of the strain is CCTCC No: M2011406; the preservation organization is China Center for Type Culture Collection; the preservation address is Wuhan University, Wuhan Province, China; and the preservation date is 25th Nov. 2011. The viable but non culturable (VBNC) arthrobacterium DSC4 can turn the color of the reaction liquid of a Giltay culture medium to dark blue from green and generate gas. At the same time, the conversion rate of nitrate nitrogen is detected to be 54.7 percent, the total nitrogen depletion rate is 52.9 percent, and residue of ammoniacal nitrogen and nitrite nitrogen is not detected. The strain has an obvious denitrification effect.
Description
Technical field
The invention belongs to biotechnology agricultural, field of environment protection, relate to a strain use " the non-cultivation (VBNC) of work " genus arthrobacter bacterial classification that recovery can the cultivation technology be separated to (
ArthrobacterDSC4) and the method for resuscitation and the application of this bacterial classification sp..
Background technology
The biological treatment that contains ammonia-nitrogen sewage is very important technical measures of current agricultural, environmental protection industry (epi); The wherein oxidation of ammonia-state nitrogen; The oxidation of nitrite nitrogen; Need the participation of multiple mikrobe in the key techniques such as the reduction of nitric nitrogen and denitrogenation, the inorganic and organic nitrogen of Degradation and Transformation.The nitrite bacteria of general traditional separable simple function as
Nitrosomonas, NitrosococcusBacterial classification in the genus, nitrifier as
Nitrobacter, NitrococcusThe bacterial classification that belongs to, the denitrification denitrogenation bacterium as
Micrococcus denitrificansDeng being autotrophic bacteria, nitration denitrification effect required time is long, efficient is low.Allotrophic nitrobacteria bacterial classification and quantization function that screening possesses the nitration denitrification function are the hot topics of studying at present.
Bacterium in the nature ecotope can only have a 0.01-10% through what traditional partition method was obtained, and the overwhelming majority is in non-cultivation the (VBNC) alive, is similar to dormant state.With regard to the environmental microorganism resource; The resource bacterial classification that obtains occurring in nature 90-99.99% comprises the innovation that autotrophy and the heterotrophic nitrification flora in the above-mentioned ammonia-nitrogen sewage processing links needs the new approaches novel method, could excavate, develop, utilize these unknown Microbial resources.
The separation of VBNC state bacterium can utilize some distinctive signal molecule recoveries to promote it to be in the cultivated change of dormant state bacterium with screening.(Ding Linxian; Su Xiaomei; Horizontal field is bright. live but the progress of non-cultivation the (VBNC) state bacterium and use prospect. " mikrobe journal " .2011,51 (7): formation mechanism, transformation and kind, recovery, Research Significance and the application thereof of 858-862.) having set forth VBNC state bacterium are looked forward to.And reported in the period of Ding Linxian etc. is surplus ten to the recovery that is in VBNC state bacterium in the ecotope, can cultivation, some achievements in research of aspects such as phyletic evolution relation and potential function, the exploitation of intending to Microbial resources provides new scientific basis with application.And disclose bring back to life to promote the factor (Rpf, resuscitation promotingfactor) be by the gamboge coccus (
M. luteus) a kind of protein that can make this bacterium that is in the VBNC state recover its growth and breeding ability again of excretory, molecular weight is about 16-17 kDa.Mukamolova has reported that Rpf can recover and has been in the VBNC gram-positive microorganism in period
MycobacteriumThe mycobacterium tuberculosis that belongs to (
M. tuberculosis),
Mycobacterium bovis (
M. bovis), mycobacterium kansasii (
M.kansasii), M. smegmatics (
M. smegmatis) and mycobacterium avium (
M. avium) etc.And the gene similar with Rpf also comes to light in the gram-positive microorganism of GC such as height such as streptomycete, tubercule bacillus, coryneform bacteria grade.In addition, people such as Mukamolova have reported that also Rpf adopts the mode secretion signal of autocrine or paracrine, and the mikrobe of dormancy is brought back to life, the growth that can also stimulate normal bacterium, and the reproductive process of ability regulating cell.
At present domestic for the technology of in Sewage treatment systems, separating VBNC state nitrifying bacteria community and recovery can cultivation the nitrobacteria of VBNC state do not see that as yet public reported is arranged.
Summary of the invention
According to the digging utilization of VBNC Microbial resources and to ammonia-nitrogen sewage biological treatment need the social demand of high-level efficiency nitration denitrification bacterium resource badly; One of the object of the invention provides the VBNC Arthrobacter DSC4 bacterial strain that a kind of recovery can cultivation; Two of the object of the invention provides the activation medium of above-mentioned Arthrobacter DSC4 bacterial strain, and three of the object of the invention provides the denitrification denitrogenation that above-mentioned VBNC Arthrobacter DSC4 bacterial strain is applied to contain nitrate salt matrix.
In order to realize first above-mentioned purpose, the technical scheme below the present invention has adopted:
Recovery can cultivation VBNC Arthrobacter DSC4 (
ArthrobacterSp. bacterial strain DSC4), the deposit number of this bacterial strain is CCTCC No:M2011406, depositary institution is: Chinese typical culture collection center, the preservation address is: Chinese Wuhan Wuhan University, preservation date is: on November 21st, 2011.Classification called after: Arthrobacter DSC4
ArthrobacterSp. DSC4.As separation source, the utilization recovery can the technological VBNC resource bacterium of obtaining of cultivation with the VBNC state flora in the sewage biological treatment system for above-mentioned bacterial strain.This bacterial strain is (this patent activation culture based formulas) on activation medium, cultivates 2d for 30 ℃, and colony diameter 2-3mm is light grey or faint yellow, glossy; The cell atrichia, do not move; Gram positive bacterium.
In order to realize second above-mentioned purpose, the technical scheme below the present invention has adopted:
A kind of activation medium that is used for above-mentioned Arthrobacter DSC4 bacterial strain, this substratum have following component to constitute by weight percentage:
Nutrient broth medium 1.0 ~ 3.0g; Yeast extract paste 0.2 ~ 0.5g;
Glucose 1.5 ~ 5.0 g; Agar 1.0 ~ 4.0%;
Contain and bring back to life the recovery nutrient solution 2 ~ 20% that promotes factor R pf, deionized water 1000ml;
Above-mentioned per-cent is the volume percent that accounts for the substratum total amount.
As preferably, above-mentioned containing brings back to life and promotes the recovery nutrient solution of factor R pf to be made up of substratum base fluid, bacterium liquid and liquid soil; Described bacterium liquid is cultured to the logarithmic phase later stage by the micrococcus luteus bacterial classification on the bacterial classification liquid nutrient medium, extract through centrifugal removal bacterial classification liquid nutrient medium and obtain protein, contains to bring back to life to promote factor R pf, and bacterium liquid is the 1-10% of substratum base fluid by volume; Described liquid soil adds tap water by through garden mould air-dry, that sieve, remove impurity, and leaching filtrating or centrifuging and taking supernatant are crossed in repeatedly sterilization, and liquid soil is the 1-5% of substratum base fluid by volume.
The activation medium compound method of above-mentioned Arthrobacter DSC4 bacterial strain; This method comprises the steps: to take by weighing nutrient broth medium 1.0 ~ 3.0g, yeast extract paste 0.2 ~ 0.5g, glucose 1.5 ~ 5.0g; Deionized water 1000ml; PH transfers to 7.0, adds 1.0 ~ 4.0 agar, and 110 ~ 130 ℃ on warp, 10 ~ 30min autoclaving postcooling are to room temperature; Under aseptic condition, add in addition the recovery nutrient solution of 2 ~ 20 % (v/v) through the sterilization of 0.22 μ m membrane filtration, pH 7.0, stirring and evenly mixing divides to annotate and does admittedly, and it is for use to be inverted 4-10 ℃ of preservation.
As preferably, the preparation method of above-mentioned recovery nutrient solution is following:
1) take a morsel gamboge coccus bacterial classification inoculation in the bacterial classification liquid nutrient medium, 25 ~ 35 C, 100 ~ 150rpm vibrate cultivation; In bacterial growth to the logarithmic phase later stage, nutrient solution is with 3000 ~ 8000rpm, and thalline is removed in the centrifugal extracting of 10 ~ 30min; After the sterilization of 0.20 ~ 0.25 μ m membrane filtration ,-30 ~-10 ℃ of preservations are subsequent use;
2) the garden mould 1kg air-dry, that sieve, remove impurity that learns from else's experience adds tap water 1 ~ 2L, 110 ~ 130 ℃, the sterilization of 10 ~ 40min high-pressure sterilizing pot; After room temperature is treated an evening, use once again with method and sterilize; 1500 ~ 2500r/min low-speed centrifugal is got supernatant or is obtained filtrating with the filter paper filtering waste, and through 110 ~ 130 ℃, 10 ~ 30min high-pressure sterilizing pot sterilization postcooling ,-30 ~-10 ℃ of preservations are subsequent use;
3) accurately take by weighing each component of substratum base fluid, it is for use that pH transfers to 7.0,110 ~ 130 ℃, 10 ~ 30min high-pressure sterilizing pot sterilization postcooling to room temperature; The separation of the suitable most bacteriums of substratum base fluid is for particular requirement being arranged and selectively bacterium can corresponding plus-minus nutrition composition;
4) before the use, under aseptic condition, add 1-10% bacterium liquid in the substratum base fluid, add the 1-5% liquid soil again, use behind the mixing.
In order to realize the 3rd above-mentioned purpose, the technical scheme below the present invention has adopted:
Above-mentioned recovery can cultivation VBNC Arthrobacter DSC4 bacterial strain be applied to contain the denitrification denitrogenation of nitrate salt matrix.Concrete method is: with described recovery can cultivation the activated cultivation of VBNC Arthrobacter DSC4 bacterial strain, preceding cultivation after add in the sewage biological treatment system.
As preferably, the substratum that described activation culture adopts is made up of following component by weight percentage:
Nutrient broth medium 1.0 ~ 3.0g; Yeast extract paste 0.2 ~ 0.5g;
Glucose 1.5 ~ 5.0 g; Agar 1.0 ~ 4.0%;
Contain and bring back to life the recovery nutrient solution 10% that promotes factor R pf, deionized water 1000ml;
Above-mentioned per-cent is the volume percent that accounts for the substratum total amount.
As preferably, cultivate the substratum that adopts before described and constitute by following component by weight percentage:
Peptone 3.0 ~ 8.0 g, yeast extract paste 0.2 ~ 0.8 g,
Glucose 3.0 ~ 8.0 g, NaCl 1.5 ~ 5.5 g,
The 1000ml deionized water, pH transfers to 7.0.
The present invention is owing to adopted above technical scheme; The cultivated change VBNC Arthrobacter DSC4 bacterial strain that obtains; Can make Giltay substratum reaction solution begin to transfer mazarine to and see the aerogenesis phenomenon by green; Explain that this bacterial classification can utilize nitrate reduction to form alkaline matter, and aerogenesis makes the indicator colour-change.Recording the nitric nitrogen transformation efficiency simultaneously is 54.7%, and the total nitrogen decrement is 52.9%, does not detect the remaining of ammonia-state nitrogen and nitrite nitrogen, has tangible denitrification denitrogenation effect.
Description of drawings
Fig. 1 is the bacterium colony figure of Arthrobacter DSC4 bacterial strain of the present invention.
Fig. 2 is an Arthrobacter DSC4 bacterial strain of the present invention
The denitrification denitrogenation design sketch.
Biomaterial preservation explanation
Recovery can cultivation VBNC Arthrobacter DSC4 (
ArthrobacterSp. bacterial strain DSC4), the deposit number of this bacterial strain is CCTCC No:M2011406, depositary institution is: Chinese typical culture collection center (CCTCC), the preservation address is: Chinese Wuhan Wuhan University, preservation date is: on November 21st, 2011.
Embodiment
The described medium preparation method of the embodiment of the invention is following:
1, NYGR substratum: take by weighing Nutrient broth 1.6g, Yeast extract 0.5g, Glucose 2.5 g, deionized water 1000ml, pH transfers to 7.0, adds 2% agar, and 121 ℃ on warp, 15min autoclaving postcooling are to room temperature; The recovery nutrient solution that under aseptic condition, adds the sterilization of 10% (v/v) warp, 0.22 μ m membrane filtration, pH 7.0 in addition, stirring and evenly mixing divides to annotate and does admittedly;
2, PYGN substratum: Peptone 5.0 g, Yeast extract 0.5 g, Glucose 5.0 g, NaCl 2.5 g, the 1000ml deionized water, pH transfers to 7.0;
3, Giltay substratum: A liquid: KNO
31.0g, l-asparagine 1.0g, 1 %BTB spirituous solution 5.0mL, zero(ppm) water 500mL; B liquid: Trisodium Citrate 8.5g, MgSO
47H
2O 1.0g, FeCl
36H
2O 0.05g, KH
2PO
41.0g, CaCl
22H
2O 0.2g, zero(ppm) water 500mL.Mix A, B two solution, regulate pH value 7.0 ~ 7.2, subsequent use through sterilization.
4, recovery nutrient solution substratum is made up of A, B, is constituted with the C composition and (be called for short: ABC recovery substratum).
The A composition:
Bacterium liquid: gamboge coccus Micrococcus luteus IAM14879 bacterial classification is cultured to the logarithmic phase later stage on the LMMD substratum, extracts through centrifugal substratum and obtains protein, contains to bring back to life to promote that factor R pf, consumption are 5% of C one-tenth partial volume.
The B composition:
Liquid soil: the garden mould 1kg air-dry, that sieve, remove impurity such as stone grass roots that learns from else's experience adds tap water 1L, 121 ℃, the sterilization of 30min high-pressure sterilizing pot; After room temperature is treated an evening, once again the sterilization, triplicate; The 2000r/min low-speed centrifugal is got supernatant (or with filter paper filtering acquisition filtrating), and consumption is 2% of a C one-tenth partial volume.
The C composition:
Bacto-tryptone (Difco company) (Bacto peptone (Difco)) 10.0 g,
Yeast extract (Yeast extract (Difco)) 5.0 g,
Malt extract (Malt extract (Difco)) 5.0 g,
(hydrolysis) casamino acids (Casamino acids (Difco)) 5.0g,
Carnis Bovis seu Bubali cream (Beef extract (Difco)) 2.0g,
Glycerine (Glycerol) 2.0g,
Tween 80 (Tween 80) 0.05g,
MAGNESIUM SULPHATE HEPTAHYDRATE 99.5 (MgSO
47H
2O) 1.0 g,
Add deionized water 1000mL, pH7.0.
Above substratum is the liquid culture based component, during like the preparation solid medium, need add agar 15-20g, and through 121 ℃, 15min autoclaving postcooling uses.
The LMMD substratum is formed in the A composition:
Ammonium chloride (NH
4Cl) 4.0 g,
Potassium primary phosphate (KH
2PO
4) 1.4 g,
Vitamin H (Biotin) 0.005 g,
L-methionine(Met) (L-Methionine) 0.02 g,
VitB1 (Thiamine) 0.04 g,
Inosine (Inosine) 1.0 g,
MAGNESIUM SULPHATE HEPTAHYDRATE 99.5 (MgSO
47H
2O) 0.07 g,
Mineral solution (Mineral solution) 1.0 ml,
L-lithium lactate (Lithium L-lactate) 10.0 g,
Zero(ppm) water (Distilled water) 1000 ml, pH 7.5.
Wherein, above-mentioned mineral solution (Mineral solution):
Copper sulfate (CuSO
4) 0.24 g,
Manganous chloride tetrahydrate (MnCl
2) 0.50 g,
Ferrous sulfate (FeSO
4) 0.10 g,
Sodium orthomolybdate (Na
2MoO
4) 0.025 g,
Zinc sulfate (ZnSO
4) 0.05 g,
Zero(ppm) water (distilled water) 1000 ml.
More than be that general bacteria resuscitation is cultivated the prescription that uses.According to the different ecological environment requirement, the C composition can be done corresponding adjustment.
Recovery culture medium preparation method:
1. take a morsel gamboge coccus bacterial classification inoculation in the LMMD liquid nutrient medium, 30 C, 120 rpm vibrate cultivation; In bacterial growth to the logarithmic phase later stage, nutrient solution is used 5000 rpm, and thalline is removed in the centrifugal extracting of 20 min; After the sterilization of 0.22 μ m membrane filtration ,-20 ℃ of preservations are subsequent use;
2. the garden mould 1kg air-dry, that sieve, remove impurity such as stone grass roots that learns from else's experience adds tap water 1L, 121 ℃, the sterilization of 30min high-pressure sterilizing pot; After room temperature is treated an evening, use once again with method and sterilize; The 2000r/min low-speed centrifugal is got supernatant (or with filter paper filtering waste acquisition filtrating), and through 121 ℃, 15min high-pressure sterilizing pot sterilization postcooling ,-20 ℃ of preservations are subsequent use;
3. accurately take by weighing each component of C composition, it is for use that pH transfers to 7.0,121 ℃, 15min high-pressure sterilizing pot sterilization postcooling to room temperature.The separation of the suitable most bacteriums of C component is for particular requirement being arranged and selectively bacterium can corresponding plus-minus nutrition composition;
4. before using, should under aseptic condition, add 5% (A/C) A composition in the C composition, add 3% (B/C) B composition again, use behind the mixing.
Embodiment 1
From sewage biological treatment system; Utilize recovery to obtain VBNC Arthrobacter DSC4 bacterial strain preservation bacterial classification by the cultivation technology screening; Can cultivation VBNC Arthrobacter DSC4 strains separation from certain sewage biological treatment system; Have more than 90% general traditional separation method in the system and be difficult to obtain, be in the bacterium that non-cultivation the (VBNC) alive is similar to dormant state, can the acquisition of cultivation technology screening be in the Arthrobacter DSC4 bacterial strain of VBNC state through utilizing recovery.The deposit number of this bacterial strain is CCTCC No:M2011406, and depositary institution is: Chinese typical culture collection center, the preservation address is: Chinese Wuhan Wuhan University, preservation date is: on November 21st, 2011.Classification called after: Arthrobacter DSC4
ArthrobacterSp. DSC4, this bacterial strain generally need be stored in cryotherapy (subzero 80 degree).Must be through the DSC4 of cryotherapy preservation bacterial strain through NYGR solid medium activation culture, promptly be seeded on the NYGR solid medium 30 ℃ be inverted activation culture 2-4d after, it is subsequent use to be stored in 4 ℃ of refrigerators temporarily.
The DSC4 bacterial strain on the NYGR solid medium, 30 ℃ be inverted activation culture 2-7d after, visible diameter 2-3mm, light grey or faint yellow, glossy bacterium colony (accompanying drawing 1).
The DSC4 bacterial strain is dyed to be gram positive bacterium, and visible cell atrichia under the opticmicroscope, not move, do not have sporulation, no fluorochrome, form be quarter butt to spheric cell.
Growth temperature on Nutrient Broth substratum is 5-40 ℃, 30 ℃ of optimum temperutures; Salt tolerant scope 3-7% (w/v); PH scope 6-11, ph optimum 7.5.
Embodiment 2
Can cultivation VBNC Arthrobacter DSC4 bacterial strain to the culture identification method of the denitrification denitrogenation effect of nitrate salt matrix, comprise the steps:
1. getting can cultivation VBNC Arthrobacter DSC4 bacterial strain preservation bacterial classification, and under aseptic condition, a small amount of bacterial classification of picking is transferred on the NYGR substratum, 30 ℃ of activation culture 2-4d;
2. 1. the bacterial classification of a small amount of activated cultivation of picking is inoculated into and carries out 30 ℃, the preceding cultivation of 2d in the PYGN liquid nutrient medium;
3. in the Giltay substratum, insert 2% preceding culture bacteria liquid respectively, 30 ℃, PM 120 change shaking tables cultivate 2d after, observe ammonia-state nitrogen, nitrite nitrogen, nitric nitrogen and total nitrogen in substratum aerogenesis and colour-change situation and the mensuration nutrient solution.
Ammonia nitrogen, nitrite nitrogen, nitric nitrogen and total nitrogen adopt Nesslerization, ultraviolet spectrophotometry, GB water quality total nitrogen assay method (the mensuration alkalescence alkaline potassium per-sulfate digestion ultraviolet spectrophotometry of GB 11894-89 water quality total nitrogen) to measure respectively.The result sees Fig. 2.Observing inoculation can make Giltay substratum reaction solution begin to be transferred to mazarine and seen the aerogenesis phenomenon by green by cultivation VBNC Arthrobacter DSC4 bacterial strain, explain that this bacterial classification can utilize nitrate reduction to form alkaline matter, and aerogenesis makes the indicator colour-change.Recording the nitric nitrogen transformation efficiency simultaneously is 54.7%, and the total nitrogen decrement is 52.9%, does not detect the remaining of ammonia-state nitrogen and nitrite nitrogen.
Claims (10)
- The VBNC Arthrobacter DSC4 that 1. recovery can cultivation ( ArthrobacterSp. bacterial strain DSC4), the deposit number of this bacterial strain is CCTCC No:M2011406, depositary institution is: Chinese typical culture collection center, the preservation address is: Chinese Wuhan Wuhan University, preservation date is: on November 21st, 2011.
- 2. activation medium that is used for the described Arthrobacter DSC4 of claim 1 bacterial strain is characterized in that this substratum has following component to constitute by weight percentage:Nutrient broth medium 1.0 ~ 3.0g; Yeast extract paste 0.2 ~ 0.5g;Glucose 1.5 ~ 5.0 g; Agar 1.0 ~ 4.0%;Contain and bring back to life the recovery nutrient solution 2 ~ 20% that promotes factor R pf, deionized water 1000ml;Above-mentioned per-cent is the volume percent that accounts for the substratum total amount.
- 3. the activation medium of Arthrobacter DSC4 bacterial strain according to claim 2 is characterized in that containing the recovery nutrient solution that brings back to life promotion factor R pf and is made up of substratum base fluid, bacterium liquid and liquid soil; Described bacterium liquid is cultured to the logarithmic phase later stage by the micrococcus luteus bacterial classification on the bacterial classification liquid nutrient medium, extract through centrifugal removal bacterial classification liquid nutrient medium and obtain protein, contains to bring back to life to promote factor R pf, and bacterium liquid is the 1-10% of substratum base fluid by volume; Described liquid soil adds tap water by through garden mould air-dry, that sieve, remove impurity, and leaching filtrating or centrifuging and taking supernatant are crossed in repeatedly sterilization, and liquid soil is the 1-5% of substratum base fluid by volume.
- 4. the activation medium compound method of the described Arthrobacter DSC4 of claim 2 bacterial strain; It is characterized in that this method comprises the steps: to take by weighing nutrient broth medium 1.0 ~ 3.0g, yeast extract paste 0.2 ~ 0.5g, glucose 1.5 ~ 5.0g; Deionized water 1000ml; PH transfers to 7.0, adds 1.0 ~ 4.0 agar, and 110 ~ 130 ℃ on warp, 10 ~ 30min autoclaving postcooling are to room temperature; Under aseptic condition, add in addition the recovery nutrient solution of 2 ~ 20 (v/v) through the sterilization of 0.22 μ m membrane filtration, pH 7.0, stirring and evenly mixing divides to annotate and does admittedly, and it is for use to be inverted 4-10 ℃ of preservation.
- 5. the activation medium compound method of the described Arthrobacter DSC4 of claim 4 bacterial strain is characterized in that the preparation method of recovery nutrient solution is following:1) take a morsel gamboge coccus bacterial classification inoculation in the bacterial classification liquid nutrient medium, 25 ~ 35 C, 100 ~ 150rpm vibrate cultivation; In bacterial growth to the logarithmic phase later stage, nutrient solution is with 3000 ~ 8000rpm, and thalline is removed in the centrifugal extracting of 10 ~ 30min; After the sterilization of 0.20 ~ 0.25 μ m membrane filtration ,-30 ~-10 ℃ of preservations are subsequent use;2) the garden mould 1kg air-dry, that sieve, remove impurity that learns from else's experience adds tap water 1 ~ 2L, 110 ~ 130 ℃, the sterilization of 10 ~ 40min high-pressure sterilizing pot; After room temperature is treated an evening, use once again with method and sterilize; 1500 ~ 2500r/min low-speed centrifugal is got supernatant or is obtained filtrating with the filter paper filtering waste, and through 110 ~ 130 ℃, 10 ~ 30min high-pressure sterilizing pot sterilization postcooling ,-30 ~-10 ℃ of preservations are subsequent use;3) accurately take by weighing each component of substratum base fluid, it is for use that pH transfers to 7.0,110 ~ 130 ℃, 10 ~ 30min high-pressure sterilizing pot sterilization postcooling to room temperature; The separation of the suitable most bacteriums of substratum base fluid is for particular requirement being arranged and selectively bacterium can corresponding plus-minus nutrition composition;4) before the use, under aseptic condition, add 1-10% bacterium liquid in the substratum base fluid, add the 1-5% liquid soil again, use behind the mixing.
- 6. recovery according to claim 1 can cultivation VBNC Arthrobacter DSC4 bacterial strain be applied to contain the denitrification denitrogenation of nitrate salt matrix.
- 7. contain the denitrification method of nitrate salt matrix, it is characterized in that: this method with the described recovery of claim 1 can cultivation the activated cultivation of VBNC Arthrobacter DSC4 bacterial strain, preceding cultivation after add in the sewage biological treatment system.
- 8. the denitrification method that contains nitrate salt matrix according to claim 4 is characterized in that: the substratum that activation culture adopts is made up of following component by weight percentage:Nutrient broth medium 1.0 ~ 3.0g; Yeast extract paste 0.2 ~ 0.5g;Glucose 1.5 ~ 5.0 g; Agar 1.0 ~ 4.0%;Contain and bring back to life the recovery nutrient solution 10% that promotes factor R pf, deionized water 1000ml;Above-mentioned per-cent is the volume percent that accounts for the substratum total amount.
- 9. the denitrification method that contains nitrate salt matrix according to claim 4 is characterized in that: the substratum that preceding cultivation is adopted is made up of following component by weight percentage:Peptone 3.0 ~ 8.0 g, yeast extract paste 0.2 ~ 0.8 g,Glucose 3.0 ~ 8.0 g, NaCl 1.5 ~ 5.5 g,The 1000ml deionized water, pH transfers to 7.0.
- 10. sewage biological treatment system is characterized in that: comprise the VBNC Arthrobacter DSC4 bacterial strain that the described recovery of claim 1 can cultivation in this sewage biological treatment system.
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