CN102604870B - 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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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 21th 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 " the non-cultivation (VBNC) of living " genus arthrobacter bacterial classification (Arthrobacter sp.DSC4) that a strain application recovery can cultivation technology be separated to and method for resuscitation and the application of this bacterial classification.
Background technology
Biological treatment containing 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, in the key techniques such as the reduction of nitric nitrogen and denitrogenation, need to have the participation of multiple-microorganism, Degradation and Transformation inorganic with organic nitrogen.The nitrite bacteria of general traditional separable simple function is as the bacterial classification in Nitrosomonas, Nitrosococcus genus, the bacterial classification that nitrifier belongs to as Nitrobacter, Nitrococcus, denitrification denitrogenation bacterium as Micrococcus denitrificans etc. be autotrophic bacteria, nitration denitrification effect required time is long, efficiency is low.Allotrophic nitrobacteria bacterial classification and quantization function that screening possesses nitration denitrification function are the hot topics of studying at present.
Bacterium in nature ecotope can obtain through traditional partition method only a 0.01-10%, non-cultivate (VBNC) of the overwhelming majority in living, is similar to dormant state.With regard to environmental microorganism resource, the resource bacterial classification that obtains occurring in nature 90-99.99% comprises the innovation that autotrophy in above-mentioned ammonia-nitrogen sewage processing links and heterotrophic nitrification flora need to have new approaches novel method, could excavate, develop, utilize these unknown Microbial resources.
The isolation and screening of VBNC state bacterium can utilize some distinctive signal molecule recoveries to promote its cultivated change in dormant state bacterium.(Ding Linxian, Su Xiaomei, horizontal field is bright. live but progress and the application prospect of non-cultivation (VBNC) state bacterium. < < microorganism 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 between Ding Linxian etc. is more than ten year for the recovery in VBNC state bacterium in ecotope, can cultivation, some achievements in research of the aspect such as Phylogenetic and potential function, the exploitation that is intended to be Microbial resources and application provide new scientific basis.And Resuscitation-promoting Factor (Rpf disclosed, resuscitation promotingfactor) be that molecular weight is about 16-17kDa by a kind of protein that can make again to recover in this bacterium of VBNC state its growth and breeding ability of gamboge coccus (M.luteus) secretion.Mukamolova has reported mycobacterium tuberculosis (M.tuberculosis), Mycobacterium bovis (M.bovis), the mycobacterium kansasii (M.kansasii) that gram-positive microorganism Mycobacterium that Rpf can recover in VBNC period belongs to, M. smegmatics (M.smegmatis) and mycobacterium avium (M.avium) etc.And the gene similar with Rpf is also found in the gram-positive microorganism of streptomycete, tubercule bacillus, the contour GC of coryneform bacteria.In addition, the people such as Mukamolova have also reported that Rpf adopts the mode secretion signal of autocrine or paracrine, not only can make the microorganism of dormancy bring back to life, and can also stimulate the growth of normal bacteria, and the reproductive process of energy regulating cell.
The at present domestic technology for separated VBNC state nitrifying bacteria community in Sewage treatment systems and recovery can cultivation the nitrobacteria of VBNC state there is not yet open report.
Summary of the invention
According to the digging utilization of VBNC Microbial resources and for ammonia-nitrogen sewage, carry out a biological disposal upon and need the social demand of high-level efficiency nitration denitrification bacterial resources badly, one of the object of the invention is to provide the VBNC Arthrobacter DSC4 bacterial strain that a kind of recovery can cultivation, two of the object of the invention is to provide the activation medium of above-mentioned Arthrobacter DSC4 bacterial strain, and three of the object of the invention is to provide above-mentioned VBNC Arthrobacter DSC4 bacterial strain and is applied to the denitrification denitrogenation containing nitrate matrix.
In order to realize first above-mentioned object, the present invention has adopted following technical scheme: VBNC Arthrobacter DSC4 (Arthrobacter sp.DSC4) bacterial strain that recovery can cultivation, the deposit number of this bacterial strain is CCTCC No:M2011406, depositary institution is: Chinese Typical Representative culture collection center, preservation address is: Wuhan, China Wuhan University, preservation date is: on November 21st, 2011.Classification And Nomenclature is: Arthrobacter DSC4Arthrobacter sp.DSC4.The VBNC state flora that above-mentioned bacterial strain is usingd in sewage biological treatment system is as separation source, the VBNC resource bacterium that utilizes recovery can cultivation technology to obtain.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; Cell atrichia, do not move; Gram positive bacterium.
In order to realize second above-mentioned object, the present invention has adopted following technical scheme: a kind of activation medium for above-mentioned Arthrobacter DSC4 bacterial strain, and this substratum has following component to form by weight percentage: nutrient broth medium 1.0～3.0g; Yeast extract paste 0.2～0.5g; Glucose 1.5～5.0g; Agar 1.0～4.0%; Containing the recovery nutrient solution 2～20% of Resuscitation-promoting Factor Rpf, deionized water 1000ml; Above-mentioned per-cent is the volume percent that accounts for substratum total amount.
As preferably, the above-mentioned recovery nutrient solution containing Resuscitation-promoting Factor Rpf consists of substratum base fluid, bacterium liquid and liquid soil; Described bacterium liquid is cultured to the logarithmic phase later stage on bacterial classification liquid nutrient medium by micrococcus luteus bacterial classification, through centrifugal removal bacterial classification liquid nutrient medium, extract and obtain protein, contains Resuscitation-promoting Factor Rpf, and bacterium liquid is the 1-10% of substratum base fluid by volume; Described liquid soil by through air-dry, sieve, go deimpurity garden mould, add tap water, repeatedly sterilizing, crosses leaching filtrate or centrifuging and taking supernatant liquor, liquid soil is the 1-5% of substratum base fluid by volume.
The activation medium compound method of above-mentioned Arthrobacter DSC4 bacterial strain, the method comprises the following steps: to take nutrient broth medium 1.0～3.0g, yeast extract paste 0.2～0.5g, glucose 1.5～5.0g, deionized water 1000ml, pH is adjusted to 7.0, adds 1.0～4.0 agar, after 110～130 ℃, 10～30min autoclaving, is cooled to room temperature; Separately under aseptic condition, add 2～20% (v/v) through the recovery nutrient solution of 0.22 μ m membrane filtration sterilizing, pH7.0, stirring and evenly mixing, dispensing is solid, is inverted 4-10 ℃ of preservation stand-by.
As preferably, the preparation method of above-mentioned recovery nutrient solution is as follows: the gamboge coccus bacterial classification that 1) takes a morsel is inoculated in bacterial classification liquid nutrient medium, and 25～35 ℃, 100～150rpm vibrate cultivation; Bacterial growth is to the logarithmic phase later stage, 3000～8000rpm for nutrient solution, and thalline is removed in the centrifugal extracting of 10～30min; After 0.20～0.25 μ m membrane filtration sterilizing ,-30～-10 ℃ save backup; 2) learn from else's experience air-dry, sieve, go deimpurity garden mould 1kg, add tap water 1～2L, 110～130 ℃, the sterilizing of 10～40min high-pressure sterilizing pot; Room temperature, after an evening, is used same method sterilizing once again; 1500～2500r/min low-speed centrifugal is got supernatant liquor or is obtained filtrate with filter paper filtering waste, and cooling after 110～130 ℃, the sterilizing of 10～30min high-pressure sterilizing pot ,-30～-10 ℃ save backup; 3) accurately take each component of substratum base fluid, pH is adjusted to that to be cooled to room temperature after 7.0,110～130 ℃, the sterilizing of 10～30min high-pressure sterilizing pot stand-by; The separation of the suitable most bacteriums of substratum base fluid, for having particular requirement and selectively bacterium can corresponding plus-minus nutrition composition; 4) before using, under aseptic condition, in substratum base fluid, add 1-10% bacterium liquid, then add 1-5% liquid soil, mix rear use.
In order to realize the 3rd above-mentioned object, the present invention has adopted following technical scheme: the VBNC Arthrobacter DSC4 bacterial strain that above-mentioned recovery can cultivation is applied to the denitrification denitrogenation containing nitrate matrix.Concrete method is: after the activated cultivation of VBNC Arthrobacter DSC4 bacterial strain that can cultivation by described recovery, front cultivation, add in sewage biological treatment system.
As preferably, the substratum that described activation culture adopts consists of following component by weight percentage: nutrient broth medium 1.0～3.0g; Yeast extract paste 0.2～0.5g; Glucose 1.5～5.0g; Agar 1.0～4.0%; Containing the recovery nutrient solution 10% of Resuscitation-promoting Factor Rpf, deionized water 1000ml; Above-mentioned per-cent is the volume percent that accounts for substratum total amount.
As preferably, the substratum that described front cultivation adopts consists of following component by weight percentage: peptone 3.0～8.0g, and yeast extract paste 0.2～0.8g, glucose 3.0～8.0g, NaCl1.5～5.5g, 1000ml deionized water, pH is adjusted to 7.0.
The present invention is owing to having adopted above technical scheme, the cultivated change VBNC Arthrobacter DSC4 bacterial strain obtaining, can make Giltay substratum reaction solution start to transfer mazarine to and see aerogenesis phenomenon by green, illustrate that this bacterial classification can utilize nitrate reduction to form alkaline matter, and aerogenesis makes indicator colour-change.Record nitric nitrogen transformation efficiency is 54.7% simultaneously, and total nitrogen decrement is 52.9%, the remaining of ammonia-state nitrogen and nitrite nitrogen do not detected, has obvious denitrification denitrogenation effect.
Accompanying drawing explanation
Fig. 1 is the bacterium colony figure of Arthrobacter DSC4 bacterial strain of the present invention.
Fig. 2 is the denitrification denitrogenation design sketch of Arthrobacter DSC4 bacterial strain of the present invention.
Biomaterial preservation explanation recovery can cultivation VBNC Arthrobacter DSC4 (Arthrobacter sp.DSC4) bacterial strain, the deposit number of this bacterial strain is CCTCC No:M2011406, depositary institution is: Chinese Typical Representative culture collection center (CCTCC), preservation address is: Wuhan, China Wuhan University, preservation date is: on November 21st, 2011.
Embodiment
Substratum preparation method described in the embodiment of the present invention is as follows: 1, NYGR substratum: take Nutrient broth1.6g, Yeast extract0.5g, Glucose2.5g, deionized water 1000ml, pH is adjusted to 7.0, adds 2% agar, after 121 ℃, 15min autoclaving, is cooled to room temperature; Separately under aseptic condition, add 10% (v/v) through the recovery nutrient solution of 0.22 μ m membrane filtration sterilizing, pH7.0, stirring and evenly mixing, dispensing is solid; 2, PYGN substratum: Peptone5.0g, Yeast extract0.5g, Glucose5.0g, NaCl2.5g, 1000ml deionized water, pH is adjusted to 7.0; 3, Giltay substratum: A liquid: KNO
31.0g, l-asparagine 1.0g, 1%BTB spirituous solution 5.0mL, distilled water 500mL; B liquid: Trisodium Citrate 8.5g, MgSO
47H
2o1.0g, FeCl
36H
2o0.05g, KH
2pO
41.0g, CaCl
22H
2o0.2g, distilled water 500mL.Mix A, B two solution, regulate pH value 7.0～7.2, standby through sterilizing.
4, recovery nutrient solution substratum is comprised of A, B, forms (be called for short: ABC recovery substratum) with C composition.
A composition: bacterium liquid: gamboge coccus Micrococcus luteus IAM14879 bacterial classification is cultured to the logarithmic phase later stage on LMMD substratum, extracts and obtains protein through centrifugal substratum, contains Resuscitation-promoting Factor Rpf, and consumption is 5% of C one-tenth partial volume.
B composition: liquid soil: the garden mould 1kg air-dry, that sieve, remove the impurity such as stone grass roots that learns from else's experience, adds tap water 1L, 121 ℃, the sterilizing of 30min high-pressure sterilizing pot; Room temperature after an evening, sterilizing once again, in triplicate; 2000r/min low-speed centrifugal is got supernatant liquor (or obtaining filtrate with filter paper filtering), and consumption is 2% of C one-tenth partial volume.
C composition: bacto-tryptone (Difco company) (Bacto peptone (Difco)) 10.0g, yeast extract (Yeast extract (Difco)) 5.0g, malt extract (Malt extract (Difco)) 5.0g, (hydrolysis) casamino acids (Casamino acids (Difco)) 5.0g, extractum carnis (Beefextract (Difco)) 2.0g, glycerine (Glycerol) 2.0g, tween 80 (Tween80) 0.05g, magnesium sulfate heptahydrate (MgSO
47H
2o) 1.0g, adds deionized water 1000mL, pH7.0.
Above substratum is liquid culture based component, during as preparation solid medium, need add agar 15-20g, through 121 ℃, and cooling use after 15min autoclaving.
In A composition, LMMD substratum forms: ammonium chloride (NH
4cl) 4.0g, potassium primary phosphate (KH
2pO
4) 1.4g, vitamin H (Biotin) 0.005g, L-Methionine (L-Methionine) 0.02g, VitB1 (Thiamine) 0.04g, inosine (Inosine) 1.0g, magnesium sulfate heptahydrate (MgSO
47H
2o) 0.07g, mineral solution (Mineral solution) 1.0ml, Pfansteihl lithium (Lithium L-lactate) 10.0g, distilled water (Distilled water) 1000ml, pH7.5.
Wherein, above-mentioned mineral solution (Mineral solution): copper sulfate (CuSO
4) 0.24g, Manganous chloride tetrahydrate (MnCl
2) 0.50g, ferrous sulfate (FeSO
4) 0.10g, Sodium orthomolybdate (Na
2moO
4) 0.025g, zinc sulfate (ZnSO
4) 0.05g, distilled water (distilled water) 1000ml.
Be more than that general bacteria resuscitation is cultivated the formula using.According to the requirement of different ecological environment, C composition can be done corresponding adjustment.
The compound method of recovery substratum: the 1. gamboge coccus bacterial classification that takes a morsel is inoculated in LMMD liquid nutrient medium, 30 ℃, 120rpm vibrate cultivation; Bacterial growth is to the logarithmic phase later stage, nutrient solution 5000rpm, and thalline is removed in the centrifugal extracting of 20min; After 0.22 μ m membrane filtration sterilizing ,-20 ℃ save backup; 2. the garden mould 1kg air-dry, that sieve, remove the impurity such as stone grass roots that learns from else's experience, adds tap water 1L, 121 ℃, the sterilizing of 30min high-pressure sterilizing pot; Room temperature, after an evening, is used same method sterilizing once again; 2000r/min low-speed centrifugal is got supernatant liquor (or with filter paper filtering waste obtain filtrate), and cooling after 121 ℃, the sterilizing of 15min high-pressure sterilizing pot ,-20 ℃ save backup; 3. accurately take each component of C composition, pH is adjusted to that to be cooled to room temperature after 7.0,121 ℃, the sterilizing of 15min high-pressure sterilizing pot stand-by.The separation of the suitable most bacteriums of C component, for having particular requirement and selectively bacterium can corresponding plus-minus nutrition composition; 4. before using, should under aseptic condition, in C composition, add 5% (A/C) A composition, then add 3% (B/C) B composition, mix rear use.
Embodiment 1 is from sewage biological treatment system, utilize recovery to obtain VBNC Arthrobacter DSC4 bacterial strain preservation of bacteria strain by cultivation technology screening, can cultivation VBNC Arthrobacter DSC4 strains separation from certain sewage biological treatment system, in system, have more than 90% general traditional separation method and be difficult to the bacterium obtaining, the non-cultivation (VBNC) in living is similar to dormant state, through utilizing recovery to obtain in the Arthrobacter DSC4 of VBNC state bacterial strain by cultivation technology screening.The deposit number of this bacterial strain is CCTCC No:M2011406, and depositary institution is: Chinese Typical Representative culture collection center, preservation address is: Wuhan, China Wuhan University, preservation date is: on November 21st, 2011.Classification And Nomenclature is: Arthrobacter DSC4Arthrobacter sp.DSC4, this bacterial strain generally need be stored in superfreeze (subzero 80 degree).Must be through NYGR solid medium activation culture through the DSC4 of superfreeze preservation bacterial strain, be seeded on NYGR solid medium 30 ℃ and be inverted after activation culture 2-4d, can be stored in 4 ℃ of refrigerators standby temporarily.
DSC4 bacterial strain, on NYGR solid medium, is inverted after activation culture 2-7d for 30 ℃, and visible diameter 2-3mm is light grey or faint yellow, glossy bacterium colony (accompanying drawing 1).
DSC4 bacterial strain is dyed is gram positive bacterium, visible cell atrichia under opticmicroscope, do not move, without sporulation, without fluorochrome, form, be that quarter butt is to spherical 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, optimal pH 7.5.
Embodiment 2 can the culture identification method of cultivation VBNC Arthrobacter DSC4 bacterial strain to the denitrification denitrogenation effect of nitrate matrix, comprising the following steps: 1. to get can cultivation VBNC Arthrobacter DSC4 bacterial strain preservation of bacteria strain, under aseptic condition, the a small amount of strain transfer of picking to 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 in PYGN liquid nutrient medium, carries out cultivating before 30 ℃, 2d; 3. in Giltay substratum, access respectively 2% front cultivation bacterium liquid, 30 ℃, per minute 120 turn shaking table to be cultivated after 2d, observes ammonia-state nitrogen, nitrite nitrogen, nitric nitrogen and total nitrogen in substratum aerogenesis and colour-change situation and mensuration nutrient solution.
Ammonia nitrogen, nitrite nitrogen, nitric nitrogen and total nitrogen adopt respectively Nesslerization, ultraviolet spectrophotometry, GB water quality total nitrogen assay method (the mensuration alkalescence alkaline potassium per-sulfate digestion ultraviolet spectrophotometry of GB11894-89 water quality total nitrogen) to measure.The results are shown in Figure 2.Observing inoculation can make Giltay substratum reaction solution start to be transferred to mazarine and seen aerogenesis phenomenon by green by cultivation VBNC Arthrobacter DSC4 bacterial strain, illustrate that this bacterial classification can utilize nitrate reduction to form alkaline matter, and aerogenesis makes indicator colour-change.Record nitric nitrogen transformation efficiency is 54.7% simultaneously, and total nitrogen decrement is 52.9%, the remaining of ammonia-state nitrogen and nitrite nitrogen do not detected.
Claims (2)
- Arthrobacter ( arthrobactersp.) DSC4 bacterial strain, the deposit number of this bacterial strain is CCTCC No:M2011406, depositary institution is: Chinese Typical Representative culture collection center, preservation address is: Wuhan, China Wuhan University, preservation date is: on November 21st, 2011.
- 2. a sewage biological treatment system, is characterized in that: this sewage biological treatment system comprises the VBNC Arthrobacter DSC4 bacterial strain that recovery claimed in claim 1 can cultivation.
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| CN116286364B (en) * | 2022-09-08 | 2023-11-10 | 杭州秀川科技有限公司 | Compound for promoting anaerobic microorganism separation and culture and application thereof |
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