CN103555633B - The facultative chemoheterotrophy bacterium of the synchronous metabolism sulfide of one strain and nitrate - Google Patents
The facultative chemoheterotrophy bacterium of the synchronous metabolism sulfide of one strain and nitrate Download PDFInfo
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Abstract
The facultative chemoheterotrophy bacterium of the synchronous metabolism sulfide of one strain and nitrate, relates to a strain chemoheterotrophy bacterium. The invention provides the facultative chemoheterotrophy bacterium of the synchronous metabolism sulfide of a strain and nitrate, there is the ability of the organic matter of metabolism simultaneously, sulfide and nitrate. Does the facultative chemoheterotrophy bacterium of the synchronous metabolism sulfide of one strain and nitrate belong to pseudomonad X2(Pseudomonas? sp.X2), be deposited in Chinese Typical Representative culture collection center (CCTCC), preservation address is Luojiashan, Wuchang, Wuhan City, Hubei Province, preservation date is on August 5th, 2013, does is preserving number CCTCC? NO:M2013362. The effect of pseudomonad X2 of the present invention metabolism simultaneously organic matter, sulfide and nitrate, is applied in alternative various bacteria associating metabolism in sewage disposal system.
Description
Technical field
The present invention relates to a strain chemoheterotrophy bacterium.
Background technology
Many industrial trades use the Organic Ingredients that contains sulphur, nitrogen in process of production in a large number, cause having produced and contain in a large number sulphurThe organic wastewater of nitrogen adds that the sanitary sewage discharging in people's daily life has also produced a large amount of nitrogen-containing pollutants simultaneously, thisIf a little sewage is unprocessed, be directly discharged in natural environment, can produce all many-sided harm to it, and carry out a biological disposal uponThe advantages such as it is low that technology has operating cost, non-secondary pollution, are subject to extensive use in the world.
Process in the procedure of sulfur-bearing, nitrogenous effluent both at home and abroad, conventionally utilize the associating metabolism of various bacteria. I.e. profitUse heterotrophic denitrification bacterium, using organic matter as electron donor, nitrate anion carries out redox reaction as electron acceptor. ?In this process, remove organic matter and nitrate simultaneously. Utilize autotrophy desulfurization denitrifying bacteria, using sulfide as electron donor,Nitrate anion carries out redox reaction as electron acceptor. In this process, remove sulfide and nitrate simultaneously. But hereinReason method relies on multiple-microorganism synergy, metabolic process complexity, microbiologic population's poor stability, poor impact resistance, dirtWater treatment facilities stable operation is very difficult.
Summary of the invention
The invention provides a strain chemoheterotrophy bacterium, there is the ability of the organic matter of metabolism simultaneously, sulfide and nitrate.
The present invention is the facultative chemoheterotrophy bacterium of the synchronous metabolism sulfide of a strain and nitrate, and it is PseudomonasSp.X2, is preserved in Chinese Typical Representative culture collection center (CCTCC), and preservation address is Luojiashan, Wuchang, Wuhan City, Hubei Province,Preservation date is on August 5th, 2013, and preserving number is CCTCCNO:M2013362.
Pseudomonassp.X2(pseudomonad X2 of the present invention) be Gram-negative bacteria, be shaft-like, length is 1.0～1.5μ m, wide is 0.5～1.0 μ m, without gemma, single-stage flagellum, growth temperature range is 10 DEG C～40 DEG C, growth pH value scopeBe 7～10; The rounded projection of bacterium colony on LB solid medium, regular edges, surrounding clear, colorless, it is yellow that center is,Colony diameter is 1mm～2mm.
Pseudomonassp.X2(pseudomonad X2 of the present invention) can utilize dusty yeast, starch or acetic acid etc. as carbon sourceAnd utilize nitrate anion, ammonium ion to grow as nitrogenous source.
Pseudomonassp.X2(pseudomonad X2 of the present invention) the catalase positive, oxidase positive, anti-nitration reactionThe positive, hydrogen sulfide produces reaction negative.
Pseudomonassp.X2 of the present invention is by 16SrDNA gene order compare of analysis, with pseudomonas(Pseudomonas) homology of 16SrDNA sequence is the highest, and similitude is 95%, by conjunction with thalli morphology spyLevy, growth conditions, Physiology and biochemistry qualification result determine that pseudomonad X2 belongs to pseudomonas (Pseudomonas), forPseudomonassp.X2(pseudomonad X2) bacterial strain.
Pseudomonassp.X2(pseudomonad X2 of the present invention) can tachymetabolism organic matter, sulfide and nitrate,Pseudomonassp.X2(pseudomonad X2) can under aerobic conditions, breathe acquisition energy using organic matter as substrateMeasure and grow; Under oxygen free condition, using organic matter and sulfide as substrate, breathe and obtain using nitrate anion as electron acceptorObtain energy growth. Growing environment and condition are extensive, are conducive to keep biomass.
Pseudomonassp.X2(pseudomonad X2 of the present invention) be applied in sewage disposal system and can while metabolism haveMachine thing, sulfide and nitrate, can be at the organic carbon of 6h intracellular metabolite 61.8%, 32.1% sulfide and 57.1% nitreHydrochlorate, at the organic carbon of 12h intracellular metabolite 73%, 83% sulfide and 99% nitrate, with existing sewage disposal systemMiddle various bacteria associating metabolism is compared, and has simplified the complexity of metabolic process, Pseudomonassp.X2 of the present invention(pseudomonad X2) has actual application value for sulfur-bearing nitrogenous effluent; In microbiologic population's stability and resistance to impactAspect, Pseudomonassp.X2(pseudomonad X2 of the present invention) become dominant microflora Hou Shi microbiologic population stabilityHigh by 20% with various bacteria in the more existing sewage disposal system of resistance to impact.
Pseudomonassp.X2(pseudomonad X2 of the present invention) in sewage disposal system, tame and docile through activated sludge is directedChange Pseudomonassp.X2(pseudomonad X2) become dominant microflora, it has the organic matter of metabolism simultaneously, sulfideWith the ability of nitrate, institute is so that operation of sewage disposal system parameter is simple, and system is more stable, through high load capacity Sewage shockRear system is more easily recovered, and has better application prospect.
Pseudomonassp.X2 of the present invention belongs to pseudomonas (Pseudomonas), is preserved in Chinese Typical Representative and cultivatesThing preservation center (CCTCC), preservation address is Luojiashan, Wuchang, Wuhan City, Hubei Province, preservation date is August 5 in 2013Day, preserving number is CCTCCNO:M2013362.
Brief description of the drawings
Fig. 1 is the scanning electron microscope image of pseudomonad X2 of the present invention, and multiplication factor is 25000 times;
Fig. 2 be carry out sequence analysis structure by the 16SrDNA gene order of pseudomonad X2 and close bacterial strain beSystem is grown tree graph.
Detailed description of the invention
Technical solution of the present invention is not limited to following cited detailed description of the invention, also comprise between each detailed description of the invention arbitrarilyCombination.
Detailed description of the invention one: the facultative chemoheterotrophy bacterium of the present embodiment synchronous metabolism sulfide of one strain and nitrate, itFor Pseudomonassp.X2, be preserved in Chinese Typical Representative culture collection center (CCTCC), preservation address is Hubei Province's forceChinese city loujia hill belongs, preservation date is on August 5th, 2013, preserving number is CCTCCNO:M2013362.
The Pseudomonassp.X2(pseudomonad X2 of present embodiment) be Gram-negative bacteria, be shaft-like, length is1.0～1.5 μ m, wide is 0.5～1.0 μ m, without gemma, single-stage flagellum, growth temperature range is 10 DEG C～40 DEG C, growth pHValue scope is 7～10; The rounded projection of bacterium colony on LB solid medium, regular edges, surrounding clear, colorless, centerBe yellow, colony diameter is 1mm～2mm.
Reference " the outstanding Bacteria Identification handbook of uncle " the 8th edition and " common bacteria system identification handbook " are to Pseudomonassp.X2(pseudomonad X2) carries out Physiology and biochemistry qualification: the catalase positive, oxidase positive, the anti-nitration reaction positive, sulfurationHydrogen produces reaction negative.
The Pseudomonassp.X2(pseudomonad X2 of present embodiment) can tachymetabolism organic matter, sulfide and nitreHydrochlorate, pseudomonad X2 can breathe using organic matter as substrate and obtain energy growth under aerobic conditions; In nothingUnder oxygen condition, using organic matter and sulfide as substrate, breathe using nitrate anion as electron acceptor and obtain energy growth.Growing environment and condition are extensive, are conducive to keep biomass.
The Pseudomonassp.X2(pseudomonad X2 of present embodiment) being applied in can be contemporary in sewage disposal systemThank organic matter, sulfide and nitrate, combine metabolism with various bacteria in existing sewage disposal system and compare, simplifiedThe complexity of metabolic process; Aspect microbiologic population's stability and resistance to impact, pseudomonad X2 of the present invention becomes excellentIn gesture flora Hou Shi microbiologic population's stability and the more existing sewage disposal system of resistance to impact, various bacteria is high by 20%.
The Pseudomonassp.X2(pseudomonad X2 of present embodiment) fixed through activated sludge in sewage disposal systemTo domestication, pseudomonad X2 becomes dominant microflora, has the ability of the organic matter of metabolism simultaneously, sulfide and nitrate, instituteSimple to be operation of sewage disposal system parameter, system is more stable, and after high load capacity Sewage shock, system is more easily recovered,There is better application prospect.
The Pseudomonassp.X2(pseudomonad X2 of present embodiment) optimum growth temperature: 30 DEG C, the most suitable growthpH：7～8
Detailed description of the invention two: the pseudomonad X2 of present embodiment is obtained by screening in activated sludge. Screening is by following stepSuddenly carry out:
(1) preparation of bacteria suspension: the 100mL activated sludge that taking-up is gone from sewage treatment plant's secondary sedimentation tank is put into and detestedIn oxygen bottle, then add fluid nutrient medium in anaerobism bottle, under the condition of 30 DEG C, with 120r/min preculture 48h,To bacteria suspension;
Wherein said fluid nutrient medium is by containing 0.75gNa in every 1L fluid nutrient medium2S，1gKNO3，1gCH3COONa，1gNH4Cl，1.5gKH2PO4，3gNa2HPO4，0.1gMgSO4；
(2) bacteria suspension gradient dilution: under aseptic technique, get the bacteria suspension 1mL that step (1) obtains and joinThe concentration of making bacteria suspension in the anaerobism pipe that contains 9mL sterilized water is 10-1, then carry out the gradient of 10 times with same methodDilution, until the concentration of dilution is 10-8Till;
(3) static constant temperature culture: under aseptic technique, by outstanding the bacterium that obtains each extension rate in step (2)Liquid respectively takes out 1ml, puts into respectively the anaerobism pipe that contains solid medium, makes the solid of bacteria suspension wall built-up to anaerobism inside pipe wallOn culture medium, remove in anaerobism pipe after remaining bacteria suspension, obtain the anaerobism pipe that contains bacteria suspension wall built-up, will contain bacteria suspensionThe anaerobism bottle of wall built-up is put into incubator, and static constant temperature culture 72-96h under the condition of 30 DEG C, obtains single bacterium colony, observes singleThe size of bacterium colony and its characteristic morphology;
The wherein said anaerobism pipe that contains solid medium, by being prepared as follows: discharge the air in anaerobism pipe, oftenSolid agar culture before adding 7ml to solidify in anaerobism pipe, covers tightly rubber plug, is under the condition of 121 DEG C in temperatureSterilizing 15min, adopts Heng Gaite anaerobism to roll pipe method and prepares the anaerobism pipe that contains solid medium;
Wherein said Solid agar culture is formulated by fluid nutrient medium and agar step (1) Suo Shu, whereinThe mass volume ratio of agar and fluid nutrient medium is 15g:1L;
(4) shake constant temperature culture: under aseptic technique, the single bacterium colony transfer needle obtaining in step (3) is accessedIn sterilized fluid nutrient medium, in constant temperature air bath shaking table, be that 30 DEG C, shaking speed are 120r/min in temperatureCondition under cultivate;
(5) repeat to cultivate: under aseptic technique, getting the OD600 value that step (4) obtains is 1～1.5 bacteria suspensionIt is 10 that 1mL joins the concentration of making bacteria suspension in the anaerobism pipe that contains 9mL sterilized water-1, then carry out with same methodThe gradient dilution of 10 times, until the concentration of dilution is 10-8Till, then repeating step (3) and step (4), until fine jadeThe morphological feature of all single bacterium colonies of growing on fat solid medium is: rounded projection, and regular edges, surrounding clear, colorless,It is yellow that center is, and obtains pseudomonad X2.
The pseudomonad X2 that screening is obtained carries out Molecular Identification, carries out according to the following steps:
(1) utilize bacterial genomes to extract kit and extract total DNA of pseudomonad X2 pure culture, adopt bacterial 16 SRDNA universal primer F27, R1492, carry out pcr amplification taking genomic DNA as template. Then utilize glue to reclaim examinationAgent box (buying from Tian Gen biochemical technology Co., Ltd) reclaims purified pcr product, clones afterwards, transforms, screeningPositive colony daughter colony is entrusted the order-checking of Hua Da genome company after expanding cultivation.
The 16SrRNA gene order length of pseudomonad X2 is 1411bp, and its sequence is committed to GenBank, to determineThe race relation of bacterial strain. Homology analysis result shows, the 16SrDNA of this sequence and pseudomonas (Pseudomonas)The homology of sequence is the highest, and similitude is 95%, by identifying in conjunction with morphological features, growth conditions, Physiology and biochemistryResult determines that pseudomonad X2 belongs to pseudomonas (Pseudomonas), is the false unit cell of Pseudomonassp.X2(Bacterium X2) bacterial strain.
PCR primer is bought from Hua Da genome company, and other reagent are purchased from precious bioengineering Co., Ltd.
To the Pseudomonassp.X2(pseudomonad X2 of present embodiment) carry out functional verification:
Pseudomonassp.X2(pseudomonad X2) efficacy detection of metabolism organic matter, sulfide and nitrate, concreteMethod is as follows:
1. with distilled water preparation efficacy detection culture medium, described efficacy detection culture medium is by every 1L efficacy detection culture mediumIn contain 0.75gNa2S，1gKNO3，1gCH3COONa，1gNH4Cl，1.5gKH2PO4，3gNa2HPO4，0.1gMgSO4；
2. get the efficacy detection culture medium 200mL that 1. step configure and put into anaerobism bottle, after emptying the oxygen in anaerobism bottle,At 121 DEG C, under 15min condition, the anaerobism bottle containing efficacy detection culture medium is carried out to sterilizing, obtain examining containing effect after sterilizingSurvey the anaerobism bottle of culture medium;
3. contain efficacy detection culture medium by what cultivate that the bacteria suspension 5mL that is 1～1.5 to OD600 value joins that 2. step obtainAnaerobism bottle in, then by it at 30 DEG C, under 120r/min condition, cultivate, cultivating after 0h, 6h and 12h, use DionexICS-3000 type ion chromatograph is measured Pseudomonassp.X2(pseudomonad X2) organic in the culture medium of growingThe variation of thing, sulfide and nitrate, wherein the running parameter of DionexICS-3000 type ion chromatograph is: adoptIonPacAG4AAS4A-SC4mm analytical column, leacheate composition is 1.8mmol/LNa2CO3、1.7mmol/LNaHCO3, flow velocity is 1cm3/ min; Obtain table 1:
Table 1 is for cultivating Pseudomonassp.X2(pseudomonad X2 after 0h, 6h and 12h) the bacterium culture medium of growingThe variation of middle organic matter, sulfide and nitrate
Note: unit (mg/L)
As can be seen from Table 1: pseudomonad X2(Pseudomonassp.X2) can be at 12h intracellular metabolite 73% organicCarbon, 83% sulfide and 99% nitrate, have actual application value for sulfur-bearing nitrogenous effluent.
Claims (1)
1. the facultative chemoheterotrophy bacterium of the synchronous metabolism sulfide of a strain and nitrate, is characterized in that it isThiopseudomonasdenitrificansX2, is deposited in Chinese Typical Representative culture collection center, and preservation date is 2013 8The moon 5, preserving number is CCTCCNO:M2013362.
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| CN101993833A (en) * | 2010-02-05 | 2011-03-30 | 哈尔滨工业大学 | Heterotrophy denitrifying bacteria for synchronously degrading sulfide, nitrate and organic carbon source |
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| CN101993833A (en) * | 2010-02-05 | 2011-03-30 | 哈尔滨工业大学 | Heterotrophy denitrifying bacteria for synchronously degrading sulfide, nitrate and organic carbon source |
Non-Patent Citations (2)
| Title |
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| Electricity harvest from nitrate/sufide-containing wastewaters using microbial fuel cell with autotrophic denitrifier,Pseudomonas sp.C27;Chin-Yu Lee,et al;《International Journal of Hydrogen Energy》;20121031;第37卷(第20期);15827-15832 * |
| 一株铁/硝酸盐还原菌分离、特征鉴定及其与铁氧化物作用;汪国威;《中国优秀硕士学位论文全文数据库》;20140315(第3期);A006-198 * |
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