CN101307297A - Separating, purifying and identifying method with anaerobic humus respiration capability reducing bacterium - Google Patents
Separating, purifying and identifying method with anaerobic humus respiration capability reducing bacterium Download PDFInfo
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- CN101307297A CN101307297A CNA2007101646088A CN200710164608A CN101307297A CN 101307297 A CN101307297 A CN 101307297A CN A2007101646088 A CNA2007101646088 A CN A2007101646088A CN 200710164608 A CN200710164608 A CN 200710164608A CN 101307297 A CN101307297 A CN 101307297A
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Abstract
The invention discloses a separation purification and identification method for a reduction bacterium with an anaerobic humus respiratory capability. That is, the method comprises the following: taking different carbon sources as an electron donor, taking a humus type anthraquinone-2,6-disulfonate as an electron acceptor, acclimating an enrichment bacterium with the humus respiratory capability, and determining the bacterium as the humus reduction bacterium based on the capacity of the humus type anthraquinone-2,6-disulfonate and identifying the taxonomic status of the bacterium. The method adopts a culture medium suitable for the growth of the humus reduction bacterium and adds sodium formate or sodium acetate or glucose into a basic inorganic salt culture medium. Firstly a strain is purified to determine the anaerobic humus reduction capability of the strain, and then a bacterial spawn is identified. The method obtains a pure flora with the anaerobic humus respiratory capability, thereby supplying the spawn for microbiological study and industrialized application, and proving the physiological biochemical characteristics of the strain for easy microbial preservation and volume amplification with simple separation and identification processes and reliable results.
Description
Technical field
The present invention relates to a kind of separation and purification and authentication method with anaerobic humus respiration capability reducing bacterium.
Background technology
Anaerobic humus respiration (Anaerobic humus respiration) reported first was in 1996, it is one is electron donor with the organic carbon source, with soil ulmin (quinones substance, as: anthraquinone-2,6-disulfonate AQDS, anthraquinone-2-sulfonic acid salt AQS, humic acid) be unique terminal electron acceptor, the generation of coupling energy, the complex biological chemical reaction process of support thalli growth, the reaction product of this process is for (for example: acetate going back the ortho states soil ulmin
-+ 4H
2O+4AQDS → 4AH
2DS+2HCO
3 -+ H
+, lactate
-+ 2H
2O+2AQDS → 2AH
2DS+acetate
-+ HCO
3 -+ H
+)
[1]Under anaerobic, certain micro-organisms can soil ulmin as unique electron acceptor(EA), the different pollution substances in the well-oxygenated environment.In the microbial respiratory field, it has broken through traditional soil ulmin to the nonactive theory of microbial metabolism, people grind brand-new soil ulmin/collaborative reducing metal of soil ulmin reduction bacterium and the degraded organic or inorganic pollutent technology created based on this, as soil ulmin/collaborative reducing metal Fe of soil ulmin reduction bacterium
3+, soil ulmin/soil ulmin reduction bacterium Synergistic degradation azoic dyestuff and soil ulmin/soil ulmin reduction bacterium Synergistic degradation toluene etc.These soil ulmin/related microorganism of soil ulmin reduction bacterium synergy is a heterotrophic organism, soil ulmin then is a kind of complicated organism of difficult microbiological deterioration, and it goes back ortho states can be reduced bacterium reduction and realize the oxidation state soil ulmin and go back between the ortho states soil ulmin circulating by soil ulmin again after oxidized.Soil ulmin/soil ulmin reduction bacterium synergy not only adds organic carbon source outside and exists down, the reducing heavy metal hardly degraded organic substance can constantly circulate, and can as electron donor it be degraded by some organic pollutant under the situation of additional carbon not having, consider soil ulmin and extensive existence, can provide foundation as the original position reparation of anaerobism contaminate environment with microorganism of humus respiration capability.
In natural ecosystems, because humic matter extensively exists, and the extensive existence that can be used as the organic matter of microbe carbon source, usually take place soil ulmin also pathogenic microorganism with organic carbon source as electron donor, be the phenomenon of humus respiration with soil ulmin as the electron transport of unique acceptor.Now prove, in the active sludge of abundant settling, Contaminated soil and wastewater treatment of organic content, all can detect anaerobic humus respiration
[2-10]Organic content abundant natural habitat sample or artificial habitat sample be the good separation source of anaerobic humus reduction bacterium.
Up to now, the reduction bacterium of the tool anaerobic humus respiration capability of bibliographical information has many kinds, belongs to respectively in the dissimilar reduction bacterium, as Fe (III) reduction bacterium [(Geobacter spp.)
[3], nitrate reduction bacterium (Shewanellaputrefaciens)
[4], sulphur reduction bacterium (Desulfurom onas spp.)
[5], fermenting bacteria (Propionibacterium freudenreichii)
[6], thermophilic product methane archeobacteria or the like (M ethanococcusthermolithotrophicus)
[7]These anaerobic humus reduction bacterium all are to utilize the bacterial classification that adopts traditional method to separate to obtain to identify and be defined as having the reduction bacterium of humus respiration capability through the soil ulmin reduction again.
Reference:
[1] Lovley D, Coates J, Blunt-Harris EL, et al. soil ulmin is used for microbial respiratory as electron acceptor(EA). nature (Humic substancesas electron acceptors for microbial respiration.Nature,) 1996,382:445-448.
[2] Maurice G, Sander A, Bas Godschalk, et al. chlorine is breathed bacterium and associated biomolecule to the anaerobic reduction of quinones substance and oxidation and to the reduction (Anaerobic reduction and oxidation of quinone moieties and the reduction of oxidized metalsby halorespiring and related organisms.FEMS Microbiol Ecol.) 2004 of oxidation state metal, 49:145-150.
[3] Coates J, Bhupathiraju V, Achenbach L, et al. ground bar is had a liking for the hydrogen bacterium, ground bar chapellei and ground bar grbiciae, three kinds of novel strictly anaerobic alienation Fe (III) reduction bacterium. (Geobacter hydrogenophilus, Geobacter chapellei and Geobacter grbiciae, three new, strictly anaerobic, dissimilatory Fe (III)-reducers) .Int J Syst Evol Microbiol, 2001,51:581-588.
[4] Coates J, Ellis D, Blunt-Harris E, et al. derives from the enrichment of the soil ulmin reduction bacterium of multiple environment, applied environment microorganism (Recovery of Humic-Reducing Bacteria from a Diversity of Environments.Appl Environ Microbiol), 1998,64 (4): 1504-1509.
[5] Lovley D, Fraga J, Blunt-Harris E, et al. humic matter is used for the microorganism catalysis reducing metal as mesosome, metal catalytic reduction (Humic Substances as a Mediator for Microbially Catalyzed Metal Reduction.) 1998,26 (3): 152-157.
[6] Benz M, Schink B, Brune A. propionibacterium freudenreichii and other fermenting bacterias reduction humic acid, applied environment microorganism (Humic Acid Reduction by Propionibacterium freudenreichii and Other Fermenting Bacteria.Appl EnvironMicrobiol.) 1998,64 (11): 4507-4512.
[7] Lovley D, Kashefi K, Vargas M, super thermophilic microorganism reduction humic matter of et al. and Fe (III), geochemistry (Reduction ofhumic substances and Fe (III) by hyperthermophilic microorganisms.Chem Geol), 2000,169 (3-4): 289-298.
[8] CervantesF, Dijksma W, Duong-dac T, et al. is at quinone and soil ulmin domestication settling anaerobism mineralising toluene during as electron acceptor(EA), applied environment microorganism (Anaerobic mineralization of toluene by enriched sediments with quinones and humus asterminal electron acceptors.Appl Environ Microbiol.) 2001,67 (10): 4471-4478.
[9] Cervantes F, van der Velde S, Lettinga G, et al. quinone promotes microbiological anaerobic oxidation aldehydes matter as the terminal electronic acceptor, biological degradation (Quinones as terminal electron acceptors for anaerobic microbial oxidation of phenolic compounds.Biodegradation) .2000,11:313-321.
[10] Cervantes F, Duong-Dac T, Ivanova A, et al. with quinones substance as terminal electronic acceptor selective enrichment ground bacillus from anaerobic grain sludge. biotechnology wall bulletin (Selective enrichment of Geobacter sulfurreducens from anaerobic granular sludgewith quinones as terminal electron acceptors.Biotechnol Lett,) 2003,25:39-45.
[11] eastern elegant pearl, Cai Miaoying, etc. common bacteria system identification handbook. Science Press, 2001.
[12] Holt J., Krieg N., Sneath P. et al. uncle Jie Shi Bacteria Identification handbook (the 9th edition), William Si, Louis Wilkins, (Bergey ' s manualof determinative bacteriology (9th edition) Williams; Wilkins) 1994:427-455.
[13] Staley J, Bryant M, PfennigN and Holt J. uncle Jie Shi bacterial system handbook (the 3rd volume), William Si, Louis Wilkins (Bergey ' smanual of systematic bacteriology (Vol.3) .Williams; Wilkins) 1989:1807-1835.
Summary of the invention
The invention provides a kind of separation and purification and authentication method with anaerobic humus respiration capability reducing bacterium.
Separation purification method with anaerobic humus respiration capability reducing bacterium comprises the steps:
1) choose the natural habitat and the sample conduct of artificial habitat of being rich in organic matter and have the separation source of the reduction bacterium of anaerobic humus respiration capability, the natural habitat sample is a bottom mud in lake, and artificial habitat sample is municipal sewage plant's anaerobic digester mud;
2) in separation source, add liquid nutrient medium as enrichment matrix, contain the 6mM sodium formiate in the liquid nutrient medium, 3mM sodium acetate or 1mM glucose, also contain soil ulmin pattern thing anthraquinone-2, the basic inorganic salt liquid of 6-disulfonate, added the 6mM sodium formiate again every 5～7 days, 3mM sodium acetate or 1mM glucose and basic inorganic salt liquid, and oxygenation is with the orange red ortho states anthraquinone-2 of going back, the 6-disulfonate is oxidized to lurid anthraquinone-2, the 6-disulfonate, with nitrogen unnecessary oxygen is driven out of then, repeat 7～9 times domestication enrichment anaerobic humus reduction granule sludge so continuously;
3) get enrichment and get anaerobic humus reduction granule sludge, use ultrasonic disruption, make elementary bacteria suspension, stepwise dilution is made serial bacteria suspension again;
4) with serial bacterial suspension inoculation in the solid medium that melts, under 28-30 ℃, carry out anaerobism and roll the cultivation of pipe method, with kapillary anaerobism being rolled the orange red single bacterium colony that grows on the pipe chooses in the liquid nutrient medium, cultivated 7-10 days 28-30 ℃ of following anaerobism, after the cultivation, adopt anaerobism to roll the pipe method again and separate single bacterium colony, so repeat 4～5 times, when the bacterial cell form of examining under a microscope is consistent, obtain to have the pure flora of humus respiration capability.
Described liquid culture based formulas is: NH
4Cl 1500mg/L, NaH
2PO
4600mg/L, CaCl
2.2H
2O 100mg/L, KCl 100mg/L, MgCl
2.6H
2O 2mg/L, MnCl
2.4H
2O 5mg/L, NaMoO
4.2H
2O 1mg/L, anthraquinone-2,6-disulfonate 1mM, 6mM sodium formiate, 3mM sodium acetate or 1mM glucose.
Described solid medium is for adding agar 20g/L again in the aforesaid liquid substratum.
Authentication method with reduction bacterium of anaerobic humus respiration capability comprises the steps::
1) will have the pure flora of humus respiration capability, at the centrifugal results somatic cells of 5000rpm, make the thalline suspension with liquid nutrient medium, under anaerobic condition, soil ulmin pattern thing anthraquinone-2, the 6-disulfonate is reduced to the orange red ortho states anthraquinone-2 of going back, and the ability of 6-disulfonate judges whether this bacterial strain is soil ulmin reduction bacterium;
2) determine the classification position of bacterial strain with thalli morphology and 16S rDNA sequence, thalli morphology comprises observation and the observation of cultivating thalline and the observation of bacterial cell structure of cultivating bacterium colony, the poly-extraction strain gene group DNA that comprises of the mensuration step of 16S rDNA sequence, pcr amplification 16S rDNA, purifying 16S rDNA, measure the 16SrDNA sequence,, determine the classification position of bacterial strain existing 16SrDNA sequence alignment among the 16S rDNA sequence that records and the GenBank (www.ncbi.com).
Advantage of the present invention: 1) can obtain the pure growth of anaerobic humus reduction bacterium, for microbiological research and industrial applications provide bacterial classification; 2) physio-biochemical characteristics of the separated bacterial strain of understanding are convenient to culture presevation and a large amount of amplification; 3) qualification program is simple, credible result.
Embodiment
Below in conjunction with specific embodiment the present invention is further described, but protection scope of the present invention is not limited in this.
Embodiment 1
The step of anaerobic humus reduction bacterium isolation identification is as follows:
1, this example is chosen the separation source of bottom mud in lake as the reduction bacterium of anaerobic humus respiration capability.
2, to add the 6mM sodium formiate and to contain 1mMAQDS (anthraquinone-2, the 6-disulfonate) basic inorganic salt liquid nutrient medium is as enrichment matrix, adopt the mode of batch culture, cultivate anaerobic humus reduction granule sludge, added 6mM sodium formiate and basic inorganic salt liquid again every 5～7 days, and oxygenation is with the orange red ortho states anthraquinone-2 of going back, the 6-disulfonate is oxidized to lurid anthraquinone-2, the 6-disulfonate, with nitrogen unnecessary oxygen is driven out of then, repeat 7～9 times domestication enrichment anaerobic humus reduction granule sludge so continuously;
3, get enrichment and get anaerobic humus reduction granule sludge, use ultrasonic disruption, make bacteria suspension, serial dilution is made bacteria suspension step by step again.
4, with serial bacterial suspension inoculation in the solid medium that melts, under 28-30 ℃, carry out anaerobism and roll the cultivation of pipe method, with kapillary anaerobism being rolled the orange red single bacterium colony that grows on the pipe chooses in the liquid nutrient medium, cultivated 7-10 days 28-30 ℃ of following anaerobism, after the cultivation, adopt anaerobism to roll the pipe method again and separate single bacterium colony, so repeat 4～5 times, when the bacterial cell form of examining under a microscope is consistent, obtain to have the pure flora of humus respiration capability.
5, with liquid nutrient medium amplification pure growth, centrifugal results somatic cells, make the thalline suspension with the basic inorganic salt substratum, add AQDS and sodium formiate, (go back ortho states anthraquinone-2 under the anaerobic condition AQDS is reduced to orange-red AH2QDS, the 6-disulfonate) ability judges whether this bacterial strain has anaerobic humus respiration capability.
6,, determine the classification position of bacterial strain, concrete reference literature according to the morphological specificity and the molecular biological characteristics of pure training thing
[12,13]Carry out.
1) morphological specificity comprises observation and the observation of cultivating thalline and the observation of bacterial cell structure of cultivating bacterium colony.Document is pressed in the bacterial cell morphologic observation
[11]The method dyeing of introducing adopts German Leica research microscope (DMLB+QCOLite) to observe.
2) molecular biological characteristics mainly is a 16S rDNA sequence, and it is measured to go on foot to gather and is:
A) adopt DNA of bacteria extraction agent box to extract the also genomic dna of the separated bacterium of purifying.
B) with total DNA of bacterium for touching plate, adopt the universal primer of bacterial 16 S rDNA to carry out pcr amplification.Forward primer 27F:5-GTTTGATCCTGGCTCA G-3 '; Reverse primer 1492R:5 '-GGTTACCTTGTTACGACTT-3 '.The amplification system of PCR is: 10 * PCR Buffer, 2.0 μ l, 10mmol/L dNTPs 2.0 μ l, 25mmol/L MgCl
20.8 μ l, 5U/ μ l Taq DNA enzyme 0.3 μ l, each 1.0 μ l of forward primer and reverse primer, the template DNA 3 μ l of 17ng/ μ l, ddH
2O 9.9 μ l, the reaction cumulative volume is 20 μ l.Amplified reaction: behind 94 ℃ of pre-sex change 5min; 94 ℃ of sex change 1min, 55 ℃ of annealing 1min, 72 ℃ are extended 2min, totally 35 circulations; In 72 ℃ of flat 7min of benefit, final temperature is 4 ℃ at last.The agarose gel electrophoresis of employing 1.5% detects the pcr amplification product of 16S rDNA, adopts the automatic gel images analytical system picked-up of the JS-380A amplification collection of illustrative plates of Shanghai Peiqing Science Co., Ltd.
C) purifying 16S rDNA entrusts bio tech ltd to measure 16S rDNA sequence.
D), determine the classification position of isolated strains with the comparison of existing sequence among the 16S rDNA sequence that records and the GenBank (www.ncbi.com)
Embodiment 2
The step of anaerobic humus reduction bacterium isolation identification is as follows:
1, this example is chosen bottom mud in lake as having the separation source of the reduction bacterium of anaerobic humus respiration capability.
2, with the basic inorganic salt liquid nutrient medium that contains 1mMAQDS that adds the 3mM sodium acetate as enrichment matrix, adopt the mode of batch culture, cultivate anaerobic humus reduction granule sludge, added 3mM sodium acetate and basic inorganic salt liquid again every 5～7 days, and oxygenation is with the orange red ortho states anthraquinone-2 of going back, the 6-disulfonate is oxidized to lurid anthraquinone-2, the 6-disulfonate, with nitrogen unnecessary oxygen is driven out of then, repeated 7～9 times so continuously, domestication enrichment anaerobic humus reduction granule sludge;
3, get enrichment and get anaerobic humus reduction granule sludge, use ultrasonic disruption, make bacteria suspension, serial dilution is made bacteria suspension step by step again.
4, with serial bacterial suspension inoculation in the solid medium that melts, under 28-30 ℃, carry out anaerobism and roll the cultivation of pipe method, with kapillary anaerobism being rolled the orange red single bacterium colony that grows on the pipe chooses in the liquid nutrient medium, cultivated 7-10 days 28-30 ℃ of following anaerobism, after the cultivation, adopt anaerobism to roll the pipe method again and separate single bacterium colony, so repeat 4～5 times, when the bacterial cell form of examining under a microscope is consistent, obtain to have the pure flora of humus respiration capability.
5, with liquid nutrient medium amplification pure growth, centrifugal results somatic cells is made the thalline suspension with the basic inorganic salt substratum, and interpolation AQDS and sodium acetate are under the anaerobic condition to be reduced to AQDS orange-red AH
2The ability of QDS judges whether this bacterial strain has anaerobic humus respiration capability.
6,, determine the classification position of bacterial strain, concrete reference literature according to the morphological specificity and the molecular biological characteristics of pure training thing
[12,13]Carry out.
1) morphological specificity comprises observation and the observation of cultivating thalline and the observation of bacterial cell structure of cultivating bacterium colony.Document is pressed in the bacterial cell morphologic observation
[11]The method dyeing of introducing adopts German Leica research microscope (DMLB+QCOLite) to observe.
2) molecular biological characteristics mainly is a 16S rDNA sequence, and it is measured to go on foot to gather and is:
A) adopt DNA of bacteria extraction agent box to extract the also genomic dna of the separated bacterium of purifying.
B) with total DNA of bacterium for touching plate, adopt the universal primer of bacterial 16 S rDNA to carry out pcr amplification.Forward primer 27F:5 '-GTTTGATCCTGGCTCA G-3 '; Reverse primer 1492R:5 '-GGTTACCTTGTTACGACTT-3 '.The amplification system of PCR is: 10 * PCR Buffer, 2.0 μ l, 10mmol/L dNTPs 2.0 μ l, 25mmol/L MgCl
20.8 μ l, 5U/ μ l Taq DNA enzyme 0.3 μ l, each 1.0 μ l of forward primer and reverse primer, the template DNA 3 μ l of 17ng/ μ l, ddH
2O 9.9 μ l, the reaction cumulative volume is 20 μ l.Amplified reaction: behind 94 ℃ of pre-sex change 5min; 94 ℃ of sex change 1min, 55 ℃ of annealing 1min, 72 ℃ are extended 2min, totally 35 circulations; In 72 ℃ of flat 7min of benefit, final temperature is 4 ℃ at last.The agarose gel electrophoresis of employing 1.5% detects the pcr amplification product of 16S rDNA, adopts the automatic gel images analytical system picked-up of the JS-380A amplification collection of illustrative plates of Shanghai Peiqing Science Co., Ltd.
C) purifying 16S rDNA entrusts bio tech ltd to measure 16S rDNA sequence.
D), determine the classification position of isolated strains with the comparison of existing sequence among the 16S rDNA sequence that records and the GenBank (www.ncbi.com).
Present embodiment is compared with embodiment 1 and changed sodium formiate into sodium acetate, and is same separable to the reduction bacterium with anaerobic humus respiration capability.
Embodiment 3
The step of anaerobic humus reduction bacterium isolation identification is as follows:
1, this example is chosen bottom mud in lake as having the separation source of the reduction bacterium of anaerobic humus respiration capability.
2, with the basic inorganic salt liquid nutrient medium that adds 1mM glucose and contain 1mMAQDS as enrichment matrix, adopt the mode of batch culture, cultivate anaerobic humus reduction granule sludge, added 1mM glucose and basic inorganic salt liquid again every 5～7 days, and oxygenation is with the orange red ortho states anthraquinone-2 of going back, the 6-disulfonate is oxidized to lurid anthraquinone-2, the 6-disulfonate, with nitrogen unnecessary oxygen is driven out of then, repeated 7～9 times so continuously, domestication enrichment anaerobic humus reduction granule sludge;
3, get enrichment and get anaerobic humus reduction granule sludge, use ultrasonic disruption, make bacteria suspension, serial dilution is made bacteria suspension step by step again.
4, with serial bacterial suspension inoculation in the solid medium that melts, under 28-30 ℃, carry out anaerobism and roll the cultivation of pipe method, with kapillary anaerobism being rolled the orange red single bacterium colony that grows on the pipe chooses in the liquid nutrient medium, cultivated 7-10 days 28-30 ℃ of following anaerobism, after the cultivation, adopt anaerobism to roll the pipe method again and separate single bacterium colony, so repeat 4～5 times, when the bacterial cell form of examining under a microscope is consistent, obtain to have the pure flora of humus respiration capability.
5, with liquid nutrient medium amplification pure growth, centrifugal results somatic cells is made the thalline suspension with the basic inorganic salt substratum, and interpolation AQDS and glucose are under the anaerobic condition to be reduced to AQDS orange-red AH
2The ability of QDS judges whether this bacterial strain has anaerobic humus respiration capability.
6,, determine the classification position of bacterial strain, concrete reference literature according to the morphological specificity and the molecular biological characteristics of pure training thing
[12,13]Carry out.
1) morphological specificity comprises observation and the observation of cultivating thalline and the observation of bacterial cell structure of cultivating bacterium colony.Document is pressed in the bacterial cell morphologic observation
[11]The method dyeing of introducing adopts German Leica research microscope (DMLB+QCOLite) to observe.
2) molecular biological characteristics mainly is a 16S rDNA sequence, and it is measured to go on foot to gather and is:
A) adopt DNA of bacteria extraction agent box to extract the also genomic dna of the separated bacterium of purifying.
B) with total DNA of bacterium for touching plate, adopt the universal primer of bacterial 16 S rDNA to carry out pcr amplification.Forward primer 27F:5 '-GTTTGATCCTGGCTCA G-3 '; Reverse primer 1492R:5 '-GGTTACCTTGTTACGACTT-3 '.The amplification system of PCR is: 10 * PCR Buffer, 2.0 μ l, 10mmol/L dNTPs 2.0 μ l, 25mmol/L MgCl
20.8 μ l, 5U/ μ l Taq DNA enzyme 0.3 μ l, each 1.0 μ l of forward primer and reverse primer, the template DNA 3 μ l of 17ng/ μ l, ddH
2O 9.9 μ l, the reaction cumulative volume is 20 μ l.Amplified reaction: behind 94 ℃ of pre-sex change 5min; 94 ℃ of sex change 1min, 55 ℃ of annealing 1min, 72 ℃ are extended 2min, totally 35 circulations; In 72 ℃ of flat 7min of benefit, final temperature is 4 ℃ at last.The agarose gel electrophoresis of employing 1.5% detects the pcr amplification product of 16S rDNA, adopts the automatic gel images analytical system picked-up of the JS-380A amplification collection of illustrative plates of Shanghai Peiqing Science Co., Ltd.
C) purifying 16S rDNA entrusts bio tech ltd to measure 16S rDNA sequence.
D), determine the classification position of isolated strains with the comparison of existing sequence among the 16S rDNA sequence that records and the GenBank (www.ncbi.com).
Present embodiment is compared with embodiment 1, embodiment 2 and changed sodium formiate, sodium acetate into glucose, and is same separable to the reduction bacterium with anaerobic humus respiration capability.
Embodiment 4
The step of anaerobic humus reduction bacterium isolation identification is as follows:
1, this example is chosen municipal sewage plant's anaerobic digester mud as having the separation source of the reduction bacterium of anaerobic humus respiration capability.
2, with the basic inorganic salt liquid nutrient medium that adds the 6mM sodium formiate and contain 1mMAQDS as enrichment matrix, adopt the mode of batch culture, cultivate anaerobic humus reduction granule sludge, added 3mM sodium acetate and basic inorganic salt liquid again every 5～7 days, and oxygenation is with the orange red ortho states anthraquinone-2 of going back, the 6-disulfonate is oxidized to lurid anthraquinone-2, the 6-disulfonate, with nitrogen unnecessary oxygen is driven out of then, repeated 7～9 times so continuously, domestication enrichment anaerobic humus reduction granule sludge;
3, get enrichment and get anaerobic humus reduction granule sludge, use the glass homogenizer fragmentation, make bacteria suspension, serial dilution is made bacteria suspension step by step again.
4, with serial bacterial suspension inoculation in the solid medium that melts, under 28-30 ℃, carry out anaerobism and roll the cultivation of pipe method, with kapillary anaerobism being rolled the orange red single bacterium colony that grows on the pipe chooses in the liquid nutrient medium, cultivated 7-10 days 28-30 ℃ of following anaerobism, after the cultivation, adopt anaerobism to roll the pipe method again and separate single bacterium colony, so repeat 4～5 times, when the bacterial cell form of examining under a microscope is consistent, obtain to have the pure flora of humus respiration capability.
5, with liquid nutrient medium amplification pure growth, centrifugal results somatic cells is made the thalline suspension with the basic inorganic salt substratum, adds AQDS and sodium formiate, under the anaerobic condition AQDS is reduced to the ability of orange-red AH2QDS, judge whether this bacterial strain has anaerobic humus respiration capability.
6,, determine the classification position of bacterial strain, concrete reference literature according to the morphological specificity and the molecular biological characteristics of pure training thing
[12,13]Carry out.
1) morphological specificity comprises observation and the observation of cultivating thalline and the observation of bacterial cell structure of cultivating bacterium colony.Document is pressed in the bacterial cell morphologic observation
[11]The method dyeing of introducing adopts German Leica research microscope (DMLB+QCOLite) to observe.
2) molecular biological characteristics mainly is a 16S rDNA sequence, and it is measured to go on foot to gather and is:
A) adopt DNA of bacteria extraction agent box to extract the also genomic dna of the separated bacterium of purifying.
B) with total DNA of bacterium for touching plate, adopt the universal primer of bacterial 16 S rDNA to carry out pcr amplification.Forward primer 27F:5 '-GTTTGATCCTGGCTCA G-3 '; Reverse primer 1492R:5 '-GGTTACCTTGTTACGACTT-3 '.The amplification system of PCR is: 10 * PCR Buffer, 2.0 μ l, 10mmol/L dNTPs 2.0 μ l, 25mmol/L MgCl
20.8 μ l, 5U/ μ l Taq DNA enzyme 0.3 μ l, each 1.0 μ l of forward primer and reverse primer, the template DNA 3 μ l of 17ng/ μ l, ddH
2O 9.9 μ l, the reaction cumulative volume is 20 μ l.Amplified reaction: behind 94 ℃ of pre-sex change 5min; 94 ℃ of sex change 1min, 55 ℃ of annealing 1min, 72 ℃ are extended 2min, totally 35 circulations; In 72 ℃ of flat 7min of benefit, final temperature is 4 ℃ at last.The agarose gel electrophoresis of employing 1.5% detects the pcr amplification product of 16S rDNA, adopts the automatic gel images analytical system picked-up of the JS-380A amplification collection of illustrative plates of Shanghai Peiqing Science Co., Ltd.
C) purifying 16S rDNA entrusts bio tech ltd to measure 16S rDNA sequence.
D), determine the classification position of isolated strains with the comparison of existing sequence among the 16S rDNA sequence that records and the GenBank (www.ncbi.com).
Compare with embodiment 1, present embodiment adopts different separation source and different anaerobic humus respiration sludge crushing modes can be separated to the reduction bacterium with anaerobic humus respiration capability equally.
Embodiment 5, the contrast experiment
The step of anaerobic humus reduction bacterium isolation identification is as follows:
1, this example is chosen bottom mud in lake as having the separation source of the reduction bacterium of anaerobic humus respiration capability.
2, with the basic inorganic salt liquid nutrient medium that do not contain AQDS as enrichment matrix, adopt the mode of batch culture, cultivate anaerobic humus reduction granule sludge, added 3mM sodium acetate and basic inorganic salt liquid again every 5～7 days, and oxygenation is with the orange red ortho states anthraquinone-2 of going back, the 6-disulfonate is oxidized to lurid anthraquinone-2, the 6-disulfonate, with nitrogen unnecessary oxygen is driven out of then, repeated 7～9 times so continuously, domestication enrichment anaerobic humus reduction granule sludge;
3, get enrichment and get anaerobic humus reduction granule sludge, use ultrasonic disruption, make bacteria suspension, serial dilution is made bacteria suspension step by step again.
4, with serial bacterial suspension inoculation in the solid medium that melts, under 28-30 ℃, carry out anaerobism and roll the cultivation of pipe method, with kapillary anaerobism being rolled the orange red single bacterium colony that grows on the pipe chooses in the liquid nutrient medium, cultivated 7-10 days 28-30 ℃ of following anaerobism, after the cultivation, adopt anaerobism to roll the pipe method again and separate single bacterium colony, so repeat 4～5 times, when the bacterial cell form of examining under a microscope is consistent, obtain to have the pure flora of humus respiration capability.
5, with liquid nutrient medium amplification pure growth, centrifugal results somatic cells is made the thalline suspension with the basic inorganic salt substratum, adds AQDS, under the anaerobic condition AQDS is reduced to the ability of orange-red AH2QDS, judge whether this bacterial strain has anaerobic humus respiration capability.
6,, determine the classification position of bacterial strain, concrete reference literature according to the morphological specificity and the molecular biological characteristics of pure training thing
[12,13]Carry out.
Morphological specificity comprises observation and the observation of cultivating thalline and the observation of bacterial cell structure of cultivating bacterium colony.Document is pressed in the bacterial cell morphologic observation
[11]The method dyeing of introducing adopts German Leica research microscope (DMLB+QCOLite) to observe.
More above-mentioned several embodiment, adopt enrichment that different carbon sources and crumbling method do not influence the reduction bacterium with anaerobic humus respiration capability and separate, if but do not add AQDS in the substratum, then can not the enrichment anaerobic humus the mixed training thing of reduction bacterium, also just can not obtain the pure training thing of the reduction bacterium of tool anaerobic humus respiration capability.
The present invention (embodiment 1,2,3,4) compares with contrast experiment (embodiment 5), and advantage is: 1) can obtain the pure growth (not containing contamination of cells) of anaerobic humus reduction bacterium, for microbiological research and industrial applications provide purebred; 2) easily verify the physio-biochemical characteristics of separated bacterial strain, be convenient to culture presevation and a large amount of amplification; 3) separate, qualification program is simple, credible result.
Claims (4)
1, a kind of separation purification method with anaerobic humus respiration capability reducing bacterium is characterized in that comprising the steps:
1) choose the natural habitat and the sample conduct of artificial habitat of being rich in organic matter and have the separation source of the reduction bacterium of anaerobic humus respiration capability, the natural habitat sample is a bottom mud in lake, and artificial habitat sample is municipal sewage plant's anaerobic digester mud;
2) in the separation source of reduction bacterium, add liquid nutrient medium as enrichment matrix, contain the 6mM sodium formiate in the liquid nutrient medium, 3mM sodium acetate or 1mM glucose, also contain soil ulmin pattern thing anthraquinone-2, the basic inorganic salt liquid of 6-disulfonate, added the 6mM sodium formiate again every 5～7 days, 3mM sodium acetate or 1mM glucose and basic inorganic salt liquid, and oxygenation is with the orange red ortho states anthraquinone-2 of going back, the 6-disulfonate is oxidized to lurid anthraquinone-2, the 6-disulfonate, with nitrogen unnecessary oxygen is driven out of then, repeat 7～9 times domestication enrichment anaerobic humus reduction granule sludge so continuously;
3) get enrichment and get anaerobic humus reduction granule sludge, use ultrasonic disruption, make elementary bacteria suspension, stepwise dilution is made serial bacteria suspension again;
4) with serial bacterial suspension inoculation in the solid medium that melts, under 28-30 ℃, carry out anaerobism and roll the cultivation of pipe method, with kapillary anaerobism being rolled the orange red single bacterium colony that grows on the pipe chooses in the liquid nutrient medium, cultivated 7-10 days 28-30 ℃ of following anaerobism, after the cultivation, adopt anaerobism to roll the pipe method again and separate single bacterium colony, so repeat 4～5 times, when the bacterial cell form of examining under a microscope is consistent, obtain to have the pure flora of humus respiration capability.
2, a kind of separation purification method with anaerobic humus respiration capability reducing bacterium according to claim 1 is characterized in that, described liquid culture based formulas is: NH
4Cl 1500mg/L, NaH
2PO
4600mg/L, CaCl
22H
2O 100mg/L, KCl 100mg/L, MgCl
26H
2O 2mg/L, MnCl
24H
2O5mg/L, NaMoO
42H
2O 1mg/L, anthraquinone-2,6-disulfonate 1mM.
3, a kind of separation purification method with anaerobic humus respiration capability reducing bacterium according to claim 1 and 2 is characterized in that, described solid medium is for adding agar 20g/L again in the aforesaid liquid substratum.
4, a kind of authentication method with reduction bacterium of anaerobic humus respiration capability is characterized in that comprising the steps::
1) will have the pure flora of humus respiration capability, at the centrifugal results somatic cells of 5000rpm, make the thalline suspension with liquid nutrient medium, under anaerobic condition, soil ulmin pattern thing anthraquinone-2, the 6-disulfonate is reduced to the orange red ortho states anthraquinone-2 of going back, and the ability of 6-disulfonate judges whether this bacterial strain is soil ulmin reduction bacterium;
2) determine the classification position of bacterial strain with thalli morphology and 16S rDNA sequence, thalli morphology comprises observation and the observation of cultivating thalline and the observation of bacterial cell structure of cultivating bacterium colony, the poly-extraction strain gene group DNA that comprises of the mensuration step of 16S rDNA sequence, pcr amplification 16S rDNA, purifying 16S rDNA, measure the 16SrDNA sequence,, determine the classification position of bacterial strain existing 16SrDNA sequence alignment among the 16S rDNA sequence that records and the GenBank (www.ncbi.com).
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| CN101892180A (en) * | 2010-04-30 | 2010-11-24 | 广东省生态环境与土壤研究所 | Corynebacterium humireducens and application thereof |
| CN105713617A (en) * | 2016-03-02 | 2016-06-29 | 广东省生态环境与土壤研究所（广东省土壤科学博物馆） | Heavy metal cadmium passivator for activating activity of rice field soil sulfur reducing bacteria and application |
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| CN105713617B (en) * | 2016-03-02 | 2017-05-03 | 广东省生态环境与土壤研究所（广东省土壤科学博物馆） | Heavy metal cadmium passivator for activating activity of rice field soil sulfur reducing bacteria and application |
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| CN110499275B (en) * | 2018-05-18 | 2021-11-05 | 天津大学 | Method for domesticating humic acid reducing bacteria under disturbance load condition based on AQS |
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