CN105907678A - Low-temperature-resisting pseudomonas fragi and application thereof - Google Patents

Low-temperature-resisting pseudomonas fragi and application thereof Download PDF

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CN105907678A
CN105907678A CN201610304708.5A CN201610304708A CN105907678A CN 105907678 A CN105907678 A CN 105907678A CN 201610304708 A CN201610304708 A CN 201610304708A CN 105907678 A CN105907678 A CN 105907678A
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temperature
pseudomonas fragi
bacterial strain
sewage
pseudomonad
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王小雨
周丹丹
范伟
毛娟
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Northeastern University China
Northeast Normal University
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    • C12R2001/00—Microorganisms ; Processes using microorganisms
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    • C12R2001/38—Pseudomonas
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    • C12N1/20—Bacteria; Culture media therefor
    • C12N1/205—Bacterial isolates
    • C—CHEMISTRY; METALLURGY
    • C02—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
    • C02F—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
    • C02F3/00—Biological treatment of water, waste water, or sewage
    • C02F3/34—Biological treatment of water, waste water, or sewage characterised by the microorganisms used
    • C—CHEMISTRY; METALLURGY
    • C02—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
    • C02F—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
    • C02F2101/00—Nature of the contaminant
    • C02F2101/30—Organic compounds
    • C02F2101/34—Organic compounds containing oxygen
    • C—CHEMISTRY; METALLURGY
    • C02—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
    • C02F—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
    • C02F2101/00—Nature of the contaminant
    • C02F2101/30—Organic compounds
    • C02F2101/38—Organic compounds containing nitrogen

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  • Purification Treatments By Anaerobic Or Anaerobic And Aerobic Bacteria Or Animals (AREA)

Abstract

The invention relates to low-temperature-resisting pseudomonas fragi and application thereof and in particular relates to temperature-resisting pseudomonas fragi which is seperated and identified. The pseudomonas fragi has a low low-temperature-resisting property and can still normally grow at 4 DEG C; the most suitable growth temperature is 10 DEG C to 16 DEG C and a growable temperature range is 4 DEG C to 28 DEG C. The pseudomonas fragi can be used for removing COD, NH3-N and TP in sewage at the same time under a low temperature condition. Under the condition of 8 DEG C, the removing rates of the COD, the NH3-N and the TP in the manually prepared sewage (with initial COD, NH3-N and TP content of 544mg/L, 14mg/L and 12mg/L respectively) are 52 percent, 41 percent and 18 percent respectively, so that the low-temperature-resisting pseudomonas fragi can be used for biologically treating the COD, the NH3-N and the TP in the sewage at a low temperature.

Description

Pseudomonad that one strain is low temperature resistant and application thereof
Technical field
The invention belongs to technical field of environmental microorganism.It is specifically related to a pseudomonas Pseudomonas fragi YB bacterial strain, and at low ambient temperatures to dirty organic pollutants and the application of nutrient removal.
Background technology
Along with fast development and the continuous quickening of urbanization process of China's economy, town sewage amount is continuously increased, and shortage of water resources and water pollute the key factor having become as the sustainable economic development of restriction China.Municipal sewage treatment is the important means preventing water from polluting, improving urban water environmental quality, to improving Urban Water Environment, ensureing that urban economy development plays the effect of key.Biological treatment is most widely used processing method in municipal sewage treatment, and it utilizes the metabolism of microorganism in nature, carries out decomposing and converting by the pollutant in sewage, reaches the purpose purified.Mesophilic micoorganism is the main body of biological method for treating waste water, and their optimum growth temperature is between 20 DEG C～37 DEG C.But, in the northern area of China, cold in winter is very long, sewage temperature is on the low side, and sewage temperature is typically even lower at about 10 DEG C.In this low temperature environment, growth and the metabolic activity of mesophilic micoorganism slow down, and metabolism is in holddown, and the ability of pollution degradation material is obstructed.Low temperature weakens the degradation capability to organic pollution largely, affects the efficiency of north of china in winter Sewage Biological Treatment, causes water outlet to be difficult to qualified discharge.At present, solution conventional in engineering of water treatment is to reduce sludge loading, increases the methods such as dwell time of sewage, but the floor space that these methods make Sewage Plant increases, and running cost increases, and treatment effect is the most not fully up to expectations.Therefore, how improving cold district winter low temperature sewage treating efficiency is practical problem urgently to be resolved hurrily in the sewage disposal in winter of north cold area.
In nature, there is a class can be considered as much the microorganism still in the extreme environment (such as high temperature, low temperature, peracid, high-alkali etc.) of life forbidden zone with indomitable vitality, is referred to as Situation of Microorganism Under Extremity Environment.Wherein, it is possible to the microorganism adapting to low temperature environment is exactly important extreme microorganism monoid.Psychrotrophs, in prolonged cold environment, defines a series of special physiological mechanism and low-temperature catalytic activity, it is ensured that they anabolism of intracellular matter and catabolic be normally carried out under cryogenic.At present, psychrophile effect in terms of waste water process has caused the interest of researchers, becomes the focus matter of science and technology of concern both at home and abroad.It is reported, pseudomonas (Pseudomonas) bacterium can utilize simple in construction and complicated organic compound, has stronger resistance to low temperature, is resistance to low microorganism more typically.Studies have reported that, pseudomonas bacterium such as Pseudomonas koreensis, Pseudomonas fragi, Pseudomonas lundensis can be so that there is corruption in deepfreeze food, show that these pseudomonas bacteriums possess good resistance to cold, can the most normally breed.But, the research about above-mentioned removal of bacteria dirt water pollutant is also rarely reported.
Summary of the invention
It is an object of the invention to for the inefficient problem of municipal sewage treatment under cryogenic conditions, it is provided that a strain removes the application of organic contamination and nutritive salt be applicable to pseudomonad (Pseudomonas fragi) the YB bacterial strain of low temperature extreme environment and this bacterium sewage.
Pseudomonad provided by the present invention (Pseudomonas fragi) YB bacterial strain, it is preserved in " China Committee for Culture Collection of Microorganisms's common micro-organisms center (being called for short CGMCC; address is: Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3 Institute of Microorganism, Academia Sinica) " on October 18th, 2013, preservation registration number is CGMCC No.8398, it is proposed that Classification And Nomenclature is pseudomonad (Pseudomonas sp.).
Pseudomonad Pseudomonas fragi YB bacterial strain provided by the present invention is to separate from the bed mud of cold water fish pond, Jilin Province (east longitude: 129.03, north latitude: 42.77).With low temperature as selection pressure, in LB culture medium, acquisition is cultivated in domestication repeatedly.Bacterial strain YB is low temperature resistant, still can be with normal growth at 4 DEG C, and optimum growth temperature is 10～16 DEG C, can growth temperature range 4 DEG C～28 DEG C.
The colony characteristics of pseudomonad Pseudomonas fragi YB bacterial strain: on LB Agar Plating, cultivates 48h, is about 2.5mm for colony diameter for 15 DEG C.Bacterium colony is circular protrusions, smooth surface, quality thickness, milky, translucent.
The cell characteristic of pseudomonad Pseudomonas fragi YB bacterial strain: bacterium cell be shaft-like, cell size be 0.53 × 1～2.87 μm.
The physiological and biochemical property of pseudomonad Pseudomonas fragi YB bacterial strain: Gram-negative, catalase, oxidizing ferment, methyl red, gelatin liquefaction, generation hydrogen sulfide production test and arginine dihydrolase reaction result are positive, and product indole test, Starch Hydrolysis test, V-P result of the test are negative.
The 16S rRNA gene sequence characteristic of pseudomonad Pseudomonas fragi YB bacterial strain: a length of 1419bp of the 16S rRNA gene order (sequence is as shown in list) of bacterial strain YB, the accession number in GeneBank is KJ496054.By reaching 99.79% with GeneBank homologous sequence comparison, bacterial strain YB and pseudomonad Pseudomonas fragi ATCC4973 homology.
With reference to " primary Jie Shi determinative bacteriology handbook " (the 8th edition), according to strain morphology feature, physiological and biochemical property, in conjunction with the 16S rRNA gene order of this bacterium comparison result in GeneBank, the bacterial strain YB of separation is accredited as pseudomonad Pseudomonas sp.
It is a further object to provide employing low temperature-resistant strains and remove dirty organic pollutants and the method for nutritive salt under cryogenic.
Protection scope of the present invention is fallen within the biologic product that pseudomonad Pseudomonas fragi YB bacterial strain is prepared for active component.
The pseudomonad Pseudomonas fragi YB bacterial strain of the present invention, is isolatable from the bed mud of cold water fish pond, Jilin Province (east longitude: 129.03, north latitude: 42.77).Pseudomonad Pseudomonas fragi YB, under 8 DEG C of cryogenic conditions, can make initial COD, NH3-N and TP is respectively the clearance of 3 kinds of pollutants in the sewage of 544mg/L, 14mg/L, 12mg/L and is respectively 52%, 41% and 18%.Therefore, the present invention provides pseudomonad Pseudomonas fragi YB bacterial strain can remove dirty organic pollutants and the effect of nutritive salt at cryogenic conditions simultaneously, possesses good application prospect.
Below in conjunction with specification drawings and specific embodiments, the invention will be further described, not limitation of the present invention.
Accompanying drawing explanation
Fig. 1 be pseudomonad Pseudomonas fragi YB bacterial strain scanning electron microscopic picture (A) and under LB flat board growth colonial morphology photo (B)
Fig. 2 is pseudomonad Pseudomonas fragi YB bacterial strain growing state (A) under the conditions of Different hypothermia and growth curve (B).
Fig. 3 be pseudomonad Pseudomonas fragi YB bacterial strain under 8 DEG C of cryogenic conditions to chemical oxygen demand in sewage COD, ammonia nitrogen NH3-N and the clearance of total phosphorus TP
Detailed description of the invention
Following it is implemented in experimental technique described in example, if no special instructions, is conventional method；Described reagent and biomaterial, if no special instructions, the most commercially buy acquisition.
Following experimental technique is conventional method if no special instructions, and the solvent in all culture mediums is distilled water.Each experiment be 3 parallel, and arrange blank experiment.
In following experimental technique, the biomass of bacterial strain YB uses OD600Characterizing, i.e. using spectrophotometry bacterial strain YB nutrient solution is the absorbance of 600nm at wavelength.
The sewage used in following experiment is artificial preparation.Initial COD, NH of sewage3-N and TP is respectively 544mg/L, 14mg/L, 12mg/L.
In the present invention, organic pollution uses COD (COD) and nutritive salt (ammonia nitrogen NH3-N and total phosphorus TP) all use national standard method to measure.Wherein COD uses dichromate titration to measure, NH3-N uses Berthelot spectrophotometry to measure, and TP uses Ammonium Molybdate Spectrophotometric Method for Determination.
The separation of embodiment 1. pseudomonad Pseudomonas fragi YB bacterial strain, purify and identify.
Consisting of of the LB fluid nutrient medium used in Psychrotrophs separation process: peptone 2g, dusty yeast 1g, NaCl 2g, agar 4g, water 200mL, pH 7.0～7.5.
Consisting of of LB solid medium: add agar 16g in aforesaid liquid culture medium.
Pseudomonad Pseudomonas fragi YB strain isolation Zi Lin saves the bed mud (east longitude: 129.03, north latitude: 42.77) in certain cold water fish pond, concrete enrichment, separate, purge process as follows:
The bed mud sample inoculation gathered by 1g is to (liquid amount is 20ml/100ml triangular flask) in physiological saline, and 15 DEG C, vibrate under 180rpm 24h.Taking 1ml suspension in centrifuge tube, 5000r/min is centrifuged 10min, abandoning supernatant, and the physiological saline adding same volume washs, and repeats above washing process 3 times.Gained precipitation being carried out a series of gradient dilution, coats on LB solid medium, under the conditions of the flat board after coating is placed in 10 DEG C, quiescent culture is until growing and being evident that bacterium colony.The psychrotolerant bacteria obtained after isolated and purified by primary dcreening operation, prepares bacteria suspension respectively, carries out the most multiple sieve, final acquisition one strain advantage Low Temperature-Resistant Strain, named YB at 8 DEG C and 5 DEG C of conditions.
Use morphologic observation, Physiology and biochemistry to identify and bacterial strain YB is identified by 16S rRNA gene sequencing
Visible milky on LB culture medium, smooth surface, quality thickness, milky, translucent bacterium colony.Electricity Microscopic observation, the laticiferous cell of bacterial strain YB be shaft-like, cell size be 0.53 × 1～2.87 μm.
Physiology and biochemistry qualification result is: Gram's staining is the positive；Catalase, oxidizing ferment, methyl red, gelatin liquefaction, generation hydrogen sulfide production test and arginine dihydrolase reaction result are positive, and product indole test, Starch Hydrolysis test, V-P test result are negative.
The 16S rRNA gene sequencing of bacterial strain and Phylogenetic Analysis.To cultivate 12h, centrifugal thalline of collecting, Eddy diffusion in inoculation after purified preservation to LB fluid nutrient medium 180r/min, use phenol-chloroform method to extract the DNA of bacterial strain, 0.8% agarose electrophoresis detects.Bacterial 16 S rDNA universal primer is used to carry out PCR amplification.Wherein, forward primer is 27f (5'-AGA GTT TGA TCC TGG CTC AG-3'), and reverse primer is 1492r (5'-GGC TAC CTT GTT ACG ACT T-3') '.PCR reaction condition is: first 94 DEG C of 6min；Then 94 DEG C of 45s, 54 DEG C of 45s, 72 DEG C of 90s, totally 30 circulations；Last 72 DEG C extend 10min.Pcr amplification product delivers to the order-checking of Shanghai Sheng Gong bioengineering limited company.The a length of 1419bp of 16RNA gene order of bacterial strain YB, the accession number in GeneBank is KJ496054.By reaching 99.79% with GeneBank homologous sequence comparison, bacterial strain YB and pseudomonad Pseudomonas fragi ATCC4973 homology.The 16S rRNA gene order of bacterial strain YB is as follows:
0001 AAGCTACACATGCAAGTCGAGCGGTAGAGAGAGTGCTTGCACCTCTTGAGAGCGGCGGAC
0061 GGGTGAGTAATACCTAGGAATCTGCCTGGTAGTGGGGGATAACGTTCGGAAACGGACGCT
0121 AATACCGCATACGTCCTACGGGAGAAAGCAGGGGACCTTCGGGCCTTGCGCTATCAGATG
0181 AGCCTAGGTCGGATTAGCTAGTTGGTGAGGTAATGGCTCACCAAGGCTACGATCCGTAAC
0241 TGGTCTGAGAGGATGATCAGTCACACTGGAACTGAGACACGGTCCAGACTCCTACGGGAG
0301 GCAGCAGTGGGGAATATTGGACAATGGGCGAAAGCCTGATCCAGCCATGCCGCGTGTGTG
0361 AAGAAGGTCTTCGGATTGTAAAGCACTTTAAGTTGGGAGGAAGGGCATTAACCTAATACG
0421 TTAGTGTCTTGACGTTACCGACAGAATAAGCACCGGCTAACTCTGTGCCAGCAGCCGCGG
0481 TAATACAGAGGGTGCAAGCGTTAATCGGAATTACTGGGCGTAAAGCGCGCGTAGGTGGTT
0541 TGTTAAGTTGAATGTGAAATCCCCGGGCTCAACCTGGGAACTGCATCCAAAACTGGCAAG
0601 CTAGAGTATGGTAGAGGGTAGTGGAATTTCCTGTGTAGCGGTGAAATGCGTAGATATAGG
0661 AAGGAACACCAGTGGCGAAGGCGACTACCTGGACTGATACTGACACTGAGGTGCGAAAGC
0721 GTGGGGAGCAAACAGGATTAGATACCCTGGTAGTCCACGCCGTAAACGATGTCAACTAGC
0781 CGTTGGGAACCTTGAGTTCTTAGTGGCGCAGCTAACGCATTAAGTTGACCGCCTGGGGAG
0841 TACGGCCGCAAGGTTAAAACTCAAATGAATTGACGGGGGCCCGCACAAGCGGTGGAGCAT
0901 GTGGTTTAATTCGAAGCAACGCGAAGAACCTTACCAGGCCTTGACATCCAATGAACTTTC
0961 CAGAGATGGATTGGTGCCTTCGGGAGCATTGAGACAGGTGCTGCATGGCTGTCGTCAGCT
1021 CGTGTCGTGAGATGTTGGGTTAAGTCCCGTAACGAGCGCAACCCTTGTCCTTAGTTACCA
1081 GCACGTAATGGTGGGCACTCTAAGGAGACTGCCGGTGACAAACCGGAGGAAGGTGGGGAT
1141 GACGTCAAGTCATCATGGCCCTTACGGCCTGGGCTACACACGTGCTACAATGGTCGGTAC
1201 AAAGGGTTGCCAAGCCGCGAGGTGGAGCTAATCCCATAAAACCGATCGTAGTCCGGATCG
1261 CAGTCTGCAACTCGACTGCGTGAAGTCGGAATCGCTAGTAATCGTGAATCAGAATGTCAC
1321 GGTGAATACGTTCCCGGGCCTTGTACACACCGCCCGTCACACCATGGGAGTGGGTTGCAC
1381 CAGAAGTAGCTAGTCTAACCCTCGGGAGGACGGTACCAT
Based on colony characteristics, the form of bacterial strain, physiological and biochemical property and 16S rRNA measurement result, by the identification of strains screened named pseudomonad Pseudomonas fragi YB.This bacterial strain YB is preserved in " Chinese microorganism strain preservation centre management committee's common micro-organisms center (being called for short CGMCC; address is: Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3 Institute of Microorganism, Academia Sinica) " on October 18th, 2013, and preservation registration number is CGMCC No.8398.
Embodiment 2. is pseudomonad Pseudomonas fragi YB bacterial strain growing state under the conditions of Different hypothermia and growth curve.
For research pseudomonad Pseudomonas fragi YB bacterial strain to growing state at different temperatures, the inoculum concentration by 10% is by the LB fluid nutrient medium under bacterial suspension inoculation to condition of different temperatures, and rotating speed 180r/min cultivates, and measures the OD of nutrient solution after 48h600.The temperature set in experiment is respectively 4 DEG C, 7 DEG C, 10 DEG C, 16 DEG C, 20 DEG C, 28 DEG C and 37 DEG C.
For research pseudomonad Pseudomonas fragi YB bacterial strain growth rhythm at 5 DEG C, 8 DEG C, 10 DEG C and 15 DEG C, inoculum concentration by 10% is by the LB fluid nutrient medium under the conditions of bacterial suspension inoculation to relevant temperature, rotating speed 180r/min cultivates, experimental period is 96h, take nutrient solution every 6h, measure its OD600.Pseudomonad Pseudomonas fragi YB growth curve under cryogenic is drawn according to experimental result.
Pseudomonad Pseudomonas fragi YB bacterial strain growing state such as Fig. 2 (A) under the conditions of 4 DEG C, 7 DEG C, 10 DEG C, 16 DEG C, 20 DEG C, 28 DEG C and 37 DEG C.Test result indicate that, bacterial strain YB can tolerate cryogenic conditions, 4 DEG C still can with normal growth, but biomass is relatively low.Along with temperature is gradually increased to 7 DEG C, biomass starts to increase.When temperature is 10 DEG C～16 DEG C, the biomass of bacterial strain YB reaches maximum.When temperature is higher than 20 DEG C, bacterial strain biomass raises along with temperature, declines on the contrary, and when temperature is 37 DEG C, bacterial strain grows hardly.Therefore, the optimum growth temperature of bacterial strain YB is 10 DEG C～16 DEG C, for typical low temperature resistant bacterium.
Pseudomonad Pseudomonas fragi YB bacterial strain is shown in Fig. 2 (A) at the growth curve of 5 DEG C, 8 DEG C, 10 DEG C and 15 DEG C.The growth cycle of bacterial strain YB is generally 78h.Wherein, the laundering period is about 0～18h；18～54h is exponential phase；Stationary phase is entered after 54h.
Embodiment 3. pseudomonad Pseudomonas fragi YB bacterial strain is under cryogenic to COD, NH3The removal of-N and TP
North of china in winter treatment plant sewage temperature is generally between 8 DEG C～15 DEG C.COD of sewage, NH is removed at following all application pseudomonad Pseudomonas fragi YB3All selecting 8 DEG C in the experiment of-N and TP is low temperature test condition, initial COD, NH of sewage3-N and TP is respectively 544mg/L, 14mg/L, 12mg/L.Its bacterial strain is equipped with in the 250mL triangular flask that 100mL manually prepares sewage by the inoculum concentration with 10%, 8 DEG C, cultivate under the conditions of 180r/min, respectively 0,24,48,72,96,120,144,168, take out sample and blank sample after 192h, nutrient solution, through 8000r/min centrifugal segregation thalline, then takes supernatant and surveys its COD, NH respectively3-N, TP, calculate the every kind of bacterial strain removal effect to dirty pollutant in water.Each experiment 3 is parallel.
Pseudomonad Pseudomonas fragi YB bacterial strain under cryogenic, prepares COD, NH in sewage to artificial3The removal effect of-N and TP is shown in Fig. 3-1,3-2 and 3-3 respectively.As it can be seen, after the effect of 192h, bacterial strain YB can remove COD, NH in sewage simultaneously3-N and TP, clearance is respectively 52%, 41% and 18%.As can be seen here, the pseudomonad Pseudomonas fragi YB bacterial strain that the present invention provides can remove COD, NH in sewage at cryogenic conditions simultaneously3-N and TP, possesses good application prospect.

Claims (3)

1. the pseudomonad that a strain is low temperature resistant, this bacterial strain is pseudomonad (Pseudomonas fragi), numbered YB is preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center on October 18th, 2013, and preservation registration number is CGMCC No.8398.
2. preserving number is that the pseudomonad (Pseudomonas fragi) of CGMCC No.8398 has good low temperature tolerance characteristics, growth temperature range is 4 DEG C～28 DEG C, optimum growth temperature is 10 DEG C～16 DEG C, when temperature is 37 DEG C, bacterial strain YB grows hardly, belongs to typical low temperature resistant bacterium.
Utilize the pseudomonad (Pseudomonas fragi) that preserving number is CGMCC No.8398 the most as described in claim 2, under the conditions of the low temperature test of 8 DEG C, to COD, NH from work preparation sewage3-N and TP is removed.Sewage initial COD, NH3-N and TP is respectively 544mg/L, 14mg/L, 12mg/L, and after the reaction of 192h, bacterial strain YB can remove COD, NH in sewage simultaneously3-N and TP, clearance is respectively 52%, 41% and 18%.
CN201610304708.5A 2016-05-10 2016-05-10 Low-temperature-resisting pseudomonas fragi and application thereof Pending CN105907678A (en)

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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
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Application publication date: 20160831

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