CN106566789B - Lysinibacillus macroides and the application of one plant of rich phosphorus and degrading organic phosphor pesticides - Google Patents

Lysinibacillus macroides and the application of one plant of rich phosphorus and degrading organic phosphor pesticides Download PDF

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CN106566789B
CN106566789B CN201610950899.2A CN201610950899A CN106566789B CN 106566789 B CN106566789 B CN 106566789B CN 201610950899 A CN201610950899 A CN 201610950899A CN 106566789 B CN106566789 B CN 106566789B
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mew88
lysinibacillus
macroides
lysinibacillus macroides
organic phosphor
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CN106566789A (en
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倪红
刘诚
万娟
唐梦君
邵闯
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Shandong Shennong Ecological Technology Co.,Ltd.
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Hubei University
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Abstract

The invention discloses one plant to have rich phosphorus and degrading organic phosphor pesticidesLysinibacillus macroides, the bacterial strain isLysinibacillus macroides MEW88, is preserved in China typical culture collection center on October 18th, 2016, and deposit number is CCTCC NO:M2016574, bacterial strain of the inventionLysinibacillus macroidesRich phosphorus ability with transitory efficient, and can effectively degrade Rogor and glyphosate, have a good application prospect in terms of the biological treatment of phosphorous chemical industry waste water.

Description

One plant of rich phosphorus and degrading organic phosphor pesticidesLysinibacillus macroidesAnd Using
Technical field
The invention belongs to microorganisms technical fields, and in particular to one plant of lysine with rich phosphorus, degrading organic phosphor pesticides BacillusLysinibacillus macroidesAnd application.
Background technique
With the development and application of polyphase sort, bacillus (Bacillus sp.) classification happened very Big variation separates for many kinds from bacillus and is classified as other categories or separately builds new category.Lysine bacillus (Lysinibacillus sp.) be in recent years from bacillus (Bacillus sp.) the new category separated, because of its tool Have a unique whole cell peptidoglycan configuration A4 α-type (L-Lys-D-Asp), and by its with bacillus (Bacillus sp.) be distinguished.Lysine bacillus (Lysinibacillus sp.) classification position are as follows:Bacteria；Firmicutes；Bacilli；Bacillales；Bacillaceae；Lysinibacillus.The strain separating of the category is certainly native Earth, riverbed bed mud, paper waste, female mice enteron aisle, human cerebrospinal fluid, Populus Euphratica diversiform-leaved poplar stalk liquid, Surface Sediments of Tidal Flat, manganese The various environment such as mine factory, these bacterium sources are different, and function is also embodied in different aspects.At presentLysinibacillusBelong to packet Include 16 kinds being described:L. bronitoleram,L. sphaericus,L. fusiformis,L. parviboronicapiens,L. xylanilyticus, L. massiliensis,L. odysse,L. sinduriensis,L. macroides,L. mangiferihumi,L. meyeri,L. contaminans,L. chungkukjangi,L. manganicus,L. pakistanensis,L. tabacifolii.Lysine bacillus All strain common features be that thallus is in the shape of a rod, gemma can be generated, main fatty acid type is iso-C15:0Or anteiso-C15:0, the main quinones type that breathes is MK-7, and whole cell peptidoglycan contains lysine and aspartic acid, (G+ in DNA C) % content accounts for 35-38%.LysinibacillusThe polar lipid component of the bacterial strain of category has cardiolipin (DPG), phosphatidyl Glycerol (PG), phosphatidyl-ethanolamine (PE) and phosphate lipid (PL).
Currently, domestic and international researcher reported it is some aboutLysinibacillusBelong to grinding for the biological effect of some bacterial strains Study carefully, the dyestuff being concentrated mainly in degrading waste water, such as 103937697 A of patent CN；Generate extracellular protease and esterase intracellular such as Ethyl carbamate hydrolase, such as 103013948 A of patent CN；For reduction of hexavalent chromium ion in environment, such as patent CN 103395893 A；Negative sulfidion in degradation petrochemical wastewater, such as 102827786 A of patent CN；Dissolution or infection cyanobacteria, Such as 102965298 A of patent CN；Degradation noxious material polybrominated diphenyl ethers, such as 102363756 A of patent CN；And aboutLysinibacillusResearch in terms of category degrading organic phosphor pesticides is but rarely reported, and only a small amount of document is related to.Nivetha Mohan et al. was once appliedLysinibacillus fusiformisProcessing organophosphorus pesticide height goes out spirit, and obtains preferable effect Fruit.Teng Chunhong et al. acquires mud sample from insecticide factory's sewage draining exit of production 2,4- d butyl ester, and taming isolated 1 plant can be with 2,4- d butyl ester is the bacterium that sole carbon source and the energy are grownLysinibacillus fusiformis T2, and be resistant to highly concentrated The 2,4- d butyl ester of degree.This researchLysinibacillus macroides MEW88 can not only efficient degradation low concentration it is organic Phosphorus pesticide Rogor and glyphosate, and the Phos in waste water can also be adsorbed, therefore be applied to phosphorous chemical industry pollution environment Improvement has important practical significance.
Summary of the invention
The lysine bacillus that there is rich phosphorus, degrading organic phosphor pesticides the purpose of the present invention is to provide one plantLysinibacillusmacroidesMEW88, and the bacterium is for the first time in terms of degrading organic phosphor pesticides (glyphosate, Rogor) In report.
Of the inventionLysinibacillusmacroidesMEW88 is preserved in Chinese Typical Representative on October 18th, 2016 Culture collection preservation, deposit number are CCTCC NO:M2016574, and depositary institution address, Wuhan, China Wuhan is big It learns.
The present inventionLysinibacillus macroides The morphology and physiological and biochemical property of MEW88: it is trained in LB solid Base is supported, cultivates 48 h under the conditions of 28 DEG C, the bacterium bacterium colony is round, and partially faint yellow, opaque, surface is flat, and edge has a circle semi-transparent Bright furcella shape, colony diameter 2-4mm, observation strain cell form is rod-shaped under the microscope, generation gemma, Gram-positive, Puncture experiment display puncture line edge blurry, it was demonstrated that the bacterium have motility, the bacterium oxidase negative, nitrate reduction negative, Arginine hydrolase is negative, is unable to caseinhydrolysate, gelatin and urea.
The present inventionLysinibacillus macroides The molecular biological variety identification method of MEW88 are as follows: use 16s RDNA universal primer 27F and 1492R withLysinibacillus macroides MEW88 genome is as its 16s of template amplification PCR product is directly sent to Wuhan and holds up the sequencing of Kechuang neoformation Science and Technology Ltd., obtained as shown in sequence table by rDNA sequence 16s rDNA sequence.By sequence in the sequence and EzTaxon-e carry out it is homologous compare analysis, as a result, it has been found thatLysinibacillus macroides MEW88 withLysinibacillus macroidesThe sequence similarity of LMG 18474 (T) is 99.59%, withLysinibacillus sphaericus The sequence similarity of KCTC 3346 (T) is 98.28%, withLysinibacillus mangiferihumiThe sequence similarity of M-GX18 (T) is 97.86%, and the typical strain sequence of telomerization and the bacterial strain homology Column, with (the Molecular Evolutionary Genetics Analysis) software of MEGA 6.0 using adjacent method ( Neighbor-Joining) clustering constructs systematic evolution tree, such as Fig. 2.
In summary morphology, physiological and biochemical property, chemotaxonomy are as a result, MEW88 is accredited asLysinibacillus macroides, which is preserved in China typical culture collection on October 18th, 2016 Heart preservation, deposit number are CCTCC NO: M2016574.
Lysine bacillus provided by the invention is isolated by following method: acquisition Yichang phosphorizing treatment Neighbouring soil sample and water sample weighs 2g soil sample and the water sample enrichment culture in 100mL LB culture solution.Bacteria suspension is pressed into 10%(V/ V inoculum concentration) is inoculated in the fermentation medium containing organophosphorus pesticide, steps up the concentration of pesticide, until drawing after domestication is stablized Line fast growing and vigorous 4 plants of bacterium in minimal medium, picking plate, are inoculated in the fermented and cultured containing organophosphorus pesticide In base, organophosphorus pesticide content is measured using acetylcholine enzyme inhibition rate method (GB/T5009.199-2003), to filter out this The lysine bacillus of invention, is denoted as MEW88.
It is provided by the inventionLysinibacillus macroides MEW88 has good in terms of adsorbing Phos Using.Specific practice is: above-mentioned screening is obtainedLysinibacillus macroides MEW88 is by 10%(V/V) connect Kind of amount is inoculated into PAM culture medium (phosphorus content (in terms of P) is respectively 10 mg/L, 20 mg/L), at 28 DEG C, the condition of 150rpm Lower shaking flask culture is sampled in 0,12,24,48,72 h, measures content of inorganic phosphorus (the anti-ratio of GB 11893-89- molybdenum antimony in supernatant Color method).
It is provided by the inventionLysinibacillus macroides MEW88 is in degrading organic phosphor pesticides Rogor, glyphosate In application.Specific practice is: will after purificationLysinibacillus macroides MEW88 is inoculated in LB culture medium 28 It DEG C is activated overnight, adjusts bacterium solution OD600Be 2.0, by 4%-10%(V/V) inoculum concentration be forwarded to glyphosate containing various concentration or pleasure Organic phosphorus pesticide degradation rate (GB/T5009.199- is measured by sampling in 1d, 2d, 3d, 5d, 7d in the fermentation medium culture of fruit 2003).
Compared with prior art, the invention has the following advantages:
(1) provided by the inventionLysinibacillus macroides MEW88 has absorption Phos and degradation simultaneously Therefore organic phosphorus function may be directly applied in the processing of phosphorous chemical industry waste water.On the one hand, having in the bacterium energy degrading waste water Machine phosphorus, and there is high degradation efficiency to low concentration organophosphorus pesticide；On the other hand, which can absorb inorganic in waste water Phosphorus thus is avoided that and generates Phos pollution, can reduce the possibility of water eutrophication.Therefore, which has in phosphorous chemical industry waste water Good application prospect.
(2) present invention for the first time willLysinibacillus macroides MEW88 be applied to organophosphorus pesticide glyphosate and Rogor degradation, this provides a kind of new thinking for organic phosphorus pesticide degradation, while also laying a solid foundation for later research.
Detailed description of the invention
Fig. 1 (A) isLysinibacillus macroides MEW88 colonial morphology, Fig. 1 (B) are spore staining (400 ×) figure.
Fig. 2 isLysinibacillus macroides The 16S rDNA gene order phylogenetic tree of MEW88 bacterial strain.
Fig. 3 isLysinibacillus macroides The degradation that MEW88 changes with time to various concentration Rogor Rate.
Fig. 4 isLysinibacillus macroides The degradation that MEW88 changes with time to various concentration glyphosate Rate.
Specific embodiment
The following is specific embodiments of the present invention, is described further to technical solution of the present invention, but of the invention interior Appearance is not limited solely to range described in embodiment, all to be included in this without departing substantially from the change of present inventive concept or equivalent substitute Within the protection scope of invention.
Embodiment 1Lysinibacillus macroides MEW88 separation and identification
The soil sample and water sample near the phosphorizing treatment of Yichang are acquired, weighs 2g soil sample and water sample in 100mL LB culture solution In, enrichment culture 3d, LB culture medium prescription is as follows under conditions of 28 DEG C of 150 r/min: 10 g/L of peptone, and yeast extracts Object 5 g/L, NaCl10 g/L, water 1L, pH7.0-7.2.By above-mentioned bacteria suspension by 10%(V/V) inoculum concentration be inoculated in containing grass it is sweet The fermentation medium (2 g/L of peptone, yeast extract 1 g/L, NaCl 2 g/L, water 1L, pH7.0-7.2) of phosphine and Rogor, 28 DEG C, after 150 r/min enrichment culture 7d, it is forwarded to same medium, continues culture domestication under the same conditions, is tamed and dociled in passage Glyphosate and Rogor content are stepped up during changing, until its concentration is 800 mg/L；The bacterium solution for taming stable is lined Minimal medium (glucose10.0 g, (NH4)2SO4 0.5 g, NaCl 0.3 g, KCl 0.3g, MgSO4•7H2O 0.3g, FeSO4•7H2O 0.03 g, MnSO4•4H20.03 g of O, 200 mg/L of glyphosate, Rogor 200 mg/L, water 1L), 28 DEG C, Culture 5d is set, fast growing and vigorous 4 plants of bacterium colony in picking plate are inoculated in containing 50 mg/L pesticide of glyphosate and Rogor In fermentation medium, 28 DEG C, 150 r/min cultivate 3d, are surveyed using acetylcholine enzyme inhibition rate method (GB/T5009.199-2003) Determine organophosphorus pesticide content, obtains the bacterial strain of a high-efficiency degradation organophosphorus pesticide, named are as follows: MEW88, as this patent Bacterial strain.
Using MEW88 genome as template, universal primer 27F(5 '-AGAGTTTGATCATGGCTCAG-3 ' is used) and 1492R(5 '-GGTACCTTGTTACGACTT-3 ') amplification 16S rDNA sequence (see sequence table).By the sequence and EzTaxon-e Middle sequence carries out homologous comparisons analysis, as a result, it has been found that MEW88 andLysinibacillus macroidesLMG's 18474 (T) Sequence similarity is 99.59%, withLysinibacillus sphaericus The sequence similarity of KCTC 3346 (T) is 98.28%, withLysinibacillus mangiferihumiThe sequence similarity of M-GX18 (T) is 97.86%.In evolution, MEW88 withLysinibacillus macroidesLMG 18474 (T) leans on nearest.
The present inventionLysinibacillus macroides The morphology and physiological and biochemical property of MEW88: it is trained in LB solid Base is supported, cultivates 48 h under the conditions of 28 DEG C, the bacterium bacterium colony is round, and partially faint yellow, opaque, surface is flat, and edge has a circle semi-transparent Bright furcella shape, colony diameter 2-4mm, observation strain cell form is rod-shaped under the microscope, generation gemma, Gram-positive, Puncture experiment display puncture line edge blurry is fog-like to prove that the bacterium has motility, the bacterium oxidase negative, nitre to external diffusion Hydrochlorate reduction is negative, arginine hydrolase is negative, is unable to caseinhydrolysate, gelatin and urea.In summary morphology, physiology are raw Change feature, chemotaxonomy as a result, MEW88 is accredited asLysinibacillus macroides。
Embodiment 2Lysinibacillus macroides The test of MEW88 richness phosphorus
In Example 1Lysinibacillus macroides MEW88 is 28oMistake under conditions of 150 r/min of C Supernatant is removed in night activation, the bacterium solution centrifugation after activation, and precipitating is washed 1 time with sterilizing PBS(pH 7.2), and it is dense that bacterium solution is adjusted after resuspension Degree is OD600=1.0.By above-mentioned bacteria suspension by 10%(V/V) inoculum concentration be inoculated in phosphorus content (in terms of P) be 10 mg/L, 20 The 200mL PAM culture medium of mg/L, at 28 DEG C, shaking flask culture under conditions of 150rpm is sampled in 0,12,24,48,72 h, warp After 8000r/min is centrifuged 10min, measure supernatant content of inorganic phosphorus (GB 11893-89- molybdenum antimony resistance colorimetric method).The PAM training Support base component are as follows: Na3C6H5O74.0 g/L, NaCl0.5 g/L, (NH4)2SO42.5 g/L, CaCl20.25 g/L, MgSO40.25 G/L, KH2PO40.0878 g/L (phosphorus content 20mg/L), 0.01 g/L of maltose, water 1L, pH 7.2.
Rich phosphorus experimental result is shown in Table 1, as seen from the table, when phosphorus content is 10 mg/L, handles 24 h, culture through MEW88 Tp removal rate basically reaches 100% in base, and supernatant can't detect Phos, and reaching national grade one discharge standard, (P contains in waste water Amount is lower than 0.5 mg/L).When phosphorus content is 20 mg/L, richness phosphorus rate also reaches 78.71% when MEW88 culture 36h, in culture medium Supernatant inorganic phosphorus concentration drops to 4.24 mg/L by 20 mg/L, this result shows that MEW88 efficiently can not only quickly remove it is low Phos in concentration waste water also has preferable phosphor-removing effect to high concentration.
Table 1Lysinibacillus macroides MEW88MEW88 is in PAM
Richness phosphorus rate (%) changes over time when culture medium culture
Culture medium phosphorus content 12 h 24 h 32 h 48 h 72 h
P: 10 mg/L 49.36 ± 0.78 100.84 ±0.82 100.40 ± 1.07 100.32 ± 0.62 99.65 ± 0.96
P: 20 mg/L 12.16± 0.07 71.30 ±0.85 78.71 ± 1.09 76.24 ± 0.9 72.33 ± 0.75
Embodiment 3Lysinibacillus macroides MEW88 tests various concentration Rogor degradation effect
In Example 1Lysinibacillus macroides After MEW88 is activated overnight, with PBS(pH 7.2-7.4) Washing thalline 1 time, bacterium solution OD is adjusted after resuspension600Be 2.0, then by 4%(V/V) inoculum concentration be forwarded to Rogor containing various concentration (50 mg/L, 100 mg/L, 150 mg/L, 200 mg/L) fermentation medium culture, timing sampling measure organophosphorus pesticide Degradation rate, as a result such as Fig. 3.
From figure 3, it can be seen that MEW88 Rogor degradation capability with higher, the especially pleasure of 50 mg/L of low concentration Fruit, the 5th day strains for degrading rate have reached 97.5%, are substantially not detectable Rogor within the 7th day.Therefore, the bacterium is in Rogor detoxification and reduction Environmental pollution etc. has good application value.
Embodiment 4Lysinibacillus macroides MEW88 is to various concentration glyphosate degradation effect test
In Example 1Lysinibacillus macroides MEW88 is stayed overnight under conditions of 28 DEG C, 150 r/min Activation adjusts bacterial concentration OD with PBS(pH 7.2-7.4) washing thalline 1 time after resuspension600It is 2.0.Above-mentioned bacterium solution is pressed into 4%- 10% inoculum concentration is forwarded to the fermentation medium culture containing glyphosate 25 mg/L or 50 mg/L, and timing sampling measurement grass is sweet Phosphine degradation rate.
The result shows that the glyphosate of MEW88 processing low concentration is not only quickly but also efficient, when glyphosate concentration is 25 mg/L When, it is handled two days by MEW88, glyphosate is almost degraded, and concrete outcome is shown in Fig. 4.MEW88 is for handling physical chemistry method The low concentration glyphosate eliminated pollution is difficult to more application advantage.
<110>Hubei University
Lysinibacillus macroides and the application of<120>one plants of rich phosphorus and degrading organic phosphor pesticides
<160>3
<210>1
<211>1450
<212>DNA
<213>Lysinibacillusmacroides MEW88
<400>1
cactcttcca ccttcggcgg ctggctccaa aaggttacct caccgacttc gggtgttaca 60
aactctcgtg gtgtgacggg cggtgtgtac aaggcccggg aacgtattca ccgcggcatg 120
ctgatccgcg attactagcg attccggctt catgtaggcg agttgcagcc tacaatccga 180
actgagaacg actttatcgg attagctccc tctcgcgagt tggcaaccgt ttgtatcgtc 240
cattgtagca cgtgtgtagc ccaggtcata aggggcatga tgatttgacg tcatccccac 300
cttcctccgg tttgtcaccg gcagtcacct tagagtgccc aactaaatga tggcaactaa 360
gatcaagggt tgcgctcgtt gcgggactta acccaacatc tcacgacacg agctgacgac 420
aaccatgcac cacctgtcac cgttgccccc gaaggggaaa ctatatctct acagtggtca 480
acgggatgtc aagacctggt aaggttcttc gcgttgcttc gaattaaacc acatgctcca 540
ccgcttgtgc gggcccccgt caattccttt gagtttcagt cttgcgaccg tactccccag 600
gcggagtgct taatgcgtta gctgcagcac taaggggcgg aaacccccta acacttagca 660
ctcatcgttt acggcgtgga ctaccagggt atctaatcct gtttgctccc cacgctttcg 720
cgcctcagcg tcagttacag accagaaagt cgccttcgcc actggtgttc ctccaaatct 780
ctacgcattt caccgctaca cttggaattc cactttcctc ttctgcactc aagtccccca 840
gtttccaatg accctccacg gttgagccgt gggctttcac atcagactta aaggaccgcc 900
tgcgcgcgct ttacgcccaa taattccgga caacgcttgc cacctacgta ttaccgcggc 960
tgctggcacg tagttagccg tggctttcta ataaggtacc gtcaaggtac agccagttac 1020
tactgtactt gttcttccct tacaacagag ttttacgatc cgaaaacctt cttcactcac 1080
gcggcgttgc tccatcaggc tttcgcccat tgtggaagat tccctactgc tgcctcccgt 1140
aggagtctgg gccgtgtctc agtcccagtg tggccgatca ccctctcagg tcggctacgc 1200
atcgtcgcct tggtgagccg ttacctcacc aactagctaa tgcgccgcgg gcccatccta 1260
tagcgacagc cgaaaccgtc tttcagtctt tcaccatgaa gcaaaagaga ttattcggta 1320
ttagccccgg tttcccggag ttatcccaaa ctatagggta ggttgcccac gtgttactca 1380
cccgtccgcc gctaacgtca aaggagcaag ctccttttct gttcgctcga cttgcattat 1440
aggcacgccg 1450
<210> 2
<211>20
<212> DNA
<213>artificial sequence
<400> 2
agagtttgat catggctcag 20
<210> 3
<211> 18
<212> DNA
<213>artificial sequence
<400> 3
ggtaccttgt tacgactt 18

Claims (2)

1. one plant of rich phosphorus and degrading organic phosphor pesticidesLysinibacillus macroides, it is characterised in that: the bacterium Strain beLysinibacillus macroides MEW88 has been preserved in Chinese Typical Representative culture guarantor on October 18th, 2016 Hiding center, deposit number are CCTCC NO:M 2016574.
2. one plant of rich phosphorus as described in claim 1 and degrading organic phosphor pesticidesLysinibacillusmacroidesAnswer With, it is characterised in that: it is describedLysinibacillusmacroidesMEW88 is for being enriched with Phos and degrading organic phosphor agriculture Medicine；
The organophosphorus pesticide is glyphosate and Rogor.
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