CN107201320A - One plant can make tosca spindle lysine bacillus LZ1 and its application - Google Patents

One plant can make tosca spindle lysine bacillus LZ1 and its application Download PDF

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CN107201320A
CN107201320A CN201610158637.2A CN201610158637A CN107201320A CN 107201320 A CN107201320 A CN 107201320A CN 201610158637 A CN201610158637 A CN 201610158637A CN 107201320 A CN107201320 A CN 107201320A
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李红玉
孙莺
李洋
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Lanzhou University
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Abstract

The present invention relates to one plant of spindle lysine bacillus LZ1 (Lysinibacillus fusiformis LZ1) that can make tosca and its application, orchard is isolated from.China General Microbiological culture presevation administrative center is preserved in September in 2015 within 30th, preserving number is CGMCC 11271, entitled spindle lysine bacillus LZ1 (Lysinibacillus fusiformis LZ1), the bacterium has good tosca performance, as biological binding agent, can carry out the repairing of building tear, regeneration concrete it is cementing etc..

Description

One plant can make tosca spindle lysine bacillus LZ1 and its application
Technical field：
The invention belongs to microorganism field, and in particular to one plant can make the spindle lysine bacillus of tosca LZ1 (Lysinibacillus fusiformis LZ1) and its mixed in repairing, the regeneration of building tear as biological binding agent Coagulate the cementing application of soil.The bacterium is preserved in depositary institution on 24th in August in 2015：Chinese microorganism strain preservation management committee Member can common micro-organisms center.Address：Chinese Beijing Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3s, postcode：100101, electricity Words：010-64807355, fax：010-64807288, E-mail：Cgmcc@im.ac.cn, its preserving number is CGMCC No.11271。
Background technology：
The high energy consumption of traditional Geotechnical Engineering reinforcement material, high pollution problem becomes countries in the world focus of attention.Research and development Low, the environment-friendly Geotechnical Engineering of energy consumption reinforces new technology, has important practical significance.Microorganism and Geotechnical Engineering, which are intersected, urges Raw microorganism Geotechnical Engineering subject, with the low advantage of its environment-friendly, energy consumption, turns into microorganism Geotechnical Engineering field at present Study hotspot, 21 century important research field was defined as by national research council in 2006.Geotechnical Engineering reinforces skill Art is one of emphasis of countries in the world research, is also one of the important topic in Geotechnical Engineering field.It is big in the epoch of energy-conserving and environment-protective Under background, maximum discharge that cement, waterglass and other organic materials are brought as traditional Geotechnical Engineering reinforcement material, high energy The problems such as consuming, easily cause pollution, it is more and more of interest by people.Microorganism induction tosca (MICP) technology turns into ground The study hotspot in engineering reinforcement field.
The doped calcium based on microorganism induction of the technology, it is substantial amounts of high living using being produced in the activity of urease-producing bacterial metabolism Property urase, obligate hydrolysis urea produces CO3 2-, and change environmental pH, in external source Ca2+Lower generation precipitation of calcium carbonate is introduced, is played Cementing and closure effect.
The present invention relates to the spindle lysine bacillus that one plant has the efficient derivable doped calcium of urease-producing ability LZ1 (Lysinibacillus fusiformis LZ1), the strain isolation is from orchard soil.Protected within 24th in August in 2015 China Committee for Culture Collection of Microorganisms's common micro-organisms center is hidden in, preserving number is CGMCC No.11271, entitled Spindle lysine bacillus (Lysinibacillus fusiformis).
Lysinibacillus fusiformis LZ1 of the present invention feature is as follows：
(1) morphological feature：
The bacterial strain is shaft-like, and thalline size is about 1.0-2.0 × 0.4-1.0 μm, Gram-negative bacterium.
(2) cultural characteristic：
The bacterial strain is in NH4After the culture 2 days of 30 DEG C of-YE plating mediums, there are milky circular colonies, diameter about 1-2mm, The smooth moistening in bacterium colony surface.
(3) physiological and biochemical property of bacterial strain：
Oxydase reaction is positive, and esterase (C 4), lipoid esterase (C 8), a- chymotrypsins, acid phosphatase, naphthols- AS-BI- phosphohydrolases, beta galactoside enzyme, a- glucuroides are positive, gelatin hydrolysate.
Bacterial strain can produce urase, and decomposing urea forms CO32-And NH4+.When containing finite concentration calcium ion in solution, Calcium ion can be adsorbed by cell, so as to using cell as nucleus, will form the calcium carbonate knot with gelatification around bacterium It is brilliant.
The most suitable growth pH is 6.0-9.5, and optimum growth temperature is 30-37 DEG C.
(4) genetics characteristics：
The 16s rDNA gene orders of bacterial strain disclosed by the invention are：
TGGCGCGCGCTATACATGCTAGTCGAGCGAACAGAGAAGGAGCTTGCTCCTTCGACGTTAGCGGCGGACGGGTGAGT AACACGTGGGCAACCTACCTTATAGTTTGGGATAACTCCGGGAAACCGGGGCTAATACCGAATAATCTGTTTCACCT CATGGTGAAACACTGAAAGACGGTTTCGGCTGTCGCTATAGGATGGGCCCGCGGCGCATTAGCTAGTTGGTGAGGTA ACGGCTCACCAAGGCGACGATGCGTAGCCGACCTGAGAGGGTGATCGGCCACACTGGGACTGAGACACGGCCCAGAC TCCTACGGGAGGCAGCAGTAGGGAATCTTCCACAATGGGCGAAAGCCTGATGGAGCAACGCCGCGTGAGTGAAGAAG GATTTCGGTTCGTAAAACTCTGTTGTAAGGGAAGAACAAGTACAGTAGTAACTGGCTGTACCTTGACGGTACCTTAT TAGAAAGCCACGGCTAACTACGTGCCAGCAGCCGCGGTAATACGTAGGTGGCAAGCGTTGTCCGGAATTATTGGGCG TAAAGCGCGCGCAGGTGGTTTCTTAAGTCTGATGTGAAAGCCCACGGCTCAACCGTGGAGGGTCATTGGAAACTGGG AGACTTGAGTGCAGAAGAGGATAGTGGAATTCCAAGTGTAGCGGTGAAATGCGTAGAGATTTGGAGGAACACCAGTG GCGAAGGCGACTATCTGGTCTGTAACTGACACTGAGGCGCGAAAGCGTGGGGAGCAAACAGGATTAGATACCCTGGT AGTCCACGCCGTAAACGATGAGTGCTAAGTGTTAGGGGGTTTCCGCCCCTTAGTGCTGCAGCTAACGCATTAAGCAC TCCGCCTGGGGAGTACGGTCGCAAGACTGAAACTCAAAGGAATTGACGGGGGCCCGCACAAGCGGTGGAGCATGTGG TTTAATTCGAAGCAACGCGAAGAACCTTACCAGGTCTTGACATCCCGTTGACCACTGTAGAGATATGGTTTCCCCTT CGGGGGCAACGGTGACAGGTGGTGCATGGTTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAG CGCAACCCTTGATCTTAGTTGCCATCATTTAGTTGGGCACTCTAAGGTGACTGCCGGTGACAAACCGGAGGAAGGTG GGGATGACGTCAAATCATCATGCCCCTTATGACCTGGGCTACACACGTGCTACAATGGACGATACAAACGGTTGCCA ACTCGCGAGAGGGAGCTAATCCGATAAAGTCGTTCTCAGTTCGGATTGTAGGCTGCAACTCGCCTACATGAAGCCGG AATCGCTAGTAATCGCGGATCAGCATGCCGCGGTGAATACGTTCCCGGGCCTTGTACACACCGCCCGTCACACCACG AGAGTTTGTAACACCCGAAGTCGGTGAGGTAACCTTTGGAGCCAGCCGCTCGAAGTGGATG
In http:On //blast.ncbi.nlm.nih.gov/Blast.cgi, with BLAST softwares by spindle lysine Bacillus LZ1 (Lysinibacillus fusiformis LZ1) 16s rDNA gene orders and GenBank known sequence Row are compared.As a result show spindle lysine bacillus LZ1 (Lysinibacillus fusiformis LZ1) with relying Propylhomoserin bacillus (Lysinibacillussp.E4 16S ribosomal RNA gene, partial sequence) Sequence homology reaches 99%, it was demonstrated that spindle lysine bacillus LZ1 (the Lysinibacillus fusiformis LZ1 lysine bacillus) is belonged to.
Spindle lysine bacillus LZ1 (Lysinibacillus fusiformis LZ1) of the present invention can produce Raw urase, decomposing urea forms CO32-And NH4+.When containing finite concentration calcium ion in solution, calcium ion can be inhaled by cell It is attached, so that using cell as nucleus, the calcium carbonate crystal with gelatification will be formed around bacterium.The bacterium has good Tosca performance, with control strain Bacillus pasteurii (Sporosarcina pasteurii, S.pasteuri, ATCC11859 the comparison of tosca amount) is carried out, LZ1 is 11.57g/L, and S.pasteuri is 10.55g/L.LZ1 than conventional High 10 ﹪ of carbonate mineralized bacterium S.pasteurii.Can be as biological binding agent, repairing, the regeneration for carrying out building tear are mixed Solidifying soil it is cementing etc..
Brief description of the drawings
Fig. 1 spindle lysine bacillus LZ1 (Lysinibacillus fusiformis LZ1) Gram's staining
Fig. 2 spindle lysine bacillus LZ1 (Lysinibacillus fusiformis LZ1) transmission electron microscope picture (multiplication factor:7000 times, scale：200nm, Electronic Speculum model：FEI TECNAI G2TF20)
Fig. 3 spindle lysine bacillus LZ1 (Lysinibacillus fusiformis LZ1) deposition calcium carbonate is brilliant The X-ray diffractogram of body
Fig. 4 spindle lysine bacillus LZ1 (Lysinibacillus fusiformis LZ1) deposition calcium carbonate is brilliant The scanning electron microscope (SEM) photograph of body
Fig. 5 spindle lysine bacillus LZ1 (Lysinibacillus fusiformis LZ1) deposited crystal EDX collection of illustrative plates
Fig. 6 spindle lysine bacillus LZ1 (Lysinibacillus fusiformis LZ1) and control strain S.pasteurii tosca amount compares
Embodiment：
Embodiment 1 separates spindle lysine bacillus LZ1 (Lysinibacillus from soil with dilution-plate method fusiformis LZ1)
(1) collection of soil sample：Soil sample gathers the orchard from Laizhou City of Shandong Province Cheng Guo towns in March, 2014.
(2) sample dilution and culture：Soil sampling 10g, 10 are diluted to sterilized water-1、10-2、10-3、10-4、10-5、10-6Altogether 6 concentration, take dilution to be coated on urea flat board (L-1:5g NaCl,2g KH2PO4,1g glucose, 0.012g is phenol red, 0.2g Peptone, 20g urea, 15g agar, pH 6.8) in 30 DEG C of cultures.
(3) separation, the purifying of bacterial strain：After 2-3 days, after flat board grows bacterium colony, the single bacterium that picking color reddens is fallen within 30 DEG C of constant-temperature shaking cultures on shaking table in 10ml urea liquid culture mediums.Purified using plate streaking partition method, by Shaking culture Thing is lined to be separated on urea liquid solid medium, solid-liquid alternate culture 2-3 times, until purifying obtains single bacterium Fall, obtain spindle lysine bacillus LZ1 (Lysinibacillus fusiformis LZ1).The bacterium culture exists NH4Be creamy white circular colonies in-YE solid cultures, diameter about 1-2mm, the smooth moistening in bacterium colony surface.
(4) preservation mode：The spindle lysine bacillus LZ1 (Lysinibacillus isolated and purified Fusiformis LZ1) with -80 DEG C of preservations of 20% glycerine.
The bacterial strain is preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms on the 24th in August in 2015 The heart, preserving number is CGMCC No.11271, entitled spindle lysine bacillus (Lysinibacillus fusiformis)。
The spindle lysine bacillus LZ1 (Lysinibacillus fusiformis LZ1) of embodiment 2 feature mirror It is fixed
Thalli morphology is observed and staining reaction：
Spindle lysine bacillus LZ1 (Lysinibacillus fusiformis LZ1) is inoculated in liquid training Support after 30 DEG C of the base incubated suitable time, dyeing microscopic examination.
Dyeing and microscopy result (Fig. 1)：Spindle lysine bacillus LZ1 (Lysinibacillus fusiformis LZ1 it is) bacillus, Gram's staining result is shown as Gram-negative bacteria, and thalline length is about 2.0-3.0 × 0.8-1.5 μ M (Fig. 2).
(2) compared using 16s rDNA and analyze and identify spindle lysine bacillus LZ1 (Lysinibacillus fusiformis LZ1)：
Amplification and sequencing primer：
Forward primer：27F 5’-AGAGTTTGATCCTGGCTCAG-3’
Reverse primer：1492R 5’-GGTTACCTTGTTACGACTT-3’
Extract the total of spindle lysine bacillus LZ1 (Lysinibacillus fusiformis LZ1) bacterial strain DNA, the 16s rDNA genes that performing PCR expands the bacterial strain are entered with primer 2 7F and 1492R, are sequenced with primer 2 7F and 1492R, The 16s rDNA gene orders of the bacterial strain are：
TGGCGCGCGCTATACATGCTAGTCGAGCGAACAGAGAAGGAGCTTGCTCCTTCGACGTTAGCGGCGGACGGGTGAGT AACACGTGGGCAACCTACCTTATAGTTTGGGATAACTCCGGGAAACCGGGGCTAATACCGAATAATCTGTTTCACCT CATGGTGAAACACTGAAAGACGGTTTCGGCTGTCGCTATAGGATGGGCCCGCGGCGCATTAGCTAGTTGGTGAGGTA ACGGCTCACCAAGGCGACGATGCGTAGCCGACCTGAGAGGGTGATCGGCCACACTGGGACTGAGACACGGCCCAGAC TCCTACGGGAGGCAGCAGTAGGGAATCTTCCACAATGGGCGAAAGCCTGATGGAGCAACGCCGCGTGAGTGAAGAAG GATTTCGGTTCGTAAAACTCTGTTGTAAGGGAAGAACAAGTACAGTAGTAACTGGCTGTACCTTGACGGTACCTTAT TAGAAAGCCACGGCTAACTACGTGCCAGCAGCCGCGGTAATACGTAGGTGGCAAGCGTTGTCCGGAATTATTGGGCG TAAAGCGCGCGCAGGTGGTTTCTTAAGTCTGATGTGAAAGCCCACGGCTCAACCGTGGAGGGTCATTGGAAACTGGG AGACTTGAGTGCAGAAGAGGATAGTGGAATTCCAAGTGTAGCGGTGAAATGCGTAGAGATTTGGAGGAACACCAGTG GCGAAGGCGACTATCTGGTCTGTAACTGACACTGAGGCGCGAAAGCGTGGGGAGCAAACAGGATTAGATACCCTGGT AGTCCACGCCGTAAACGATGAGTGCTAAGTGTTAGGGGGTTTCCGCCCCTTAGTGCTGCAGCTAACGCATTAAGCAC TCCGCCTGGGGAGTACGGTCGCAAGACTGAAACTCAAAGGAATTGACGGGGGCCCGCACAAGCGGTGGAGCATGTGG TTTAATTCGAAGCAACGCGAAGAA CCTTACCAGGTCTTGACATCCCGTTGACCACTGTAGAGATATGGTTTCCCCTTCGGGGGCAACGGTGACAGGTGGTG CATGGTTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCTTGATCTTAGTTGCCA TCATTTAGTTGGGCACTCTAAGGTGACTGCCGGTGACAAACCGGAGGAAGGTGGGGATGACGTCAAATCATCATGCC CCTTATGACCTGGGCTACACACGTGCTACAATGGACGATACAAACGGTTGCCAACTCGCGAGAGGGAGCTAATCCGA TAAAGTCGTTCTCAGTTCGGATTGTAGGCTGCAACTCGCCTACATGAAGCCGGAATCGCTAGTAATCGCGGATCAGC ATGCCGCGGTGAATACGTTCCCGGGCCTTGTACACACCGCCCGTCACACCACGAGAGTTTGTAACACCCGAAGTCGG TGAGGTAACCTTTGGAGCCAGCCGCTCGAAGTGGATG
In http:On //blast.ncbi.nlm.nih.gov/Blast.cgi, with BLAST softwares by spindle lysine Bacillus LZ1 (Lysinibacillus fusiformis LZ1) 16s rDNA gene orders and GenBank known sequence Row are compared.As a result show spindle lysine bacillus LZ1 (Lysinibacillus fusiformis LZ1) with relying Propylhomoserin bacillus 16SrDNA genes (Lysinibacillussp.E4 16S ribosomal RNA gene, partial Sequence sequence homology) reaches 99%, it was demonstrated that the spindle lysine bacillus LZ1 (Lysinibacillus Fusiformis LZ1) belong to lysine bacillus.
The spindle lysine bacillus LZ1 (Lysinibacillus fusiformis LZ1) of embodiment 3 calcium carbonate Deposition properties
2% spindle lysine bacillus LZ1 (Lysinibacillus fusiformis LZ1) bacterium solution is with compareing Bacterial strain S.pasteurii, after cultivating 1 day, takes bacterium solution 10ml, adds 140ml Urea-CaCl2Culture medium (1L：3gNB； 2.12gNaHCO3；20gNH4Cl, surplus is distilled water, adjusts after pH to 6.5, autoclaving, adds the urea of filtration sterilization And CaCl2, final concentration is 1M), 150rpm is cultivated 7 days in 30-37 DEG C of shaking table, generates the precipitation of white.It is heavy that LZ1 is produced Form sediment and carry out XRD (Fig. 3), SEM (Fig. 4) and EDX analyses (Fig. 5) after drying.As a result show that this is precipitated as calcium carbonate, crystal type For calcite.
LZ1 and the comparison (Fig. 6) of S.pasteurii tosca amounts are carried out, it is 11.57g/L as a result to show LZ1, S.pasteuri is 10.55g/L.LZ1 than conventional high 10 ﹪ of carbonate mineralized bacterium S.pasteurii.Doped calcium performance is better than S.pasteurii.
Knowable to summary experimental result, spindle lysine bacillus LZ1 (Lysinibacillus Fusiformis LZ1) there is stronger tosca performance, in urea and exogenous Ca2+In the presence of, carbonic acid can be generated Calcium, its crystal type is most stable of calcite, and doped calcium performance is higher than control strain S.pasteurii, can be used as biological slime Tie agent, carry out the repairing of building tear, regeneration concrete it is cementing etc..
。

Claims (5)

1. one plant can make the bacterium of tosca, it is characterised in that the bacterium is spindle lysine bacillus LZ1 (Lysinibacillus fusiformis LZ1), is preserved in China General Microbiological culture presevation administrative center, preserving number is CGMCC 11271。
2. bacterium according to claim 1, it is characterised in that：
(1) morphological feature：The bacterial strain is shaft-like, and thalline size is about 2.0-3.0 × 0.8-1.5 μm, Gram-negative bacterium；
(2) cultural characteristic：The bacterial strain on tryptose soya agar plating medium 30 DEG C culture 2 days after, there is milky Circular colonies, diameter about 1-2mm, the smooth moistening in bacterium colony surface；
(3) physiological and biochemical property of bacterial strain：Oxydase reaction is positive, esterase, lipoid esterase, Chymetin, acid phosphatase Enzyme, naphthols-AS-BI- phosphohydrolases, beta galactoside enzyme, alpha-glucosidase are positive, gelatin hydrolysate.Urase can be produced, The most suitable growth pH is 6.0-9.5, and optimum growth temperature is 30-37 DEG C；
The induction tosca characteristic of bacterial strain：Bacterial strain can produce urase, and decomposing urea forms CO32-And NH4+, when in solution During containing finite concentration calcium ion, calcium ion can be adsorbed by cell, so as to using cell as nucleus, will form tool around bacterium There is the calcium carbonate crystal of gelatification；In tosca experiment, deposition reaches 11.57g/L, and crystal type is calcite.
(4) the 16S rDNA gene orders such as Seq ID No of bacterial strain:Shown in 1.
3. application of the bacterium as claimed in claim 1 or 2 in biological binding agent is prepared.
4. bacterium repairing as claimed in claim 1 or 2 mends the application in building tear.
5. application of the bacterium as claimed in claim 1 or 2 in cementing regeneration concrete.
CN201610158637.2A 2016-03-18 2016-03-18 Lysinibacillus fusiformis LZ1 capable of depositing calcium carbonate and application thereof Active CN107201320B (en)

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CN108558260A (en) * 2018-05-24 2018-09-21 中交四航工程研究院有限公司 A kind of self union concrete complex enzyme microencapsulation material and preparation method thereof
CN108558260B (en) * 2018-05-24 2020-12-01 中交四航工程研究院有限公司 Self-healing concrete complex enzyme microcapsule material and preparation method thereof
CN111592991A (en) * 2019-02-21 2020-08-28 中国科学院南京土壤研究所 Efficient calcium mineralization bacterium and application thereof
CN110054444A (en) * 2019-04-30 2019-07-26 东南大学 It is a kind of suitable for the cement-based material self repairing agent of low temperature environment and its application
CN114921369A (en) * 2022-05-19 2022-08-19 中国水产科学研究院黑龙江水产研究所 Bacterial strain capable of producing protease and application thereof
CN114921369B (en) * 2022-05-19 2022-11-04 中国水产科学研究院黑龙江水产研究所 Bacterial strain capable of producing protease and application thereof
CN115095305A (en) * 2022-05-27 2022-09-23 中国石油大学(北京) Profile control oil displacement method based on microorganisms

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