CN101935633B

CN101935633B - Bacillus amyloliquefacien BZ6-1 strain for producing biosurfactant and application thereof in oil sludge elution

Info

Publication number: CN101935633B
Application number: CN2010102178034A
Authority: CN
Inventors: 刘五星; 骆永明; 王小兵; 王殿玺
Original assignee: Institute of Soil Science of CAS
Current assignee: Institute of Soil Science of CAS
Priority date: 2010-07-06
Filing date: 2010-07-06
Publication date: 2011-10-19
Anticipated expiration: 2030-07-06
Also published as: CN101935633A

Abstract

The invention provides a bacillus amyloliquefacien BZ6-1 strain for producing a biosurfactant and application thereof in oil sludge elution. In the invention, a separated biosurfactant produced by microorganisms is used for treating sludge containing oil so as to achieve the purposes of recovering crude oil deposited in the sludge and reducing oil sludge damages. The strain is preserved in a preservation unit appointed by the State Intellectual Property Office (SIPO) on January 22, 2009, the name of the preservation unit is China General Microbiological Culture Collection Center, and the preservation number is CGMCC No.2892. When the biosurfactant extracted from microorganisms is used for eluting the sludge containing the oil, the average recovery rate of the oil is 87.7 percent, and the content of residual oil in lower sludge is 4.1 percent; and while water is used for contrast treatment, the average recovery rate of the oil is 13.0 percent, and the content of the oil in the lower sludge is 80.6 percent. In addition, the color of the oil sludge treated by the biosurfactant obviously becomes white.

Description

Produce the bacillus amyloliquefaciens BZ6-1 bacterial strain and the application in the greasy filth wash-out thereof of thing tensio-active agent

One, technical field

The invention belongs to contaminated soil recovery technique field, relate in particular to a kind of bacillus amyloliquefaciens BZ6-1 bacterial strain and application in the greasy filth wash-out thereof that produces the thing tensio-active agent.

Two, background technology

Bio-surfactant is the compounds that the microorganism synthetic has amphiphilic (while possess hydrophilic property and lipophilicity group in the molecular structure).The bio-surfactant of microorganisms comprises many different kinds, as glycolipid, lipopeptid, polysaccharide-composite of lipid, phosphatide, lipid acid and neutral fat etc.Compare with chemical surfactant, bio-surfactant have reduce surface tension, stable emulsion, nontoxic, can biological degradation etc. characteristics.

Oily sludge (abbreviation greasy filth) is the oil-containing solids refuse that produces in oil production, transportation, refining and oily water treatment process.General oil length is 10～50% in the greasy filth, and water ratio is 40～90%.In the China's oil chemistry industry, produce 800,000 tons of jar bed muds, pond bed mud every year on average approximately.Contain hundreds of poisonous and harmful compounds in the greasy filth, some compound wherein has teratogenesis, carcinogenic and mutagenic effect.Therefore, Environmental Protection Agency classifies some material wherein as priority pollutant, and the discharging of these materials is had strict restriction, and China also lists greasy filth in national Hazardous wastes register.Therefore reclaim and reduce greasy filth harm and have great economy and society and be worth being deposited on crude oil in the mud.

Three, summary of the invention

Goal of the invention: the invention provides a kind of bacillus amyloliquefaciens BZ6-1 bacterial strain and application in the greasy filth wash-out thereof that produces the thing tensio-active agent.Utilize the Bio-surface active that separates a strain microorganisms that obtains to handle oily sludge, realize reclaiming crude oil that is deposited in the mud and the purpose that reduces greasy filth harm.

Technical scheme: a strain produces bacillus amyloliquefaciens (B.amyloliquefaciens) the BZ6-1 bacterial strain of thing tensio-active agent, this bacterial strain is in the preservation of specified depositary institution of State Intellectual Property Office, preservation date is on 01 22nd, 2009, depositary institution's title: China Committee for Culture Collection of Microorganisms common micro-organisms center, deposit number: CGMCC No.2892.

The application of above-mentioned bacterial strains in the greasy filth wash-out, step comprises: microorganism culturing: bacillus amyloliquefaciens BZ6-1 inoculation is cultivated in fermention medium, and 20～25 ℃ of following 150～300rpm cultivate 15～20h; The preparation of bio-surfactant: 4 ℃ of following centrifugal thalline that go of above-mentioned fermented liquid, supernatant liquor is transferred pH to 2.0, flocks appears, 4 ℃ of standing over night, centrifugal collecting precipitation again, with the acid rinsing of pH 2.0 once, subsequently precipitation is dissolved in NaOH solution, making whole pH value is 7.0, and lyophilize gets light brown loose shape solid bio-surfactant crude product; Purify: crude product is put CH ₂Cl ₂Middle extracting, back evaporated under reduced pressure, NaOH solution dissolves, and forms many bubble liquid, filter paper filtering, filtrate adds HCl accent pH to 2.0 once more, and the precipitation that obtains is centrifugal, gets the beige throw out, and vacuum-drying is the pure product of tensio-active agent after removing residual moisture; The greasy filth wash-out: the sodium-chlor vibration that adds the tensio-active agent that accounts for greasy filth quality 1% and account for greasy filth quality 1～2% in greasy filth, standing demix is finished wash-out.

Above-mentioned fermentative medium formula is: Trisodium Citrate 10g, MgSO ₄0.2g, K ₂HPO ₄1.0g, KH ₂PO ₄1.0g, NH ₄NO ₃1.0g, FeSO ₄0.05g, CaCl ₂0.02g, distilled water 1000mL, pH 7.2.

Beneficial effect: use the bio-surfactant wash-out oily sludge that from this microorganism, extracts, oil recovery rate average out to 87.7%, residual oil-contg is 4.1% in lower floor's mud.And in handling in contrast with water, oil recovery rate average out to 13.9%, residual oil-contg is 80.6% in lower floor's mud.In addition, handling back greasy filth color through tensio-active agent also obviously bleaches.

Four, description of drawings

The Gram-stained Photomicrograph of Figure 1B Z6-1 bacterial strain (* 1000);

Fig. 2 BZ6-1 bacterial strain transmission electron microscope;

The MS/MS/CID fragment mass spectrum of Fig. 3 m/z 1034 quasi-molecular ions;

The MS/MS/CID fragment mass spectrum of Fig. 4 m/z 1048 quasi-molecular ions;

The MS/MS/CID fragment mass spectrum of Fig. 5 m/z 1468 quasi-molecular ions;

The MS/MS/CID fragment mass spectrum of Fig. 6 m/z 1482 quasi-molecular ions;

The MS/MS/CID fragment mass spectrum of Fig. 7 m/z 1509.96 quasi-molecular ions.

Five, embodiment

The invention will be further described below in conjunction with example:

Embodiment 1:

1. bacterial strain

Produce thing tensio-active agent bacterial strain BZ6-1 and obtain (in China Committee for Culture Collection of Microorganisms's common micro-organisms center preservation, deposit number is CGMCC No.2892) for this laboratory separation.The laboratory is stored in the VGPA culture medium slant.

2.BZ6-1 the evaluation of bacterial strain

Cultural characteristic and thalli morphology are observed: Gram-positive (G ⁺) (Fig. 1), rod-short, size is the μ m (Fig. 2) of (0.6-0.7) μ m * (2.0-2.5), produces gemma.

16S rDNA gene sequencing and analysis: with the BZ6-1 strain gene group DNA is template, universal primer with bacterium carries out pcr amplification, amplified production is rich inferior order-checking through Shanghai, record 16S rDNA expanding fragment length and be 1458bp (concrete sequence sees below), the big or small basically identical of the product of this and PCR.Use the BLASTN program to compare and 16S rDNA gene order similarity is further analyzed at National Center Biotechnology Information (NCBI), show that it is a class that BZ6-1 bacterial strain and Bacillus spp. gather, and with the number of landing be that the Bacillus amyloliquefaciens bacterial strain sibship of NC009725 is nearest, homology surpasses 99%.

In conjunction with the morphological specificity of BZ6-1 bacterial strain, be bacillus amyloliquefaciens (B.amyloliquefaciens) with BZ6-1 bacterial strain preliminary evaluation.

CGACTTCACCCCAATCATCTGTCCCACCTTCGGCGGCTGGCTCCATAAAGGTTACCTCAC

CGACTTCGGGTGTTACAAACTCTCGTGGTGTGACGGGCGGTGTGTACAAGGCCCGGGA

ACGTATTCACCGCGGCATGCTGATCCGCGATTACTAGCGATTCCAGCTTCACGCAGTCGA

GTTGCAGACTGCGATCCGAACTGAGAACAGATTTGTGGGATTGGCTTAACCTCGCGGTT

TCGCTGCCCTTTGTTCTGTCCATTGTAGCACGTGTGTAGCCCAGGTCATAAGGGGCATGA

TGATTTGACGTCATCCCCACCTTCCTCCGGTTTGTCACCGGCAGTCACCTTAGAGTGCCC

AACTGAATGCTGGCAACTAAGATCAAGGGTTGCGCTCGTTGCGGGACTTAACCCAACAT

CTCACGACACGAGCTGACGACAACCATGCACCACCTGTCACTCTGCCCCCGAAGGGGA

CGTCCTATCTCTAGGATTGTCAGAGGATGTCAAGACCTGGTAAGGTTCTTCGCGTTGCTT

CGAATTAAACCACATGCTCCACCGCTTGTGCGGGCCCCCGTCAATTCCTTTGAGTTTCAG

TCTTGCGACCGTACTCCCCAGGCGGAGTGCTTAATGCGTTAGCTGCAGCACTAAGGGGC

GGAAACCCCCTAACACTTAGCACTCATCGTTTACGGCGTGGACTACCAGGGTATCTAATC

CTGTTCGCTCCCCACGCTTTCGCTCCTCAGCGTCAGTTACAGACCAGAGAGTCGCCTTC

GCCACTGGTGTTCCTCCACATCTCTACGCATTTCACCGCTACACGTGGAATTCCACTCTC

CTCTTCTGCACTCAAGTTCCCCAGTTTCCAATGACCCTCCCCGGTTGAGCCGGGGGCTTT

CACATCAGACTTAAGAAACCGCCTGCGAGCCCTTTACGCCCAATAATTCCGGACAACGC

TTGCCACCTACGTATTACCGCGGCTGCTGGCACGTAGTTAGCCGTGGCTTTCTGGTTAGG

TACCGtCAaGGTGCCGCCCTATTTGAACGGCACTTGTTCTTCCCTAACAACAGAGCTTTAC

GATCCGAAAACCTTCATCACTCACGCGGCGTTGCTCCGTCAGACTTTCGTCCATTGCGG

AAGATTCCCTACTGCTGCCTCCCGTAGGAGTCTGGGCCGTGTCTCAGTCCCAGTGTGGC

CGATCACCCTCTCAGGTCGGCTACGCATCGTCGCCTTGGTGAGCCGTTACCTCACCAACT

AGCTAATGCGCCGCGGGTCCATCTGTAAGTGGTAGCCGAAGCCACCTTTTATGTCTGAAC

CATGCGGTTCAGACAACCATCCGGTATTAGCCCCGGTTTCCCGGAGTTATCCCAGTCTTA

CAGGCAGGTTACCCACGTGTTACTCACCCGTCCGCCGCTAACATCAGGGAGCAAGCTCC

CATCTGTCCGCTCGACTTGCATGTATAGC

3. substratum

The VGPA solid medium: glucose 10g, peptone 5g, yeast extract paste 5g, agar 20g, distilled water 1000mL, pH 7.2.

Fermention medium: Trisodium Citrate 10g, MgSO ₄0.2g, K ₂HPO ₄1.0g, KH ₂PO ₄1.0g, NH ₄NO ₃1.0g, FeSO ₄0.05g, CaCl ₂0.02g, distilled water 1000mL, pH 7.2.

Above substratum is all at 121 ℃ of sterilization 20min.

4. the fermentative production of bio-surfactant

Be inoculated into fermention medium from slant tube picking one ring of preserving bacillus amyloliquefaciens (B.amyloliquefaciens), the 100mL/250mL triangular flask, 22 ℃ of following 200rpm cultivate 18h.The fermented liquid of this moment promptly contains bio-surfactant.

5. the extraction of bio-surfactant, purifying and evaluation

4 ℃ of following 10000r min of fermented liquid ^-1Centrifugal 10min removes thalline, and supernatant liquor is transferred pH to 2.0 with dense HCl, flocks occurs, 4 ℃ of standing over night, 10000r min ^-1Centrifugal 10min collecting precipitation is dissolved in precipitation NaOH solution once, subsequently with the acid rinsing of pH 2.0, and making whole pH value is 7.0, lyophilize, light brown loose shape solid bio-surfactant crude product.

During purifying, crude product is put CH ₂Cl ₂Middle extracting, back evaporated under reduced pressure, dilute NaOH solution dissolving, form many bubble liquid, use the WhatmanNo.4 filter paper filtering, filtrate adds HCl once more and transfers pH to 2.0, the precipitation that obtains is centrifugal, get the beige throw out, vacuum-drying is the pure product of tensio-active agent after removing residual moisture.This material is identified the lipopeptid class material that comprises surfactivity element (surfactin) and fragrant shepherd's purse element (fengycin) through HPLC-MS.

Expert evidence

Surfactin is a kind of lipopeptide antibiotic substance, and it is made up of the little peptide of beta-hydroxy fatty acid and 7 amino-acid residues, and the carboxyl on the 7th amino acids of peptide chain and the beta-hydroxy condensation of lipid acid form ring texture (Peypoux etc., 1994).When studies show that 7 amino acids are Leu on the peptide chain, the feature fragment ion peak of m/z707 is arranged in the CID spectrogram according to document (Sun Lijun, 2006), it can be interpreted as [LeuLeuValAspLeuLeu+H ₂O+Na] ⁺, can judge surfactin[7Leu with this] and the characteristic ion peak of homologue.M/z1034 in this research (Fig. 3) and 1048 (Fig. 4), Surfactin is more similar to lipopeptid class material, analyzes by the MS/CID spectrum, and in second order ms, all there are m/z 707 feature fragment ion peaks in two kinds of materials.In addition, also have m/z 463 and m/z 594 feature fragment ion peaks, can infer that thus 1034 and 1048 is that 7 amino acids are the Surfactin of Leu on the peptide chain.

The structure of Surfactin A

The structure of Surfactin B

Lipopeptid fengycin linear structure is analyzed

Research such as Liu Huimin etc. (2000) and Chen Jing (2002) thinks that the synthetic of antibacterial lipopeptid is the huge mixture NRPS (non-ribosomal peptide synthetases) that forms owing to a plurality of peptide synthetases, and the mode of " multichip carrier sulphur template mechanism (Multiple Carrier Thiotemplate Mechanism) " is come synthetic by a kind of being called.Sun Lijun (2006) utilizes this principle to infer the intermediate that Fengycin A, the linear structure lipopeptid.At m/z 1468,1482, y type characteristic ion fragments (Fig. 5～7) such as 1098 and 984 are all arranged in 1509.96 in this research, they can be interpreted as respectively having lost lipid acid and adjacent Glu from N-terminal in the molecular structure, and Glu-Orn.While 1468,1482 differs 14 between 1509.96, show in the fatty acid chain to differ-CH ₂-.Can infer that thus these three kinds of materials are the linear structure lipopeptid of 15C, 16C and 18C lipid acid.

Linear lipopeptid molecular structure CID analyzes:

6. the mensuration of the collection of greasy filth and oil-contg

Derive from Shengli Oil Field for formation testing mud.Adopt the oleaginousness in the gravimetric determination greasy filth.Concrete grammar is: with air-dry greasy filth and the isopyknic anhydrous Na of crossing the 2mm sieve of 5g ₂SO ₄Mixing then with the bottled 70mL of the going into trichloromethane of eggplant type of weighing, extracts 24h through Soxhlet under 75 ℃.With extract evaporated under reduced pressure on the decompression Rotary Evaporators, weighing eggplant type bottle calculates total oil-contg again then.

7. the wash-out of greasy filth experiment

Get fresh greasy filth 20g (greasy filth character sees Table 1), add tensio-active agent crude product 0.2g, 2wt% sodium chloride solution 150mL puts into the 250mL triangular flask, and 35 ℃ of 200rpm vibration 72h leave standstill 1h then.Mud, oil, the liquid surface of finding this moment to shake in the bottle are separated obviously, occur going up clearly (oil), in (water), descend (mud) three layers of a large amount of leaching oil float on liquid level, recovery swims in the crude oil of liquid level, simultaneously lower floor's mud is taken out wherein oil-contg of air-dry mensuration, and elute effect sees Table 2.Through this method wash-out oily sludge, oil recovery rate average out to 87.7%, residual oil-contg is 4.1% in lower floor's mud.And in handling in contrast with water, oil recovery rate average out to 13.9%, residual oil-contg is 80.6% in lower floor's mud.In addition, handling back greasy filth color through tensio-active agent also obviously bleaches.

The adding of tensio-active agent and sodium-chlor has reduced adhesive power and the electrostatic forcing of silt particle to oil, makes particle separation such as oil and silt particle, thus the purpose that realizes improving the crude oil recovery rate and reduce the greasy filth pollution.

The used greasy filth character of table 1 experiment

Fresh greasy filth	Weight in wet base (g)	Dry weight (g)	Oleaginousness (g)	Oil length (%)
					1	20	11.58	3.59	31.0
2	20	11.62	4.60	39.5
					3	20	11.42	3.53	30.9
Mean value	20	11.54	3.91	33.9

Table 2 different treatment is to the elute effect of greasy filth

The rate of recovery=upper strata weight of oil/experiment greasy filth total oil content amount * 100%

Residual oil content=lower floor's greasy filth oleaginousness/lower floor's greasy filth dry weight * 100%

Claims

1. a strain produces bacillus amyloliquefaciens (B.amyloliquefaciens) the BZ6-1 bacterial strain of thing tensio-active agent, this bacterial strain is in the preservation of specified depositary institution of State Intellectual Property Office, preservation date is on 01 22nd, 2009, depositary institution's title: China Committee for Culture Collection of Microorganisms common micro-organisms center, deposit number: CGMCC No.2892.

2. the application of the described bacterial strain of claim 1 in the greasy filth wash-out is characterized in that step comprises:

A. microorganism culturing: bacillus amyloliquefaciens BZ6-1 inoculation is cultivated in fermention medium, 20～25 ℃ down 150～300rpm cultivate 15～20h;

B. the preparation of bio-surfactant: 4 ℃ of following centrifugal thalline that go of above-mentioned fermented liquid, supernatant liquor is transferred pH to 2.0, flocks appears, 4 ℃ of standing over night, centrifugal collecting precipitation again, with the acid rinsing of pH 2.0 once, subsequently precipitation is dissolved in NaOH solution, making whole pH value is 7.0, and lyophilize gets light brown loose shape solid bio-surfactant crude product;

C. purify: crude product is put CH ₂Cl ₂Middle extracting, back evaporated under reduced pressure, NaOH solution dissolves, and forms many bubble liquid, filter paper filtering, filtrate adds HCl accent pH to 2.0 once more, and the precipitation that obtains is centrifugal, gets the beige throw out, and vacuum-drying is the pure product of bio-surfactant after removing residual moisture;

D. greasy filth wash-out: add the sodium-chlor vibration that accounts for the pure product of bio-surfactant of greasy filth quality 1% and account for greasy filth quality 1～2% in greasy filth, standing demix is finished wash-out.

3. the application of bacterial strain according to claim 2 in the greasy filth wash-out is characterized in that described fermentative medium formula is: Trisodium Citrate 10g, MgSO ₄0.2g, K ₂HPO ₄1.0g, KH ₂PO ₄1.0g, NH ₄NO ₃1.0g, FeSO ₄0.05g, CaCl ₂0.02g, distilled water 1000mL, pH 7.2.