CN104845902B - Applications of achromobacter (Achromobacter sp.) the MT H in diisooctyl phthalate of degrading - Google Patents

Abstract

The invention belongs to environment pollutant biological treatment technical field, specifically disclose achromobacter (AchromobacterSp.) applications of the MT H in diisooctyl phthalate of degrading, the bacterial strain were deposited in China typical culture collection center on January 22nd, 2015（CCTCC）, deposit number is CCTCC M 2015058.The bacterial strain has very high degradation efficiency to DEHP, and the DEHP that can preferably be degraded in wider pH and temperature range, it was demonstrated that in terms of MT H can be applied to the biological treatment of DEHP pollutions as excellent DEHP degradation bacterias.

Description

Achromobacter (Achromobacter sp.) MT-H is different in degraded phthalic acid two Application in monooctyl ester

Technical field

The present invention relates to environment pollutant biological treatment technical field, in particular it relates to achromobacter (AchromobacterSp.) applications of the MT-H in diisooctyl phthalate of degrading.

Background technology

Phthalic acid ester, also known as phthalate ester, abridge PAEs, is the general designation for the ester that phthalic acid is formed.Wherein, adjacent benzene Dioctyl phthalate di-isooctyl（DEHP）It is a kind of typical phthalate material, purposes is very wide in people's daily life It is general, especially as plastic additive, up to 20~50% in weight.Because DEHP is easily transferred into ring in plastic products Border, so as to be widely present in air, water body, soil and organism.DEHP in phthalate Substance Intoxication most By force, can enter by all means in organism, severe jamming its endocrine, reproduction and nervous system, and with extremely strong Teratogenesis, mutagenicity, carcinogenicity.At present, DEHP has turned into one of most common pollutant in the whole world, and by American National ring Guarantor office and China Environmental Monitoring General Station are classified as priority pollutants.

DEHP hydrolysis, photodissociation and rate of volatilization are very slow in natural environment, and biodegradation turns into it from environment The main path of disappearance.But because DEHP side chain is longer, although many DEHP that can degrade strain in soil be present, it is certainly Right degradation capability is also extremely weak, far can not meet the needs of DEHP environmental pollution improvements.Therefore, the processing to DEHP needs In seeking efficient degrading bacteria.

At present for microbial degradation DEHP, numerous studies are carried out both at home and abroad.For example, the screening such as Kim is isolated One plant of Fusarium oxysporum, and one plant of Rhodococcus sp isolating such as Chao and Cheng is respectively provided with good degraded effect to DEHP Fruit.But the bacterial strain reported is not met by the requirement of actual contamination control, and relevant energy degraded to DEHP degradation rate DEHP achromobacter germ plasm resource is rarely reported.

The content of the invention

The present invention is in order to overcome the above-mentioned deficiency of prior art, there is provided one plant of new diisooctyl phthalate degraded Bacterium.

To achieve these goals, the present invention is achieved by following scheme：

One plant of achromobacter (AchromobacterSp.) bacterial strain MT-H, the bacterial strain were deposited on January 22nd, 2015 China typical culture collection center（CCTCC）, deposit number is CCTCC M 2015058, and preservation address is：Wuhan City, Hubei Province Wuhan University of city, strain classification are named asAchromobactersp. MT-H。

Achromobacter of the present invention (AchromobacterSp.) bacterial strain MT-H is dirty from Guangzhou great Tan Shachen City city The returned sludge of water treatment plant, through artificial enrichment culture, isolate and purify to obtain.The bacterial strain there is very high degraded to imitate DEHP Rate, and the DEHP that can preferably be degraded in wider pH and temperature range, it was demonstrated that MT-H bacterial strains can be as excellent DEHP In terms of degradation bacteria is applied to the biological treatment of DEHP pollutions.

Achromobacter (AchromobacterSp.) bacterium colony that bacterial strain MT-H is cultivated seven days on LB culture mediums is circle, Microprotrusion, faint yellow, moistening, translucent, neat in edge are smooth.Under ESEM be mostly spherical or ellipsoid, a diameter of 0.5 ~ 1μm。

Achromobacter (AchromobacterSp.) bacterial strain MT-H 16S rDNA gene orders such as SEQ ID NO:1 institute Show, pass through NCBI official websites（http://www.ncbi.nlm.nih.gov/）Blast program with other logged in bacterium Bacterial strain 16S rDNA sequences are compared, the results showed that the bacterial strain withAchromobacterSp. similitude highest, homology reach 99%。

Achromobacter (AchromobacterSp.) applications of the MT-H in diisooctyl phthalate of degrading；It is described Bacterial strain was deposited in China typical culture collection center on January 22nd, 2015（CCTCC）, deposit number is CCTCC M 2015058。

Achromobacter (AchromobacterSp.) MT-H answering in diisooctyl phthalate pollution medium is repaired With；The bacterial strain was deposited in China typical culture collection center on January 22nd, 2015（CCTCC）, deposit number CCTCC M 2015058。

Preferably, the medium is soil, water or air.

Preferably, bacterial strain MT-H is prepared into bacteria suspension and is used for diisooctyl phthalate of degrading.

Preferably, the OD of the bacteria suspension_{600 nm} = 0.8~1.2。

Achromobacter (AchromobacterSp.) MT-H is preparing the biological agent of degraded diisooctyl phthalate In application；The bacterial strain was deposited in China typical culture collection center on January 22nd, 2015（CCTCC）, deposit number For CCTCC M 2015058.

Compared with prior art, the invention has the advantages that：

The invention provides new strains-achromobacter of a high-efficiency degradation DEHP (Achromobacter sp.)MT- H, the germplasm resource bank of phthalic acid ester degradation bacteria is enriched, and the bacterium can be preferably in wider pH and temperature range Degrade DEHP on ground, it was demonstrated that in terms of MT-H bacterial strains can be applied to the biological treatment of DEHP pollutions as excellent DEHP degradation bacterias.

Brief description of the drawings

Fig. 1 is the growthform feature that MT-H bacterium are cultivated 7 days on LB culture mediums.

Fig. 2 is the stereoscan photograph of MT-H bacterium.

Fig. 3 is the 16S rDNA of MT-H bacterium phylogenetic tree.

Fig. 4 is degradation effect of the MT-H bacterium to the DEHP of various concentrations.

Fig. 5 be MT-H bacterium at different conditions（PH, temperature and rotating speed）To DEHP degradation effect.

Embodiment

The present invention is further elaborated on reference to Figure of description and specific embodiment.Following examples of the present invention For the preferable embodiment of the present invention, the present invention mainly illustrates the bacterial strain and the application thought based on the bacterial strain, implements The replacement of simple parameter can not repeat in embodiment one by one in mode, but therefore not limit the scope of the invention, its His any Spirit Essence without departing from the present invention with made under principle change, modification, replacement, combine, simplification, should be considered as The substitute mode of effect, within protection scope of the present invention.

Culture medium prescription is as follows described in embodiment：

Inorganic salts nutrient solution（MSM, g/L）：K₂HPO₄：5.8, KH₂PO₄：4.5, (NH₄)₂SO₄：2.0, MgCl₂：0.16, CaCl₂：0.02, Na₂MoO₄·2H₂O：0.0024, FeCl₃：0.0018, MnCl₂·2H₂O：0.0015.Final pH is 7.5.

Beef-protein medium（LB）：The g of dusty yeast 5.0, the g of peptone 10.0, the g of sodium chloride 10.0, add ultra-pure water To 1L, pH=7.0 are adjusted；121 DEG C of sterilizing 20min.Solid plate then adds 1.5%（w/v）Agar powder.

The separation and identification of the bacterial strain of embodiment 1

The returned sludge of Guangzhou great Tan Sha municipal sewage plants is gathered, weighs 5 g activated sludge samples in containing 50 In 150 mL triangular flasks of mL sterilized waters, at 30 DEG C, 140 rpm take 5 mL sludge suspensions to be added to 100 mL to contain after cultivating 3 days There is DEHP（50 mg L^-1）Above-mentioned MSM culture mediums in.Through 30 DEG C, after 140 rpm are cultivated 7 days, 1 mL inoculation is pressed every time The continuous enrichment of amount, switching 10 times, and the content of DEHP in culture medium is accordingly improved to 1000 mg L^-1.Then will transfer 10 times Nutrient solution dilution 10³~10⁵It is coated on again on LB solid plates, 30 DEG C are inverted culture 1 ~ 3 day.After single bacterium colony is grown on flat board, Picking single bacterium colony is repeatedly rule purifying, and separation obtains one plant of bacterium, numbering MT-H.Then by inoculation on LB solid plates 30 DEG C are inverted culture 7 days, observe its colonial morphology.For the bacterium colony of LB flat boards culture 7 days into circle, microprotrusion is faint yellow, moistening, Translucent, neat in edge is smooth（See Fig. 1）.

Scanning electron microscope example prepares and observation：Bacterial strain MT-H after purification is accessed into the LB fluid nutrient mediums containing 10 mL It is activated overnight.Draw 800 μ L bacterium solutions and centrifuge 3 ~ 5 min through 8000 rpm, remove supernatant, add 500 μ L PBS and wash bacterium 3 times. 2.5% glutaraldehyde that 1 mL is added in the bacterial sediment of harvest fully mixes, and 4 DEG C stand overnight.Then again through 8000 rpm 3 ~ 5 min are centrifuged, remove supernatant, 500 μ L PBS is added and washes bacterium 3 times.Then by thalline respectively 30%, 50%, 70%, 85%, It is dehydrated 2 times in 90% and 100% graded ethanol, each gradient about soaks 15 min, and supernatant is removed in then 8000 rpm centrifugations, most Ethanol is replaced with isoamyl acetate 2 times, every time 20 min, the same ethanol dehydration of method afterwards.By CO₂Film-making is observed after drying.Sweep Retouch under Electronic Speculum it is observed that the bacterium is in spherical or ellipsoid, diameter is about 0.5 ~ 1 μm（See Fig. 2）.

The 16S rDNA Molecular Identifications of bacterial strain：Bacteria total DNA is extracted, with bacterial 16 S rDNA universal primers to the bacterium gene Group enters performing PCR amplification.PCR primer is through sequencing（Sequencing is completed by Shanghai life work）Afterwards, sequence such as SEQ ID NO：Shown in 1, it will survey The 16S rDNA sequences reported in sequence result and GenBank carry out sequence analysis, and if choosing dry strain and doing chadogram point Analysis.As shown in figure 3, the present invention isolates and purifies obtained bacterial strain MT-H 16S rDNA sequences and achromobacter （Achromobactersp. D-12）For homology up to 99%, evolutionary distance is nearest.Its cultural characteristic and scanning electron microscopic observation feature With achromobacter（Achromobactersp.）Also it is the most similar, therefore the degradation bacteria that present invention screening obtains is achromobacter （Achromobactersp.）.

Therefore, the bacterial strain was deposited in China typical culture collection center by inventor on January 22nd, 2015 （CCTCC）, deposit number is CCTCC M 2015058, and preservation address is：Wuhan City, Hubei Province Wuhan University, strain classification life It is entitledAchromobactersp. MT-H。

Embodiment 2AchromobacterSp.MT-H tests to DEHP degradation effect

S1. prepared by bacteria suspension：LB fluid nutrient medium of the strain MT-H accesses containing 10 mL after purification is activated overnight training Support to logarithmic phase, centrifuging 10 min through 5000 rpm collects thalline, is resuspended after washing bacterium 3 times with PBS, adjusts OD_{600 nm} = 1.0 As bacteria suspension.

S2. degradation property determines：Contain various concentrations DEHP to 100mL respectively（50、100、200、400、600 mg L^-1） MSM nutrient solutions in meet the mL of bacterium 1, not connect bacterium as control, and adjust pH as 8.0, every group of three repetitions.At 30 DEG C, 140 Rpm constant-temperature tables culture 9 days, periodically sampling, the degraded situation through GC/MS measure DEHP.

Chromatographic condition：Using Shimadzu Corporation's QP2010 Plus type GC/MS tandem mass spectrometers.Chromatographic column is Agilent HP-5 Pillar (0.25 μm × 0.25 mm × 30 m), injector temperature are 250 DEG C, ion gun（EI）Temperature is 220 DEG C, using not The μ L of split sampling 1, carrier gas is high-purity helium.Heating schedule is：Initial temperature is 100 DEG C, keeps 2 min, 15 DEG C/min ladders Degree rises to 129 DEG C, is then warming up to 280 DEG C with 40 DEG C/min, keeps 5min.

Quality control：Standard curve is made using external standard method and six point calibration standard substances.6 kinds of PAEs mix target matrix and added It is 92.5 ~ 110.2% to mark average recovery rate, and relative deviation is less than 10.5%, and instrument detection is limited to 0.12-0.45 ug g^-1.The party Method meets trace organic substance quantitative analysis requirement.

As a result as shown in figure 4,AchromobacterSp. MT-H is very slow to DEHP degradation rate in Initial stage of culture Slowly, as the extension of incubation time, its degradation also increase therewith；It is less than 200 mg L in DEHP concentration^-1When, DEHP in 7d It is almost degradable；When DEHP concentration is higher, though its degradation rate is substantially suppressed；When DEHP concentration up to 600 mg L^-1When, cultivate to the 9th day, its degradation rate is also up to more than 50%.

This example demonstrates that resulting separationAchromobacterSp. MT-H can by the use of DEHP as sole carbon source and The energy carries out growth and breeding, and when DEHP concentration is relatively low, the bacterium has efficient degradation DEHP ability.

The pH of embodiment 3, temperature and rotating speed pairAchromobacterSp. MT-H degradeds DEHP influence

PH pairsAchromobacterSp.MT-H degradeds DEHP influence：Respectively to 100mL differences pH (5,6,7,8,9, 10) contain DEHP（500mg/L）MSM nutrient solutions in meet bacterium 1mL, using do not connect bacterium as control, every group of three repetitions.30 DEG C, after 140 rpm constant-temperature table cultures 48h, DEHP degraded situation is determined through GC/MS.

Temperature pairAchromobacterSp.MT-H degradeds DEHP influence：Contain DEHP to 100mL（500mg/L）'s Bacterium 2.5mL is met in MSM nutrient solutions, using do not connect bacterium as control, every group of three repetitions, respectively temperature be 20 DEG C, 25 DEG C, 30 DEG C, 35 DEG C, cultivate 48h through 140 rpm in 40 DEG C of constant-temperature table after, DEHP degraded situation is determined through GC/MS.

Rotating speed pairAchromobacterSp. MT-H degradeds DEHP influence：Contain DEHP to 100mL（500mg/L）'s Bacterium 2.5mL is met in MSM nutrient solutions, using do not connect bacterium as control, every group of three repetitions, respectively rotating speed be 80,110,140, 170th, the constant-temperature table of 200 rpm rotating speeds determines DEHP degraded situation through GC/MS after 30 DEG C of culture 48h.

Experimental result as shown in figure 5, fromAchromobacterSp. degraded of the MT-H bacterium under condition of different pH to DEHP Design sketch can be seen thatAchromobacterSp. MT-H bacterium are different to DEHP degradation capability under condition of different pH.In pH In the range of 6 ~ 9, the bacterium reaches more than 80% to DEHP degradation rate；Especially pH is that degradation efficiency can in the range of 7 ~ 8 Up to more than 90%；When pH is 5, degradation rate is suppressed by larger.FromAchromobacterSp. MT-H bacterium are in different temperatures bar DEHP degradation effect figure can be seen that when temperature is in the range of 30 ~ 40 DEG C under part, the bacterium is reachable to DEHP degradation rate More than 80%；But at a lower temperature, its degradation rate is substantially inhibited.FromAchromobacterSp. MT-H bacterium are in difference Can be seen that rotating speed under speed conditions on DEHP degradation effect figure influences not being very aobvious on the DEHP degradation efficiencies of the bacterium Write, approximate trend is that rotating speed is more high more be advantageous to the bacterium and degrade DEHP, but when rotating speed reaches 170rpm, its degradation rate becomes substantially In constant.

This example demonstrates that bacterial strainAchromobacterSp. MT-H is in neutral and meta-alkalescence wider pH and temperature model The interior DEHP that can preferably degrade is enclosed, guarantee is provided for its application in different complex environments.

SEQUENCE LISTING

<110>Ji'nan University

<120>Applications of achromobacter (Achromobacter sp.) MT-H in diisooctyl phthalate of degrading

<130>

<160> 1

<170> PatentIn version 3.3

<210> 1

<211> 1419

<212> DNA

<213>The 16S rDNA sequences of MT-H bacterial strains

<400> 1

tgagacgatc taccgtggta tcgccctcct tgcggttagt gctaactact tctggtaaaa 60

cccactccca tggtgtgacg ggcggtgtgt acaagacccg ggaacgtatt caccgcgaca 120

tgctgatccg cgattactag cgattccgac ttcacgcagt cgagttgcag actgcgatcc 180

ggactacgat cgggtttctg ggattggctc cccctcgcgg gttggcgacc ctctgtcccg 240

accattgtat gacgtgtgaa gccctaccca taagggccat gaggacttga cgtcatcccc 300

accttcctcc ggtttgtcac cggcagtctc attagagtgc cctttcgtag caactaatga 360

caagggttgc gctcgttgcg ggacttaacc caacatctca cgacacgagc tgacgacagc 420

catgcagcac ctgtgttccg gttctcttgc gagcactccc aaatctcttc rggattccag 480

acatgtcaag ggtaggtaag gtttttcgcg ttgcatcgaa ttaatccaca tcatccaccg 540

cttgtgcggg tccccgtcaa ttcctttgag ttttaatctt gcgaccgtac tccccaggcg 600

gtcaacttca cgcgttagct gcgctaccaa ggtccgaaga ccccaacagc tagttgacat 660

cgtttagggc gtggactacc agggtatcta atcctgtttg ctccccacgc tttcgtgcat 720

gagcgtcagt gttatcccag gaggctgcct tcgccatcgg tgttcctccg catatctacg 780

catttcactg ctacacgcgg aattccacct ccctctgaca cactctagcc cggtagttaa 840

aaatgcagtt ccaaagttaa gctctgggat ttcacatctt tctttccgaa ccgcctgcgc 900

acgctttacg cccagtaatt ccgattaacg cttgcaccct acgtattacc gcggctgctg 960

gcacgtagtt agccggtgct tattctgcag gtaccgtcag ttccgcgggg tattaacccg 1020

cgacgtttct ttcctgccaa aagtgcttta caacccgaag gccttcatcg cacacgcggg 1080

atggctggat cagggtttcc cccattgtcc aaaattcccc actgctgcct cccgtaggag 1140

tctgggccgt gtctcagtcc cagtgtggct ggtcgtcctc tcaaaccagc tacggatcgt 1200

cgccttggtg agccgttacc ccaccaacta gctaatccga tatcggccgc tccaatagtg 1260

caaggtcttg cgatcccctg ctttcccccg tagggcgtat gcggtattag ctacgctttc 1320

gcgtagttat cccccgctac tgggcacgtt ccgatacatt actcacccgt tcgccactcg 1380

ccaccagacc gaagttgcgt gctgccgttc gactgcatg 1419

Claims (6)

1. achromobacter (AchromobacterSp.) applications of the MT-H in diisooctyl phthalate of degrading；

The bacterial strain was deposited in China typical culture collection center on January 22nd, 2015（CCTCC）, deposit number is CCTCC M 2015058。

2. achromobacter (AchromobacterSp.) MT-H answering in the medium for repairing diisooctyl phthalate pollution With；

The bacterial strain was deposited in China typical culture collection center on January 22nd, 2015（CCTCC）, deposit number is CCTCC M 2015058。

3. application according to claim 2, it is characterised in that the medium is soil, water or air.

4. according to the application described in claim 1,2 or 3, it is characterised in that bacterial strain MT-H is prepared into bacteria suspension and is used to degrade Diisooctyl phthalate.

5. application according to claim 4, it is characterised in that the OD of the bacteria suspension_{600 nm} = 0.8~1.2。

6. achromobacter (AchromobacterSp.) MT-H is in the biological agent for preparing degraded diisooctyl phthalate Application；

The bacterial strain was deposited in China typical culture collection center on January 22nd, 2015（CCTCC）, deposit number is CCTCC M 2015058。