CN103087965A

CN103087965A - Poly beta-hydroxybutyrate production bacterium and application thereof

Info

Publication number: CN103087965A
Application number: CN2013100413688A
Authority: CN
Inventors: 崔中利; 冯丽; 孙志斌; 黄艳
Original assignee: Nanjing Agricultural University
Current assignee: Nanjing Agricultural University
Priority date: 2013-02-01
Filing date: 2013-02-01
Publication date: 2013-05-08
Anticipated expiration: 2033-02-01
Also published as: CN103087965B

Abstract

The invention belongs to the field of applied microbiology, and relates to a poly beta-hydroxybutyrate production bacterium and an application thereof. The poly beta-hydroxybutyrate production bacterium Aquabacterium sp. A7-Y is collected in China Center for Type Culture Collection on a collection date of August 27th, 2012. The bacterium has a collection number of CCTCC NO: M2012316, and is named as A7-Y strain for short. The poly beta-hydroxybutyrate production bacterium Aquabacterium sp. A7-Y provided by the invention can be applied in poly beta-hydroxybutyrate production with starch as a sole carbon source.

Description

Poly-beta-hydroxy-butanoic acid ester bacterium and application thereof are produced in one strain

Technical field

The invention belongs to the using microbe field, relate to a strain and produce poly-beta-hydroxy-butanoic acid ester bacterium and application thereof.

Background technology

The plastics in tradition petroleum products source are widely used, but discarded Xylonite has brought serious " white pollution " owing to not decomposed by microorganism.Poly-beta-hydroxy-butanoic acid (PHB) ester is a kind of a kind of high molecular polymer that can be degraded by microorganisms that utilizes the conventional carbon source accumulation in the parts of fine mycetocyte, is intracellular energy and carbon source material.PHB can processing treatment as a kind of biosynthesizing plastics, the characteristic that not only has the chemosynthesis plastics, also have the advantages such as height biodegradable, biological tissue's consistency, piezoelectricity and anticoagulant property, be expected to obtain to use in high-tech sectors such as electronics, optics, biomedicines.

The microbial profile that can syntheticly gather β monohydroxy butyric acid is extremely wide, comprises that luminous energy and chemoautotrophy microorganism and heterotrophic microorganism amount to 65 and belong to 300 multiple-microorganisms.These microorganisms can utilize the different synthetic poly-β monohydroxy butyric esters of carbon source such as the hydrolyzate of glucose, sucrose or starch.But a lot of carbon source price comparisons are expensive, the cost of producing poly-β monohydroxy butyric ester is raise, must find more cheap in order to reduce the cost of producing PHB, carbon source material more easily, and starch source is extensive, major part comes from plant, and is with low cost, can satisfy for a long time the needs of producing poly-β monohydroxy butyric ester.Therefore, the microorganism that produces poly-β monohydroxy butyric ester take starch as single carbon source has higher potential using value in the production of biological plastics.

Summary of the invention

The purpose of this invention is to provide the production poly beta-hydroxy-butanoic acid ester new strains A quabacterium sp.A7-Y of a strain take starch as sole carbon source.

Another object of the present invention is to provide the application of this bacterial strain.

The poly-beta-hydroxy-butanoic acid ester of one strain produces bacterium Aquabacterium sp.A7-Y, is preserved in Chinese Typical Representative culture collection center, and preservation date is on August 27th, 2012, and deposit number is CCTCC NO:M2012316, hereinafter to be referred as the A7-Y bacterial strain.

Poly-beta-hydroxy-butanoic acid ester of the present invention produces the application of bacterium Aquabacterium sp.A7-Y in producing poly-beta-hydroxy-butanoic acid ester, the particularly application in the poly-beta-hydroxy-butanoic acid ester take starch as sole carbon source production.

Utilize described poly-beta-hydroxy-butanoic acid ester to produce the method that bacterium Aquabacterium sp.A7-Y produces poly-beta-hydroxy-butanoic acid ester, utilize the HV substratum of improvement that poly-beta-hydroxy-butanoic acid ester generation bacterium Aquabacterium sp.A7-Y claimed in claim 1 is fermented, the composition of the HV substratum of described improvement is: starch 10g/L, NaNO ₃0.5g/L, KCl1.5g/L, Na ₂HPO ₄0.75g/L, MgSO ₄0.5g/L, FeSO ₄0.02g/L, CaCl ₂0.02g/L, pH=6.5-7.0.

Beneficial effect:

The invention provides a strain and produce the bacterial strain of poly-β monohydroxy butyric ester, in the present invention, the production of poly-β monohydroxy butyric ester can be take starch as sole carbon source, it is simple that fermentative production is cultivated formula, raw material sources are extensive, price is low, fermentation period is short, consume energy low, thereby significantly reduced poly-β monohydroxy butyric ester production cost.

Description of drawings

Fig. 1 is based on the Aquabacterium.sp A7-Y systematic evolution tree of 16s rDNA sequence

Fig. 2 Aquabacterium.sp A7-Y grows at the HV flat board: transmission electron microscope form (5000 *)

Produce PHB situation (4000 *) under the dull and stereotyped growth of Fig. 3 Aquabacterium.sp A7-Y HV transmission electron microscope

Biomaterial preservation information

Aquabacterium sp.A7-Y is preserved in Chinese Typical Representative culture collection center, and preservation date is on August 27th, 2012, the preservation address: Wuhan, China Wuhan University, deposit number is CCTCC NO:M2012316.

Embodiment

Embodiment 1

1. strain separating

At first collecting soil sample scalps veneer of soil with sampling in Anhui Province, gets with sampler the soil sample that the degree of depth is 10-30cm, in the kraft bag of packing into.Air-dry 10d under room temperature, taking 5 gram soil joins in the 100mL sterilized water and makes soil supension, add 6% yeast extract paste and 0.05%SDS and shaking culture 20min on 40 ℃ of shaking tables in Soil Slurry, get 1mL and dilute respectively 10 times, 100 times and 1000 times, get respectively each dilution diluent coating improvement HV of 0.1mL dull and stereotyped (starch 10g/L, NaNO ₃0.5g/L, KCl1.5g/L, Na ₂HPO ₄0.75g/L, MgSO ₄0.5g/L, FeSO ₄0.02g/L, CaCl ₂0.02g/L, agar 16g/L, pH=6.5-7.0), being placed in 27 ℃ of incubators cultivated 5 days, single bacterium colony on flat board in time is transferred on the HV plate culture medium with the aseptic inoculation ring, then continues to cultivate, wait to turn out single bacterium colony, the dyeing of use sudan black, what after picking dyeing, cell was black is to produce the PHB bacterial strain.Determine that according to the filling degree of PHB in its born of the same parents wherein strains A 7-Y produces PHB ability bacterial strain preferably.Adopt the HV liquid nutrient medium to cultivate A7-Y, 37 ℃ of concussions were cultivated after 2 days, the centrifugal collection thalline of 10000rpm, adopt common chloroform extraction method to extract PHB that A7-Y produces, through nucleus magnetic resonance (NMR), the means such as infrared spectra, gas chromatography-mass spectrometry machine mensuration determine that intracellular polymer that A7-Y produces is PHB.

2. strain identification

(1) 16S rRNA genetic analysis

Extract strains A 7-Y genomic dna, the 16S r DNA universal primer of employing bacterium (27f:5 '-AGAGTTTGATCCTGGCTCAG-3 '; 1492r:5 '-TACCTTGTTACGACTT-3 ') amplification 16S rRNA gene and purifying order-checking, the sequencing result is carried out similarity relatively by EzTaxon server2.1.

The sequencing results shows: the nucleotides sequence of the 16S rRNA of strains A 7-Y is classified SEQ ID NO.1 as.the nucleotide sequence similarity is higher than 97%, be Aquincola tertiaricarbonis L10T(similarity 97.105%, No. genbank is DQ656489), further the 16S rDNA with this bacterial strain and higher 50 bacterial strains of homology carries out sequence alignment, utilize ClustalX (Version1.83) and MEGA4.1 software to carry out Multiple Sequence Alignment and phylogenetic tree structure, use the Neighbor-Join method, 1000 times, obtain phyletic evolution and grow tree, result shows (Fig. 1): strains A 7-Y cluster belongs to Aquabacterium, it is an one novel species, with this called after Aquabacterium sp.A7-Y.

(2) strains A 7-Y morphological specificity and physio-biochemical characteristics

Strains A 7-Y and Aquabacterium are belonged to each identified that bacterial strain compares.Morphological specificity is according to " common bacteria system identification handbook is described and carried out (Fig. 1).Physiology and biochemistry is according to ZYM, API20NE(France Mei Liai) specification sheets carries out.DNA(G+C) the mol% assay entrusts Chinese industrial microbial strains preservation administrative center to complete.

Aquabacterium sp.A7-Y is Gram-negative, bacterium colony surface drying and projection, and inter-adhesive being difficult for scatters, and slight hardly be difficult for being vaccinated ring and provoke, white (see figure 2).Thalline is shaft-like, and inclusion is arranged, and the motor capacity (see figure 3) is arranged.According to the ZYM result: utilizable alkaline phosphatase, esterase (C4), lipoid esterase (C8), lipoidase (C14), L-LEU arylamine enzyme α-amino-isovaleric acid arylamine enzyme, Gelucystine arylamine enzyme, acid phosphatase, naphthols-AS-BI-phosphohydrolase, alpha-glucosidase, beta-glucosidase; According to the API20NE experimental result: nitrate reduction is positive, oxidase positive, hydrogen peroxide enzyme positive, can be hydrolyzed Vitamin C2, gelatin, assimilation glucose, maltose etc., the 5 strain bacterium and Aquincola tertiaricarbonis L10T Physiology and biochemistry particular case such as the table 1 relatively that wherein belong to Aquabacterium.

Table 1 Pseudomonas physio-biochemical characteristics close to kinship relatively

Embodiment 2A7-Y bacterial strain produces the research of the fermentation condition of PHB

1, the cell dry-matter is measured

A7-Y bacterial strain (CCTCC NO:M2012316) is inoculated in 250ml triangular flask 50ml improvement HV liquid nutrient medium with 5% inoculum size, 37 ℃, the 180rpmmin-1 shaking table is cultivated, cultivate after 48 hours, with 12000rpm centrifugal 20 minutes, collect thalline, throw out is with distilled water and each washing of ethanol once, dry to constant weight under 60～70 ℃, measure the thalline amount of dry matter with electronic balance.

2, PHB concentration determination

Adopt the content of spectrophotometry PHB: get 1mL bacterium liquid 12, centrifugal 5 minutes of 000rpm.Remove supernatant liquor after centrifugal, add 5mL sterilized water suspension mixing in throw out, ultrasonication 5 minutes.Get the suspension 12 after the 1mL ultrasonication, the centrifugal 5min of 000rpm.Add lmL sterilized water suspension mixing in throw out, the centrifugal 5min of 12,000rpm is to remove impurity.Add 5mL chloroform alcohol mixture (v/V=2/1) to carry out extracting in the test tube throw out, then 40 ℃ of dryings.Add the 3mL vitriol oil in each test tube, 100 ℃ of heating in water bath 10min.After being cooled to room temperature, adopt ultraviolet spectrophotometer in the content of 235nm place's measurement PHB, sulfuric acid is done reference.Do three groups of parallel tests, average.Contrast PHB typical curve, according to test the sample absorbancy calculate the concentration of PHB in sample.PHB content %=PHB concentration (g/L)/dry matter weight (g/L)

3, determine optimal conditions of fermentation

With 4 levels, 16 of 4 factors+2 level 3 factors are processed and are carried out orthogonal experiment, every group of 3 parallel tests add 50ml different levels and factor substratum to cultivate according to method 1 in the Erlenmeyer flask of each 250ml, testing data adopts the dps7.05 of Zhejiang University software analysis.

Test-results such as table 2 are determined best fermention medium condition: starch 10g/L, NaNO ₃0.5g/L, KCl1.5g/L, Na ₂HPO ₄0.75g/L, MgSO ₄0.5g/L, FeSO ₄0.02g/L, CaCl ₂0.02g/L.

Table 216 is processed (4 levels, 4 factors+2 level 3 factors)

In upper table " dry cell weight " is the cell dry matter weight.

Claims

1. the poly-beta-hydroxy-butanoic acid ester of a strain produces bacterium Aquabacterium sp.A7-Y, is preserved in Chinese Typical Representative culture collection center, and preservation date is on August 27th, 2012, and deposit number is CCTCC NO:M2012316.

2. poly-beta-hydroxy-butanoic acid ester claimed in claim 1 produces the application of bacterium Aquabacterium sp.A7-Y in producing poly-beta-hydroxy-butanoic acid ester.

3. application according to claim 2 is characterized in that poly-beta-hydroxy-butanoic acid ester claimed in claim 1 produces the application of bacterium Aquabacterium sp.A7-Y in the poly-beta-hydroxy-butanoic acid ester take starch as sole carbon source production.

4. utilize poly-beta-hydroxy-butanoic acid ester claimed in claim 1 to produce the method that bacterium Aquabacterium sp.A7-Y produces poly-beta-hydroxy-butanoic acid ester, it is characterized in that utilizing the HV substratum of improvement that poly-beta-hydroxy-butanoic acid ester generation bacterium Aquabacterium sp.A7-Y claimed in claim 1 is fermented, the composition of the HV substratum of described improvement is: starch 10g/L, NaNO ₃0.5g/L, KCl1.5g/L, Na ₂HPO ₄0.75g/L, MgSO ₄0.5g/L, FeSO ₄0.02g/L, CaCl ₂0.02g/L, pH=6.5-7.0.