CN106148222A - A kind of antibacterial and the application in producing 16-dicarboxylic acid thereof - Google Patents

Abstract

The invention provides a kind of novel bacteria, forAcinetobacterBelong to, belong to microbial technology field, simultaneously, provide its application in producing 16-dicarboxylic acid, the antibacterial that the present invention provides can produce 16-dicarboxylic acid, the antibacterial using the present invention to provide produces 16-dicarboxylic acid, it is to avoid uses Oidium tropicale to produce DC in prior art₁₆The harm of bacterial strain, novel bacteria fermenting and producing DC that the present invention provides₁₆The productivity prepared also greater than other biological method in prior art of productivity.

Description

A kind of antibacterial and the application in producing 16-dicarboxylic acid thereof

Technical field

The present invention relates to efficiently produce DC₁₆Antibacterial, relate to it simultaneously and prepare DC₁₆Method, particularly relate to cultivate Method, from oil-polluted soils, generates DC to energy metabolism hexadecane hydrocarbon₁₆Antibacterial carry out being enriched with, screen, separate and identifying, and The DC that its metabolism is produced₁₆Carry out separating, purification and mensuration.

Background technology

DC₁₆Be a kind of important synthesis material, can be used to musk ambrette ketone, replace natural Moschus, be used for preparing multiple in Patent medicine.DC₁₆Not existing in nature, and chemical industry method also is difficult to synthesis, therefore, biological metabolism method becomes production DC₁₆? Effective method.Up to the present, bioanalysis produces DC₁₆Be all utilize Candida tropicalis (Candida cloacae) come Complete, the most do not find to utilize antibacterial to produce DC₁₆Report.With Candida tropicalis (Candida cloacae) fermentation method Preparation DC₁₆Report occur in the seventies the earliest, as in Japan tail et al. with cloaca candida mycoderma (Candida cloacae) MR-12 produces DC₁₆.Later, Chen Yuantong utilize candida tropicalis (Candida cloacae) mutant UH-3-9 production DC₁₆, by adding acrylic acid in reaction system, suppress the beta oxidation of dicarboxylic acids, reduce candida tropicalis (Candida cloacae) to generating DC₁₆Decomposition, improve DC₁₆Productivity, DC when fermenting 3 days₁₆Yield reach 54 g/L, are above Gao Zhongxiang (1990) and plant the yield of about 40 g/L that village (1987) reports.The public affairs such as Cao Wubo (2011) Opened utilize candida tropicalis (Candida cloacae) mutant ly-6 fermentation hexadecane production DC₁₆Patent of invention, After adding hexadecane fermentation 165 hours, DC₁₆Productivity be 170.5 g/L, conversion ratio is 88.1%.Candida tropicalis Bacterium (Candida cloacae), i.e. Oidium tropicale, belong to Cryptococeales, Cryptococcaceae, be a kind of fungus, urgency can be caused Property, subacute or chronic infection, be modal mycosis, and may result in skin, mucosa, internal organs suffer damage.

Summary of the invention

In order to solve above technical problem, the invention provides a kind of production DC₁₆Antibacterial, and provide its concrete system Preparation Method.The present invention is not only separated to a strain efficiently metabolism can produce DC₁₆Novel species bacterium, enrich microbial resources storehouse, and DC₁₆Fermentation production rate higher than research before, avoid the harm using Candida tropicalis fermentation simultaneously.

The numbered STKD-1 of antibacterial provided by the present invention, belongs to acinetobacter calcoaceticusAcinetobacter sp.Belong to, its bacterium Strain preserving number is CGMCCNo.11839, is preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center, its ground Location is in BeiChen West Road, Chaoyang District, BeiJing City one institute 3, and preservation date is December in 2015 9.

The antibacterial that the concrete present invention provides preparation process in preparing 16-dicarboxylic acid is as follows:

(1) culture medium is prepared

Fermentation medium II:

Hexadecane 1.5g/L, peptone 4.0g/L, (NH₄)₂SO₄0.15g/L, KH₂PO₄0.8g/L, NaCl 0.1g/L, Deionized water 1000mL, adjusting pH is 7.2, is dispensed in 500 mL triangular flasks, and 121 DEG C of 20min sterilizings are stand-by；

Liquid screening medium:

K₂HPO₄1.5g/L, MgSO₄ ·7H₂O 0.5g/L, NH₄NO₃ 1.5g/L, FeCl₃0.025g/L, anhydrous CaCl₂ 0.01g/L, hexadecane 1.5 g/L, regulate pH to 7.0 with the NaOH of 2M, and 121 DEG C of 20 min sterilizing is stand-by；

(2) fermentation

The preculture process of hexadecane zymogenous bacteria: by inoculation to liquid screening medium, 28 DEG C, 150 rpm/ Min condition shaken cultivation 3 d, obtains pre-culture solution；

Sweat: take pre-culture solution 20mL and be inoculated in 3L fermentation medium II, 28 DEG C, 150 rpm/min shaken cultivation, Adjusting fermentation liquid pH to 7.2 every 24 h by 6 mol/L NaOH solution, incubation time is 20 d, takes fermentation liquid after fermentation ends Carry out extraction and the mensuration of 16-dicarboxylic acid；

(3) extraction of 16-dicarboxylic acid

Fermentation liquid is heated to 85 DEG C, and adjusting pH with NaOH while stirring is 11.0, carries out film sucking filtration while hot, and filter membrane used is Nitrocellulose filter, aperture is 0.22 m, collects sucking filtration liquid；Sucking filtration liquid is stood under the conditions of 4 DEG C 12 h, takes on sucking filtration liquid Portion's liquid adds dilute sulfuric acid, adjusts under the conditions of pH is 2.0,4 DEG C and stands 12 h, collects bottom precipitation；Take sucking filtration liquid bottom precipitation Add deionized water dissolving, with dilute sulfuric acid adjustment pH value of solution to 2.0, stand 12 h under the conditions of 4 DEG C, collect bottom precipitation；Merge Two parts precipitate, and low-temperature vacuum drying obtains total 16-dicarboxylic acid；

(4) gas chromatography mass spectrometry method measures 16-dicarboxylic acid content.

The invention provides a kind of energy metabolism and produce DC₁₆Novel bacteria, it is to avoid prior art uses Oidium tropicale Produce DC₁₆The harm of bacterial strain, meanwhile, novel bacteria fermenting and producing DC that the present invention provides₁₆Productivity more than in prior art other The productivity of bioanalysis.

Accompanying drawing explanation

Fig. 1 is that gas chromatography mass spectrometry (GC-MS) measures D₁₆Result；

Fig. 2 is that high-efficiency fermenting produces DC₁₆The phylogenetic tree of bacterial strain STDK-1.

Detailed description of the invention

Below in conjunction with embodiment, the present invention is further illustrated.

Embodiment 1

1, the enrichment of antibacterial

Take being put in beaker by soil 10 g of oil pollution near petrochemical industry gas station in Huangdao District, Qingdao, then add wherein The deionized water entering 90 mL soaks and stirs, and stands 30 min, takes leachate 5 mL and joins 100mL liquid enrichment medium In, under the conditions of 28 DEG C, shaken cultivation (150 rpm/min) 5 d, obtain enrichment culture liquid；Enrichment medium composition (/L): beef Cream 3.0g, peptone 10.0g, NaCl 5.0g, adjust pH to be that 7.6,121 DEG C of 20 min sterilizing is stand-by with NaOH；

2, the screening of hexadecane zymogenous bacteria and purification

Screening culture medium composition (/L): K₂HPO₄1.5g, MgSO₄ ·7H₂O 0.5g, NH₄NO₃1.5g, FeCl₃ 0.025g, anhydrous CaCl₂0.01g, hexadecane 1.5g, agar 20g, regulate pH to 7.0 with the NaOH of 2M, 121 DEG C 20 min sterilizings, are down flat plate while hot, i.e. obtain solid plate culture medium after cooling, and semisolid screening culture medium adds agar 4g/L, Other compositions and content are constant, and liquid screening medium is not added with agar；Separation process: take above-mentioned enrichment culture liquid 20 L dropping In solid plate culture medium, then smear uniformly, quiescent culture 3 d under the conditions of 28 DEG C, 5 single bacterium colonies that picking growth is fast Carrying out streak plate cultivation the most respectively, plating medium used and condition of culture are all the same, there are 5 groups of flat boards.See respectively Examine 5 groups of flat-plate bacterial colony features, as colony characteristics is inconsistent, then continue to repeat streak culture, until colony characteristics is consistent, now put down Plate bacterium colony is single bacterium (pure bacterium).By above-mentioned the most streak culture, it be divided into and separate out the 5 pure bacterium of strain, be denoted as STKD-1, STKD-2, STKD-3, STKD-4 and STKD-5, be saved in semisolid screening culture medium respectively under the conditions of this pure bacterium of 5 strain 4 DEG C, stand-by；

3, the fermentation of antibacterial produces DC₁₆Used medium forms

(1) fermentation medium I forms (/L): hexadecane 2g, NaCl 0.5g, (NH₄)₂SO₄0.15g, MgSO₄·7H₂O 0.03g, NaNO₃ 0.1 g, NaH₂PO₄0.5g, FeCl₃0.04 g, deionized water is prepared, and adjusts pH to be 7.2 with NaOH, is dispensed into In 500 mL triangular flasks, 121 DEG C of 20min sterilizings are stand-by；

(2) fermentation medium II forms (/L): hexadecane 1.5g, peptone 4.0g, (NH₄)₂SO₄0.15g, KH₂PO₄ 0.8g, NaCl 0.1g, deionized water 1000mL, adjusting pH is 7.2, is dispensed in 500 mL triangular flasks, and 121 DEG C of 20min go out Bacterium is stand-by；

(3) fermentation medium III forms (/L): hexadecane 2.5 g, KH₂PO₄1.5g, K₂HPO₄2.0g, NH₄ NO₃ 0.5g, NaCl 0.5g, MgSO₄·7H₂O 0.01g, anhydrous CaCl₂0.01g, FeSO₄0.002g, deionized water 1000mL, Adjusting pH is 7.2, is dispensed in 500 mL triangular flasks, and 121 DEG C of 20min sterilizings are stand-by；

4, concrete sweat:

(1) the preculture process of hexadecane zymogenous bacteria: by bacterial strain STKD-1, STKD-2, STKD-3, STKD-4 and STKD-5 Being inoculated into respectively in liquid screening medium, 28 DEG C of conditions vibration (150 rpm/min) cultivate 3 d, obtain pre-culture solution；

(2) sweat: pre-culture solution 20mL taking STKD-1, STKD-2, STKD-3, STKD-4 and STKD-5 respectively connects respectively Planting in 3L fermentation medium I, fermentation medium II and fermentation medium III, 28 DEG C of vibrations (150 rpm/min) are cultivated, often Adjusting fermentation liquid pH to 7.2 every 24 h by 6 mol/L NaOH solution, incubation time is 20 d, takes fermentation liquid and enter after fermentation ends Row DC₁₆Extraction and mensuration；

5、DC₁₆Extraction process

Above-mentioned fermentation liquid is heated to 85 DEG C, and adjusting pH with NaOH while stirring is 11.0, carries out film sucking filtration, filter used while hot Film is nitrocellulose filter, and aperture is 0.22 m, collects sucking filtration liquid, is now solubility 16-dicarboxylic acid sodium (temperature in filtrate Under the conditions of degree is 85 DEG C, 16-dicarboxylic acid sodium is the dissolved).Then sucking filtration liquid is stood under the conditions of 4 DEG C 12 h, 16 Under carbon dicarboxylic acid sodium low temperature, dissolubility reduces and is deposited in bottom, processes upper liquid and bottom precipitation further respectively, Upper liquid suspense makees liquid (I), and bottom precipitation suspense precipitates (I)；

Note: remain a small amount of 16-dicarboxylic acid sodium in liquid (I)；Precipitation (I) is 16-dicarboxylic acid sodium liquid (I) processing procedure: Adding dilute sulfuric acid in liquid (I), adjusting pH is 2.0, now remains in the 16-dicarboxylic acid sodium in liquid (I) in acid condition Form DC₁₆, under the conditions of 4 DEG C, stand 12 h, DC₁₆Under cryogenic conditions, dissolubility reduces, and is deposited to container bottom with crystal form, Collect precipitation, be denoted as precipitating (II)；

Precipitation (I) processing procedure: add deionized water dissolving in precipitation (I), then adjust the pH of solution extremely with dilute sulfuric acid 12 h, DC is stood under the conditions of 2.0,4 DEG C₁₆Crystallize under cryogenic conditions at the bottom of bottle, be denoted as precipitating (III)；

Merge precipitation (II) and precipitation (III), low-temperature vacuum drying, i.e. obtain total DC₁₆, because the present invention utilizing separation pure Change 5 kinds of bacterial strains (STKD-1, STKD-2, STKD-3, STKD-4 and STKD-5) arriving respectively to three kinds of fermentation medium (I, II and III) fermenting and producing DC of hexadecane hydrocarbon is carried out₁₆, so there are 15 groups of total DC₁₆；

6, gas chromatography mass spectrometry (GC-MS) method measures DC₁₆

(1) gas chromatography mass spectrometry (GC-MS) measures DC₁₆Operational factor

GC-MS parameter sets: the post case inner equilibrium time as 0.25 min, sets maximum temperature as 325 DEG C, column temperature heating schedule Being that 0 min reaches 150 DEG C, then 6 DEG C/min is warmed up to 300 DEG C, and total run time is 35 min；Sample size is 1 L, sample Cleaning pumping velocity is 300 L/min；

(2) gas chromatography mass spectrometry (GC-MS) measures DC₁₆Result

5 bacterial strains (STKD-1, STKD-2, STKD-3, STKD-4 and STKD-5) are with above-mentioned three kinds of fermentation medium (I, II and III) Carry out fermentation respectively and produce DC₁₆, fermentation liquid is processed by crystallization, cold drying and obtains 15 groups of fermentation crystal, dissolve with methanol 15 groups of crystal, lysate is used as sample introduction, utilizes GC-MS to measure the material composition of crystalline solid, and operational factor is shown in 6 (1).Measure knot Fruit shows, 15 groups of crystalline solid appearance times are consistent, utilize data base's comparison, it is determined that the peak that 15 groups of crystalline solid occur at 7.5min For DC₁₆, such as Fig. 1；

7、DC₁₆The calculating of productivity and the determination of optimum operating condition

DC₁₆Fermentation production rate calculate according to weight.To STKD-1, STKD-2, STKD-3, STKD-4 and STKD-5 5 bacterial strain Ferment in the fluid medium of 3L 20 d respectively, by crystallization and cold drying, the DC that will obtain₁₆Weigh, according to sending out Ferment liquid amasss, and calculates DC respectively₁₆Fermentation production rate (table 1).As can be seen from Table 1, at three kinds of different fermentation medium (I, II And III) in, bacterial strain STKD-1 produces DC to hexadecane hydrocarbon fermentation₁₆Productivity be maximum, respectively 145.3g/L, 176.2 G/L and 114.7 g/L.And compare three kinds of different fermentation medium (I, II and III) to DC₁₆Fermentation production rate from the point of view of, fermentation The medium ii DC to 5 bacterial strains₁₆Fermentation production rate be maximum, so fermenting and producing DC₁₆Best of breed be fermentation medium II With bacterial strain STKD-1.According to the above results, choosing bacterial strain STKD-1 in subsequent experimental is that representative is further analyzed；

Table 15 bacterial strain D when different fermentations culture medium (I, II and III) middle fermentation 20 d₁₆Fermentation production rate (unit: g/L)

8, the taxonomy of STKD-1 is identified

5 bacterial strains (STKD-1, STKD-2, STKD-3, STKD-4 and STKD-5) fermenting and producing DC₁₆Result show, bacterial strain STKD-1 is maximum to the fermentation production rate of hexadecane hydrocarbon, therefore, chooses bacterial strain STKD-1, solves its systematics position Analysis；

(1) bacterial strain STKD-1DNA extracts

The extraction of bacterial strain STKD-1 DNA uses test kit-DP303 to complete, description in extraction step reference reagent box；

(2) bacterial strain STKD-1 DNA is expanded by PCR

Utilize forward primer PF 5-AGA GTTTGA TCC TGG CTC AG-3 and reverse primer PR 5-GGY TAC CTT GTT ACG ACT T-3 carries out PCR amplification to bacterial strain STKD-1 16S rDNA.PCR reaction system (50 μ L) including: 1 μ L Taq archaeal dna polymerase, 0.5 μ L template DNA, 5 μ L 10 × PCR buffer, 3.5 μ L MgCl₂, 0.5 μ L PF and PR, 2 μ L DNTP, 37 μ L ultra-pure waters.PCR reaction condition: 95 DEG C of denaturation 3 min, 95 DEG C of degeneration 45S, 55 DEG C of annealing 45S, 72 DEG C of extensions 90S, 32 circulations, then 72 DEG C of extension 10 min；

(3) DNA sequencing

PCR primer DNA sequencer (model: Applied Biosystems 3730XL) is checked order, the sequence that will record Being sent to DDBJ (DNA Data Bank of Japan), the accession number obtaining bacterial strain STKD-1 is LC094963.

Particular sequence is as follows:

aacacatgca agtcgagcgg agagaggtag cttgctaccg atcttagcgg cggacgggtg

agtaatgctt aggaatctgc ctattagtgg gggacaacat ttcgaaagga atgctaatac

cgcatacgtc ctacgggaga aagcagggga tcttcggacc ttgcgctaat agatgagcct

aagtcggatt agctagttgg tggggtaaag gcctaccaag gcgacgatct gtagcgggtc

tgagaggatg atccgccaca ctgggactga gacacggccc agactcctac gggaggcagc

agtggggaat attggacaat gggcgcaagc ctgatccagc catgccgcgt gtgtgaagaa

ggccttatgg ttgtaaagca ctttaagcga ggaggaggct actttagtta atacctagag

atagtggacg ttactcgcag aataagcacc ggctaactct gtgccagcag ccgcggtaat

acagagggtg caagcgttaa tcggatttac tgggcgtaaa gcgcgcgtag gcggctaatt

aagtcaaatg tgaaatcccc gagcttaact tgggaattgc attcgatact ggttagctag

agtgtgggag aggatggtag aattccaggt gtagcggtga aatgcgtaga gatctggagg

aataccgatg gcgaaggcag ccatctggcc taacactgac gctgaggtgc gaaagcatgg

ggagcaaaca ggattagata ccctggtagt ccatgccgta aacgatgtct actagccgtt

ggggcctttg aggctttagt gccgcattta acgcgataag tagaccgcct ggggagtacg

gtcgcaagac taaaactcaa atgaattgac gggggcccgc acaagcggtg gagcatgtgg

tttaattcga tgcaacgcga agaaccttac ctggccttga catagtaaga actttccaga

gatggattgg tgccttcggg aacttacata caggtgctgc atggctgtcg tcagctcgtg

tcgtgagatg ttgggttaag tcccgcaacg agcgcaaccc ttttccttat ttgccagcga

gtaatgtcgg gaactttaag gatactgcca gtgacaaact ggaggaaggc ggggacgacg

tcaagtcatc atggccctta cggccagggc tacacacgtg ctacaatggt cggtacaaag

ggttgctacc tagcgatagg atgctaatct caaaaagccg atcgtagtcc ggattggagt

ctgcaactcg actccatgaa gtcggaatcg ctagtaatcg cggatcagaa tgccgcggtg

aatacgttcc cgggccttgt acacaccgcc cgtcacacca tgggagtttg ttgcaccaga

Agtagctagc ctaactgcaa agagggcggt (SEQ ID NO:1)；

(4) establishment of phylogenetic tree

Utilize nearest neighbour method that the 16S rDNA sequence of the bacterial strain STDK-1 higher bacterial strain of its homology is carried out phylogenetic tree establishment, institute It is CustalX2.1 and Mega5 with software.From phylogenetic tree (Fig. 2) it can be seen that bacterial strain STDK-1 belongs toAcinetobacter Belong to, and withAcinetobacter calcoaceticus(X81661) 16S rDNA sequence similarity is the highest, is 98%, It is a kind of novel species bacterium, namedAcinetobactersp.STDK-1(LC094963)。

The present invention isolates energy metabolism from soil and produces DC₁₆Novel bacteria, then difference composition culture medium in add Enter the hexadecane hydrocarbon sole carbon source as antibacterial, its shaker fermentation is cultivated, obtains fermentation liquid, to DC in fermentation liquid₁₆Carry out Isolated and purified, and measure in 3L fermentation tank through 20 d fermentation after DC₁₆Productivity.It addition, energy metabolism is produced DC₁₆Thin The DNA of bacterium carries out extracting, check order and phyletic evolution position has been also carried out analyzing.Result shows, at 5 isolated and purified bacterial strains In (STKD-1, STKD-2, STKD-3, STKD-4 and STKD-5), when bacterial strain STKD-1 ferments in fermentation medium II, DC₁₆ Productivity maximum, be 176.2 g/L, produce DC than the Candida tropicalis metabolism that utilizes reported before₁₆Productivity the highest.Logical Cross Phylogenetic analysis, bacterial strain STKD-1 withAcinetobacter calcoaceticus(X81661) 16S rDNA sequence Row similarity is the highest (98%), is a kind of novel species bacterium, namedAcinetobacterSp.STDK-1, this bacterial strain logs in sequence number For LC094963.

<120>a kind of antibacterial and the application in producing 16-dicarboxylic acid thereof

<160>1

<210>1

<211>1410

<212>DNA

<213>acinetobacter calcoaceticus (Acinetobacter) is derived from

<222>(1)..(1410)

<400>1

aacacatgca agtcgagcgg agagaggtag cttgctaccg atcttagcgg cggacgggtg 60

agtaatgctt aggaatctgc ctattagtgg gggacaacat ttcgaaagga atgctaatac 120

cgcatacgtc ctacgggaga aagcagggga tcttcggacc ttgcgctaat agatgagcct 180

aagtcggatt agctagttgg tggggtaaag gcctaccaag gcgacgatct gtagcgggtc 240

tgagaggatg atccgccaca ctgggactga gacacggccc agactcctac gggaggcagc 300

agtggggaat attggacaat gggcgcaagc ctgatccagc catgccgcgt gtgtgaagaa 360

ggccttatgg ttgtaaagca ctttaagcga ggaggaggct actttagtta atacctagag 420

atagtggacg ttactcgcag aataagcacc ggctaactct gtgccagcag ccgcggtaat 480

acagagggtg caagcgttaa tcggatttac tgggcgtaaa gcgcgcgtag gcggctaatt 540

aagtcaaatg tgaaatcccc gagcttaact tgggaattgc attcgatact ggttagctag 600

agtgtgggag aggatggtag aattccaggt gtagcggtga aatgcgtaga gatctggagg 660

aataccgatg gcgaaggcag ccatctggcc taacactgac gctgaggtgc gaaagcatgg 720

ggagcaaaca ggattagata ccctggtagt ccatgccgta aacgatgtct actagccgtt 780

ggggcctttg aggctttagt gccgcattta acgcgataag tagaccgcct ggggagtacg 840

gtcgcaagac taaaactcaa atgaattgac gggggcccgc acaagcggtg gagcatgtgg 900

tttaattcga tgcaacgcga agaaccttac ctggccttga catagtaaga actttccaga 960

gatggattgg tgccttcggg aacttacata caggtgctgc atggctgtcg tcagctcgtg 1020

tcgtgagatg ttgggttaag tcccgcaacg agcgcaaccc ttttccttat ttgccagcga 1080

gtaatgtcgg gaactttaag gatactgcca gtgacaaact ggaggaaggc ggggacgacg 1140

tcaagtcatc atggccctta cggccagggc tacacacgtg ctacaatggt cggtacaaag 1200

ggttgctacc tagcgatagg atgctaatct caaaaagccg atcgtagtcc ggattggagt 1260

ctgcaactcg actccatgaa gtcggaatcg ctagtaatcg cggatcagaa tgccgcggtg 1320

aatacgttcc cgggccttgt acacaccgcc cgtcacacca tgggagtttg ttgcaccaga 1380

agtagctagc ctaactgcaa agagggcggt 1410

Claims (2)

1. an antibacterial, it is preserving number CGMCCNo.11839'sAcinetobacterBelong to bacterial strain.

2. the application in preparing 16-dicarboxylic acid of the antibacterial described in claim 1, specifically comprises the following steps that

(1) culture medium is prepared

Fermentation medium II:

Hexadecane 1.5g/L, peptone 4.0g/L, (NH₄)₂SO₄0.15g/L, KH₂PO₄0.8g/L, NaCl 0.1g/L, Deionized water 1000mL, adjusting pH is 7.2, is dispensed in 500 mL triangular flasks, and 121 DEG C of 20min sterilizings are stand-by；

Liquid screening medium:

K₂HPO₄1.5g/L, MgSO₄ ·7H₂O 0.5g/L, NH₄NO₃ 1.5g/L, FeCl₃0.025g/L, anhydrous CaCl₂ 0.01g/L, hexadecane 1.5 g/L, regulate pH to 7.0 with the NaOH of 2M, and 121 DEG C of 20 min sterilizing is stand-by；

(2) fermentation

The preculture process of hexadecane zymogenous bacteria: by inoculation to liquid screening medium, 28 DEG C, 150 rpm/ Min condition shaken cultivation 3 d, obtains pre-culture solution；

Sweat: take pre-culture solution 20mL and be inoculated in 3L fermentation medium II, 28 DEG C, 150 rpm/min shaken cultivation, Adjusting fermentation liquid pH to 7.2 every 24 h by 6 mol/L NaOH solution, incubation time is 20 d, takes fermentation liquid after fermentation ends Carry out extraction and the mensuration of 16-dicarboxylic acid；

(3) extraction of 16-dicarboxylic acid

Fermentation liquid is heated to 85 DEG C, and adjusting pH with NaOH while stirring is 11.0, carries out film sucking filtration while hot, and filter membrane used is Nitrocellulose filter, aperture is 0.22 m, collects sucking filtration liquid；Sucking filtration liquid is stood under the conditions of 4 DEG C 12 h, takes on sucking filtration liquid Portion's liquid adds dilute sulfuric acid, adjusts under the conditions of pH is 2.0,4 DEG C and stands 12 h, collects bottom precipitation；Take sucking filtration liquid bottom precipitation Add deionized water dissolving, with dilute sulfuric acid adjustment pH value of solution to 2.0, stand 12 h under the conditions of 4 DEG C, collect bottom precipitation；Merge Two parts precipitate, and low-temperature vacuum drying obtains total 16-dicarboxylic acid；

(4) gas chromatography mass spectrometry method measures 16-dicarboxylic acid content.