Bacillus jizhouensis and application thereof

Abstract

The present application relates to a salt-tolerant microorganism Jejubacter sp. and its application, and belongs to the technical field of microorganisms. The Jejubacter sp. DT01 has a preservation number of CGMCC No. 26777. The Jejubacter sp. DT01 can grow normally under the conditions of high salt concentration (0-150 g / L) and high phenolic compound concentration (0-1000 mg / L), and has substrate diversity. In addition to the ordinary carbon sources such as glucose, fructose and the like which can be directly utilized, the Jejubacter sp. DT01 can not only decompose phenolic compounds such as m-hydroxyphenylpropionic acid, phenylpropionic acid, cinnamic acid, ferulic acid, benzoic acid and p-hydroxybenzoic acid, but also can utilize the above-mentioned phenolic compounds as the sole carbon source for growth.

Description

TECHNICAL FIELD

[0001] The present application relates to a salt-tolerant microorganism Jejubacter sp. and its application, belonging to the technical field of microorganisms. BACKGROUND

[0002] Phenolic compounds refer to aromatic carboxylic acid compounds with one or more phenolic hydroxyl groups substituted on the benzene ring, and are also a class of pollutants commonly found in industrial production processes or natural environments. Generally, the degradation of plant lignin, the metabolism of aromatic amino acids, and the degradation of other aromatic compounds produce phenolic acid compounds with C6-C3 structural units, including phenylpropanoid phenolic acids and their hydroxylated derivatives, m-hydroxyphenylpropanoic acid. In addition, the root system of higher plants also produces phenolic compounds into the soil environment, including cinnamic acid, ferulic acid, benzoic acid, p-hydroxybenzoic acid, etc. These phenolic compounds, as plant autotoxic substances, can affect plant metabolism and growth through various pathways such as affecting enzyme activity, cell membrane function, photosynthesis, respiration, and ion absorption, etc., resulting in continuous cropping obstacles.

[0003] Currently, the main way to decompose phenolic compounds is biodegradation, i.e. through the phenolic compound degradation pathway possessed by microorganisms themselves to degrade and produce some substances beneficial to their growth, such as succinic acid, pyruvic acid, acetyl coenzyme A, etc. It has been reported that many microorganisms can use phenolic compounds as the sole carbon source for growth, including more than 20 genera such as Acinetobacter, Pseudomonas, Arthrobacter, Bacillus amyloliquefaciens, and Paenibacillus polymyxa, but there are few reports on salt-tolerant Jejubacter sp. degrading phenolic compounds under high salt conditions. SUMMARY

[0004] The purpose of the present application is to provide a Jejubacter sp. with salt tolerance, and to use it in the degradation of phenolic compounds.

[0005] One of the technical solutions provided by the present application is a Jejubacter sp. DT01. The strain is isolated and purified from saline-alkali soil, and has the characteristics of salt tolerance and degradation of phenolic compounds.

[0006] The Jejubacter sp. DT01 strain has been deposited with the China General Microbiological Culture Collection Center (CGMCC) on March 9, 2023, at address No. 1, Beichen West Road, Hua-yuan Community, Chaoyang District, Beijing, China, Institute of Microbiology, Chinese Academy of Sciences, with a postal code of 100101, and the deposit number is CGMCC No. 26777.

[0007] The Jejubacter sp. DT01 strain has the following physicochemical properties:

[0008] (1) Gram-negative bacteria, after 12h culture at 37℃ in LB solid medium, the colony is round, convex, transparent, smooth, white. Its morphological characteristics are rod-shaped, with flagella, length is 0.8-1.5μm, diameter is 0.4-0.8μm;

[0009] (2) it can grow in the medium with salt concentration of 0-150g / L (preferably 0-90g / L), the salt includes but is not limited to NaCl, KCl, MgCl2, MgSO4, CaCl2, NaHCO3, NH4Cl, etc.;

[0010] (3) it can grow in the medium with pH value of 3-11 (preferably 4-10);

[0011] (4) it can grow at temperature of 5-55℃ (preferably 16-40℃);

[0012] (5) motility test positive, oxidase test negative, catalase test positive, nitrate reduction test positive, glycerol test weakly positive, arabinose test positive, xylose test positive, mannose test positive, rhamnose test positive, maltose test positive, trehalose test positive, mannitol test positive, arabitol test positive, potassium gluconate test positive, cellobiose test negative, lactose test negative, starch hydrolysis test negative, inulin hydrolysis test negative, fucose test negative, raffinose test negative.

[0013] The second technical solution mentioned in the application is the application of Jejubacter sp. DT01;

[0014] Further, the application is in degrading phenolic acid compounds;

[0015] Further, the phenolic acid compounds include but are not limited to m-hydroxyphenylpropionic acid, phenylpropionic acid, cinnamic acid, ferulic acid, benzoic acid and p-hydroxybenzoic acid, etc.

[0016] Further, it can be used for relieving the autotoxicity of plants in saline-alkali land, and further degrading the lignin degradation products or phenolic acid compounds produced by plants themselves.

[0017] The gene detection analysis finds that the Jejubacter sp. DT01 genome contains the phenolic acid degradation related genes that other Jejubacter sp. does not have, thereby enabling it to degrade phenolic acids. The detected phenolic acid degradation related genes include: flavin-type hydroxylase encoding gene mhpA, dioxygenase encoding gene mhpB, meta-cleavage complex hydrolase encoding gene mhpC, 2-keto-4-pentenoic acid hydratase encoding gene mhpD, 4-hydroxy-2-ketopentanoic acid aldolase encoding gene mhpE, acetyl-CoA dehydrogenase encoding gene mhpF;

[0018] Further, the flavin-type hydroxylase encoding gene mhpA has a nucleotide sequence as shown in the sequence table SEQ ID NO. 1.

[0019] Further, the dioxygenase encoding gene mhpB has a nucleotide sequence as shown in the sequence table SEQ ID NO. 2.

[0020] Further, the meta-cleavage complex hydrolase encoding gene mhpC has a nucleotide sequence as shown in the sequence table SEQ ID NO. 3.

[0021] Further, the 2-keto-4-pentenoic acid hydratase encoding gene mhpD has a nucleotide sequence as shown in the sequence table SEQ ID NO. 4.

[0022] Further, the 4-hydroxy-2-ketopentanoic acid aldolase encoding gene mhpE has a nucleotide sequence as shown in the sequence table SEQ ID NO. 5.

[0023] Further, the acetyl-CoA dehydrogenase encoding gene mhpF has a nucleotide sequence as shown in the sequence table SEQ ID NO. 6.

[0024] Beneficial effects:

[0025] The Jejubacter sp. DT01 can grow normally under the conditions of high salt concentration (0-150 g / L) and high phenolic acid concentration (0-1000 mg / L). This enables DT01 to degrade and utilize high concentrations of phenolic acids in industrial wastewater with similar high salt concentrations or in saline-alkali soil. In addition, the DT01 strain can tolerate high salt concentration environment, enabling the strain to produce non-sterile fermentation in a high salt environment, thereby directly reducing the energy consumption cost caused by sterilization in fermentation production. Sea water can be used for medium preparation during fermentation, eliminating the need for fresh water, further reducing production costs, and reducing fresh water consumption during industrial fermentation production.

[0026] Jejubacter sp. DT01 has substrate diversity. In addition to common carbon sources such as glucose, fructose and the like, it can directly utilize carbon sources, and can not only decompose phenolic acid compounds such as m-hydroxyphenylpropionic acid, phenylpropionic acid, cinnamic acid, ferulic acid, benzoic acid and p-hydroxybenzoic acid, but also can utilize the above phenolic acid compounds as the sole carbon source for growth. BRIEF DESCRIPTION OF DRAWINGS

[0027] Figure 1 Colony morphology of Jejubacter sp. DT01 strain on LB solid medium;

[0028] Figure 2 Cell morphology of Jejubacter sp. DT01 strain for transmission electron microscopy observation;

[0029] Figure 3 Growth status of Jejubacter sp. DT01 strain under different NaCl concentrations;

[0030] Figure 4 Growth status of Jejubacter sp. DT01 strain under different pH conditions;

[0031] Figure 5 Growth status of Jejubacter sp. DT01 strain under different temperature conditions;

[0032] Figure 6 Phylogenetic tree of DT01 strain based on 16S rDNA sequence construction;

[0033] Figure 7 Growth status of Jejubacter sp. DT01 strain under different m-hydroxyphenylpropionic acid concentrations and degradation rate of m-hydroxyphenylpropionic acid;

[0034] Figure 8 Growth status of Jejubacter sp. DT01 strain under 1000 mg / L phenylpropionic acid, cinnamic acid, ferulic acid, benzoic acid and p-hydroxybenzoic acid concentrations and degradation rate thereof;

[0035] Figure 9 Growth status of Jejubacter sp. DT01 strain in high-salt sterilized fermentation medium and high-salt non-sterilized fermentation medium;

[0036] Figure 10The microscopic results of Jejubacter sp. strain DT01 cultured in high-salt sterilized fermentation medium and high-salt non-sterilized fermentation medium for different time;

[0037] Figure 11 The yield comparison chart of isobutanol produced by Jejubacter sp. strain DT01 using glucose in high-salt sterilized fermentation medium and high-salt non-sterilized fermentation medium. DETAILED DESCRIPTION:

[0038] The experimental methods used in the following examples are conventional methods unless otherwise specified.

[0039] The materials, reagents and the like used in the following examples can be obtained from commercial channels unless otherwise specified.

[0040] The following examples are further illustrations of the present application and do not limit the essential content of the present application.

[0041] Example 1: Isolation, identification and preservation of the strain

[0042] I. Isolation of DT01 strain

[0043] (1) The test saline-alkali soil sample was diluted by 0.9% physiological saline at a ratio of 1:1000 and then inoculated into brain heart infusion broth (BHI) medium, and aerobic constant temperature culture was carried out in a shaker at 30°C, at a speed of 220 rpm, for 3 days, to obtain a mixed bacterial solution;

[0044] The brain heart infusion broth (BHI) medium comprises 12.5 g / L of calf brain infusion powder, 5 g / L of beef heart infusion powder, 10 g / L of proteose peptone, 2 g / L of glucose, 5 g / L of sodium chloride, 2.5 g / L of disodium hydrogen phosphate, and the rest is water, with a pH value of about 6.8;

[0045] (2) The mixed bacterial solution was diluted and spread on LB solid medium, and after 24 h of culture at 30°C, single colonies were obtained by repeated streaking of single colonies on LB solid medium.

[0046] The LB solid medium comprises 10 g / L of tryptone, 5 g / L of yeast extract, 10 g / L of sodium chloride, and 10-20 g / L of agar, and the rest is water;

[0047] (3) The microorganism obtained in step (2) was cultured in LB medium containing different concentrations of sodium chloride;

[0048] The LB medium comprises 10 g / L of tryptone, 5 g / L of yeast extract, and X g / L of sodium chloride;

[0049] The concentration X of sodium chloride in the LB medium is set to different concentration gradients, and the specific concentration gradient is designed as 10 g / L, 30 g / L, 50 g / L, 70 g / L, 90 g / L, and 110 g / L, and the salt-tolerant strain is screened step by step.

[0050] The strain that grows well under 90 g / L of salt ions is selected and named as strain DT01.

[0051] II. Identification of DT01 strain

[0052] 1. Morphological identification of DT01 strain

[0053] The morphological identification of the DT01 strain is as follows: after 12 h of culture at 37°C on LB solid medium, the colony is round, convex, transparent, smooth, white, and has a diameter of 1-2 mm. Figure 1 Under the observation of an optical microscope, the cell body is rod-shaped under 100 times magnification. At the same time, it can be observed by transmission electron microscopy that the cell body is rod-shaped and has flagella, with a length of 0.8-1.5 μm and a diameter of 0.4-0.8 μm. Figure 2 The DT01 strain is a gram-negative bacterium.

[0054] 2. Physiological and biochemical identification of DT01 strain

[0055] The physiological and biochemical characteristics of the DT01 strain are shown in Table 1.

[0056] Table 1 Physiological and biochemical characteristics of DT01 strain

[0057]

[0058]

[0059] Note: +: positive reaction; -: negative reaction; W: weak positive reaction

[0060] 3. Performance detection of DT01 strain

[0061] The performance of the DT01 strain was detected. The single colony was obtained by streaking the LB plate from the frozen bacteria of DT01. The single colony was picked into the LB medium and cultured at 37°C for 12 h under the condition of 220 rpm shaking bed, as the seed liquid. Then the seed liquid was inoculated into the LB liquid medium at an inoculation amount of 1% (volume ratio), and cultured under different conditions for 48 h, and the OD 600 was measured, as follows:

[0062] (1) Add different concentrations of NaCl (specifically: 0, 10, 15, 30, 45, 60, 75, 90, 105, 120, 135, 150, 165 g / L) to the LB medium to carry out NaCl concentration gradient tolerance experiment. After 48 h of culture at 37℃ and pH 7, the OD 600 The results are shown in Table 1. Figure 3 The results show that the DT01 strain can grow in the medium with a salt concentration of 0-150 g / L.

[0063] (2) Adjust the pH value of the LB medium to 2-12 (specifically: 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12) to carry out pH gradient experiment. After 48 h of culture at 37℃ and NaCl concentration of 10 g / L, the OD 600 The results are shown in Table 2. Figure 4 The results show that the DT01 strain can grow in the medium with a pH value of 3-11.

[0064] (3) Adjust the culture temperature to different temperatures (specifically: 5, 10, 15, 25, 30, 37, 40, 45, 50, 55, 60℃) to carry out temperature gradient experiment. After 48 h of culture at pH 7 and NaCl concentration of 10 g / L, the OD 600 The results are shown in Table 3. Figure 5 The results show that the DT01 strain can grow at a culture temperature of 5-55℃.

[0065] The above experiments were repeated 3 times.

[0066] 4. Molecular identification of DT01 strain

[0067] The activated DT01 strain was inoculated in LB medium containing 10 g / L NaCl and cultured at 30℃ for 24 h. 2 mL of fresh culture broth was centrifuged at 4℃ and 12000 rpm for 3 min, and the bacterial cells were collected in a 2 mL centrifuge tube. The genomic DNA was extracted using a bacterial genomic DNA extraction kit.

[0068] The 16S rDNA sequence of the strain was detected, and the primers for amplifying the 16S rDNA sequence were universal primers: 16F (5'-GCGGATCCGCGGCCGCTGCAGAGTTTGATCCTGGCTCAG-3'), and 16R (5'-GGCTCGAGCGGCCGCCCGGGTTACCTTGTTACGACTT-3').

[0069] The specific sequence of the 16S rDNA of the DT01 strain is as follows (shown in SEQ ID NO. 7):

[0070]

[0071] The sequence was retrieved and aligned with the taxonomically united 16S rRNA gene database in the EZBioCloud database, and a phylogenetic tree based on the full sequence of 16S rDNA was constructed by using Mega11.0 software and the Neighbor-Joining method (see Figure 6 ), and it was determined that DT01 belongs to Jejubacter.

[0072] Based on the above identification results, the DT01 strain is a new Jejubacter, named Jejubacter sp. DT01. The strain is preserved in the China General Microbiological Culture Collection Center, and the preservation number is CGMCC No. 26777.

[0073] III. Identification of phenolic acid compound degradation-related genes of DT01 strain

[0074] The genome of the DT01 strain was sent to Azenta for PacBio third-generation sequencing, and the bacterial genome completion map of the DT01 strain was obtained. Further comparison with the genome sequence of Jejubacter calystegiae KSNA2 strain (NZ_CP040428.1) found that the Jejubacter sp. DT01 genome of the present application contains phenolic acid compound degradation-related genes that other Jejubacter do not have, thereby enabling it to degrade phenolic acid substances. The detected phenolic acid compound degradation-related genes include flavin-type hydroxylase encoding gene mhpA (shown in SEQ ID NO. 1), exadiol dioxygenase encoding gene mhpB (shown in SEQ ID NO. 2), meta-cleavage complex hydrolase encoding gene mhpC (shown in SEQ ID NO. 3), 2-keto-4-pentenoic acid hydratase encoding gene mhpD (shown in SEQ ID NO. 4), 4-hydroxy-2-ketovaleric acid aldolase encoding gene mhpE (shown in SEQ ID NO. 5), and acetyl-CoA dehydrogenase encoding gene mhpF (shown in SEQ ID NO. 6).

[0075] Example 2: Degradation of m-hydroxyphenylpropionic acid by DT01 strain

[0076] Determination of degradation ability of m-hydroxyphenylpropionic acid: single colonies were obtained by streaking LB plates from the frozen bacteria of DT01. The single colonies were picked into LB medium and cultured at 37°C for 12h at 220rpm as seed liquid. The seed liquid was inoculated into 60M9 high salt base medium containing different concentrations of m-hydroxyphenylpropionic acid (3HPP) at an inoculation amount of 1% (volume ratio), and the strain DT01 was cultured at 37°C for 48h at 220rpm, and the OD 600 and the concentration of m-hydroxyphenylpropionic acid.

[0077] The 60M9 high salt base medium consists of 6g / L disodium hydrogen phosphate, 3g / L potassium dihydrogen phosphate, 1g / L ammonium chloride, 60g / L sodium chloride, 120mg / L magnesium sulfate, 11.1mg / L calcium chloride, 10mg / mL vitamin B1, X g / L m-hydroxyphenylpropionic acid, and the rest is water;

[0078] In the above 60M9 high salt base medium, m-hydroxyphenylpropionic acid is the only carbon source, and the concentration of m-hydroxyphenylpropionic acid X is set to different concentration gradients. The specific concentration gradient is designed as 0mg / L, 50mg / L, 100mg / L, 200mg / L, 400mg / L, 600mg / L, 800mg / L, and 1000mg / L.

[0079] After the culture, the residual content of m-hydroxyphenylpropionic acid in the culture medium was detected by high performance liquid chromatography, and the degradation rate was calculated. The parameter conditions are as follows: methanol and water (0.2% TFA is added) are used as mobile phase, the flow rate is 1.0mL / min, the column temperature is 30 degrees, and the detection wavelength is 276nm. Gradient elution is used, 0-15min, methanol is increased from 5% to 30%; 15-16min, methanol is increased from 30% to 100%; 16-18min, methanol is kept at 100%; 18-19min, methanol is reduced from 100% to 5%; 19-21min, methanol is kept at 5%.

[0080] The above experiments were repeated three times.

[0081] The growth status of Jejubacter sp. DT01 strain under different concentrations of m-hydroxyphenylpropionic acid and the degradation rate of m-hydroxyphenylpropionic acid are shown in Table 1. Figure 7

[0082] The results show that when there is no m-hydroxyphenylpropionic acid added in the 60M9 high salt base medium, the strain cannot grow. When 50-1000mg / L of m-hydroxyphenylpropionic acid is added in the culture medium, the strain can grow normally. It shows that the DT01 strain can use m-hydroxyphenylpropionic acid as carbon source for growth.

[0083] ​And the strain has good degradation effect on 0-1000mg / L m-hydroxyphenylpropionic acid (see the table below), especially the degradation rate of 1000mg / L high concentration of m-hydroxyphenylpropionic acid can reach 80.3%. It shows that DT01 strain has high ability to degrade m-hydroxyphenylpropionic acid.

[0084] In addition, the concentration of sodium chloride in 60M9 high-salt basic medium is 60g / L, and DT01 strain can grow normally and degrade m-hydroxyphenylpropionic acid, which shows that DT01 can degrade phenolic compounds in extreme environments such as industrial wastewater or saline-alkali soil, and alleviate the autotoxicity of plants in saline-alkali soil.

[0085] Degradation efficiency of DT01 strain on different concentrations of m-hydroxyphenylpropionic acid

[0086] Initial concentration of hydroxyphenylpropionic acid (mg / L) Degradation rate Initial concentration of hydroxyphenylpropionic acid (mg / L) Degradation rate 50 90.3％ 600 81.7％ 100 88.3％ 800 81.7％ 200 87.3％ 1000 80.3％ 400 84.0％

[0087] Example 3: Degradation of phenylpropionic acid, cinnamic acid, ferulic acid, benzoic acid and p-hydroxybenzoic acid by DT01 strain

[0088] LB plates were streaked from the frozen bacteria of DT01 to obtain single colonies. The single colonies were picked into LB medium and cultured at 37°C for 12h with 220rpm shaking as seed liquid. Then the seed liquid was inoculated into 60M9 high-salt basic medium containing 1000mg / L of phenylpropionic acid, cinnamic acid, ferulic acid, benzoic acid and p-hydroxybenzoic acid respectively, with an inoculation amount of 1% (volume ratio), and cultured at 37°C for 48h with 220rpm shaking.

[0089] After the culture, the OD 600 and the concentrations of phenylpropionic acid, cinnamic acid, ferulic acid, benzoic acid and p-hydroxybenzoic acid were measured.

[0090] The residual contents of phenylpropionic acid, cinnamic acid, ferulic acid, benzoic acid and p-hydroxybenzoic acid in the culture medium were detected by high performance liquid chromatography, and the degradation rate was calculated. The parameter conditions were as follows: methanol and water (with 0.2% TFA) were used as mobile phase, the flow rate was 1.0mL / min, the column temperature was 30 degrees, and the detection wavelength was 276nm. Gradient elution was used, 0-15min, methanol increased from 5% to 30%; 15-16min, methanol increased from 30% to 100%; 16-18min, methanol remained at 100%; 18-19min, methanol decreased from 100% to 5%; 19-21min, methanol remained at 5%.

[0091] It was found that DT01 strain could degrade and utilize phenylpropionic acid, cinnamic acid, ferulic acid, benzoic acid and p-hydroxybenzoic acid, and the degradation rates were 78.0%, 66.5%, 77.8%, 82.5% and 78.2% respectively (see Figure 8). It is shown that the DT01 strain has substrate diversity in degrading phenolic compounds, and can utilize m-hydroxyphenylpropionic acid, phenylpropionic acid, cinnamic acid, ferulic acid, benzoic acid and p-hydroxybenzoic acid as the sole carbon source.

[0092] Example 4: Comparison of growth of Jejubacter sp. DT01 in high-salt sterilized fermentation medium and high-salt non-sterilized fermentation medium

[0093] Single colonies were obtained by streaking LB plates from the frozen bacteria of DT01. The single colonies were picked into LB medium and cultured at 37°C for 12h with 220rpm shaking as seed liquid. The seed liquid was transferred into high-salt sterilized fermentation medium and high-salt non-sterilized fermentation medium at a ratio of 1%.

[0094] Preparation of high-salt sterilized fermentation medium (1L):

[0095] Preparation of M9 salt solution: weigh 6g Na2HPO4, 3g KH2PO4, 1g NH4Cl, 60g NaCl, 4g yeast extract, dissolve in distilled water, and make up to 897mL. Sterilize at 121°C for 20min. After cooling to room temperature, add 100mL of 40% glucose solution sterilized at 115°C for 15min, 1mL of 1M MgSO4solution, 1mL of 0.1M CaCl2solution, and 1mL of 10mg / mL vitamin B1 solution to the sterilized 897mL M9 salt solution and mix well.

[0096] Preparation of high-salt non-sterilized fermentation medium (1L):

[0097] Weigh 6g Na2HPO4, 3g KH2PO4, 1g NH4Cl, 60g NaCl, 4g yeast extract, 40g glucose, and add 997mL distilled water, stirring to mix well. Then add 1mL of 1M MgSO4solution, 1mL of 0.1M CaCl2solution, and 1mL of 10mg / mL vitamin B1 solution.

[0098] Each experiment had 3 replicates, and each fermentation flask was divided into 20mL of fermentation medium.

[0099] Cultivation conditions: 30°C, 220rpm; initial pH value was 7.0.

[0100] At 8h, 16h, 24h and 48h, 1mL of sample was taken to measure the OD 600 At different time points, the culture was prepared into a specimen, and the cell morphology was observed under an optical microscope for microscopic examination.

[0101] The final OD of Jejubacter sp. DT01 in the high-salt sterilized fermentation medium and the high-salt non-sterilized fermentation medium after 48 hours 600 The values were 2.09 and 2.02, respectively (see Figure 9 The results showed that the growth trend of the strain in the high-salt sterilized fermentation medium and the high-salt non-sterilized fermentation medium was similar.

[0102] The results of microscopic examination at 8 hours, 16 hours, 24 hours and 48 hours are shown in Table 2. Figure 10 The results showed that the cell morphology in the high-salt sterilized fermentation medium and the high-salt non-sterilized fermentation medium was single, and was the Jejubacter sp. DT01 strain until 48 hours. The results showed that the strain still existed in the form of pure culture in the high-salt non-sterilized fermentation medium, and was not contaminated by other microorganisms.

[0103] The above results showed that Jejubacter sp. DT01 could grow normally in the high-salt non-sterilized fermentation medium.

[0104] Example 5: The engineered bacteria constructed by using Jejubacter sp. DT01 strain as host produce isobutanol from glucose in high-salt sterilized fermentation medium and high-salt non-sterilized fermentation medium

[0105] The plasmid carrying the genes of acetolactate synthase (AlsS) derived from Bacillus subtilis, acetolactate isomerase (IlvC) and acetohydroxy acid dehydratase (IlvD) derived from Escherichia coli, ketol-acid decarboxylase (Kivd) and alcohol dehydrogenase (AdhA) derived from Lactococcus lactis, and ampicillin resistance gene was introduced into the DT01 strain. Three single colonies were inoculated in LB medium containing ampicillin and cultured at 37°C, 220 rpm for 12 hours in a shaking incubator to obtain three tubes of seed liquid as a repeated group. The seed liquid was inoculated in the high-salt sterilized fermentation medium and the high-salt non-sterilized fermentation medium at a ratio of 1%.

[0106] The high-salt sterilized fermentation medium and the high-salt non-sterilized fermentation medium were prepared in the same way as the medium preparation method in Example 4.

[0107] Each experiment had 3 repeats, and each fermentation bottle was divided into 20 mL of fermentation medium, and 20 μL of 100 mg / mL ampicillin was added to each bottle. No additional sugar source and nitrogen source were required during the fermentation process.

[0108] Fermentation conditions: 30℃, 220rpm; the initial pH value of the fermentation system was 7.0. 1mL was sampled at 8h, 16h, 24h and 48h respectively.

[0109] Sample processing: First, 200μL of each bacterial solution was taken to measure OD 600 at 12000rpm for 10 minutes. 100μL of supernatant was added to the gas phase vial's sleeve, and 100μL of prepared 1g / L n-pentanol (final concentration 0.5g / L) was added as the detection internal standard for gas chromatography. The gas phase vial's cap was screwed on tightly and shaken thoroughly.

[0110] Standard preparation: Add 160μL of water to the gas phase vial's sleeve, and add 20μL of 10g / L n-pentanol (final concentration 1g / L) and 20μL of 10g / L isobutanol (final concentration 1g / L) respectively. Screw the gas phase vial's cap tightly and shake thoroughly.

[0111] Gas chromatography detection procedure: Isobutanol quantitative determination was performed using A91 gas chromatograph (GC) from Changzhou Pannuo Instrument Co., Ltd. and DB-FFAP capillary column (30m x 0.32mm x 0.2μm; Agilent). The GC column temperature was initially maintained at 78℃ for 1.5 minutes; then increased to 94℃ at a gradient of 40℃ per minute, maintained for 0.2 minutes; increased to 100℃ at a gradient of 10℃ per minute, maintained for 0.2 minutes; increased to 235℃ at a gradient of 115℃ per minute, maintained for 0.5 minutes. The carrier gas was nitrogen, the split ratio was 50:1, and the injector and detector temperatures were maintained at 250℃ and 280℃ respectively. The injector volume was 0.2μL, and the injection was performed using a micro-injector produced by Agilent.

[0112] The results are shown in Figure 11 After 48 hours of fermentation, the strain produced isobutanol at a yield of 2.2g / L and 2.0g / L in high-salt sterilized fermentation medium and high-salt non-sterilized fermentation medium, respectively, using glucose. The results show that the strain can obtain similar isobutanol yield in high-salt non-sterilized fermentation as in high-salt sterilized fermentation. This proves the feasibility of non-sterilized fermentation of the host DT01 strain in high-salt environment.

[0113] The above embodiments only express several implementation manners of the present application, and the description is more specific and detailed, but it should not be understood as a limitation on the patent scope. It should be noted that for ordinary skilled persons in the art, the above embodiments can also be modified, combined and improved without departing from the concept of the present patent, and these all belong to the protection scope of the present patent. Therefore, the protection scope of the present patent should be subject to the claims.

Claims

1. A Zobellia sp. characterized in that, The Jeotgalibacillus is specifically Jejubacter sp. DT01, with the accession number of CGMCC No. 26777.

2. The Zobellia jejuensis of claim 1 Jejubacter The use of Zobellia jejuensis sp. DT01, characterized in that, The application of Jejubacter in degrading phenolic acid compounds, such as m-hydroxyphenylpropionic acid, phenylpropionic acid, cinnamic acid, ferulic acid, benzoic acid or p-hydroxybenzoic acid.

3. The Zobellia jejuensis of claim 1 Jejubacter The use of Zobellia jejuensis sp. DT01, characterized in that, The application of Jejubacter in high-salt non-sterilized fermentation The high salt refers to the sodium chloride concentration of 60 g / L.

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