A strain of deep-sea campylobacter, culture, inoculant and application thereof

Abstract

The application provides a strain of deep-sea Thalassolituus, a culture, a bacterial agent and application thereof, and belongs to the technical field of microbial bacterial agents.The strain of deep-sea Thalassolituus (Thalassolituus sp.) W7 of the application has a preservation number of CGMCC No.27013.The strain of deep-sea Thalassolituus W7 of the application can secrete poly-3-hydroxybutyrate (PHB) depolymerase, effectively degrade PHB, and has a PHB degradation rate of up to 79.70+ / -0.52% in 5 days.The deep-sea Thalassolituus W7 of the application can be used for bioremediation of PHB product pollution, reduces the PHB content in the environment, provides a new biological resource for biodegradation of PHB products, has great value for environmental governance, and has objective economic benefits and good social benefits.

Description

Technical Field

[0001] This invention belongs to the field of microbial inoculant technology, specifically relating to a deep-sea Campylobacter strain, culture, inoculant, and its application. Background Technology

[0002] Polyhydroxybutyrate (PHB) is a biodegradable high-molecular-weight polyhydroxy fatty acid ester. It is a carbon and energy source stored intracellularly in prokaryotes under limiting conditions (such as excess carbon and low phosphorus, nitrogen, and oxygen). PHB is a polymer of 3-hydroxybutyric acid. Intracellular PHB is encapsulated by a protein and phospholipid layer, exists in an amorphous form, and is easily degraded; PHB extracted with organic solvents exists in the form of a crystalline polymer.

[0003] Various PHB-degrading bacteria have been isolated from the environment, including fungi from genera such as *Aspergillus*, *Penicillium*, *Mucor*, and *Paecilomyces*, and bacteria from genera such as *Cupriavidus*, *Comamonas*, *Pseudomonas*, *Stenotrophomonas*, and *Bacillus* sp. The biodegradation of PHB involves biodeterioration, biofragmentation, and assimilation. During depolymerization, microorganisms produce extracellular enzymes that degrade PHB into water-soluble small molecular fragments. The products are usually monomers, or a mixture of monomers and dimers, or oligomers. Studies have also shown that PHB degradation products include 3-hydroxybutyric acid, acetoacetic acid, and a small amount of acetic acid, while under aerobic conditions, most of it is oxidized to CO2 and water. During assimilation, small molecular fragments enter cells through biomembranes and participate in metabolic cycles.

[0004] Thalassolituus is an alkane-degrading bacterium first discovered in the marine environment in 2004. It is a dominant genus of bacteria in petroleum hydrocarbon-polluted seawater and is mostly isolated from culture media with tetradecane, hexadecane, or a mixture of petroleum hydrocarbons as the sole carbon source.

[0005] However, there are currently no reports on deep-sea Campylobacter species degrading PHB. Summary of the Invention

[0006] In view of this, the purpose of the present invention is to provide a deep-sea Campylobacter strain, a bacterial agent and its application, wherein the deep-sea Campylobacter strain W7 of the present invention can effectively degrade PHB.

[0007] This invention provides a strain W7 of the genus Thalassolituus sp., with the deposit address at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, and the deposit number CGMCC No. 27013.

[0008] The present invention also provides a culture of the deep-sea Campylobacter strain W7 described in the above-described scheme.

[0009] The present invention also provides a microbial agent comprising the deep-sea Campylobacter strain W7 described in the above-described scheme and / or the culture thereof.

[0010] Preferably, the effective viable count of Campylobacter spp. strain W7 in the bacterial agent is 2–4 × 10⁻⁶. 7 cfu / mL.

[0011] The present invention also provides the application of the deep-sea Campylobacter strain W7, the culture, or the agent described above in the degradation of poly-3-hydroxybutyrate.

[0012] Preferably, the degradation of poly-3-hydroxybutyrate includes the following steps: mixing the deep-sea Campylobacter strain W7 or the culture or the bacterial agent described in the above scheme with the material to be degraded containing poly-3-hydroxybutyrate, and then performing degradation.

[0013] Preferably, the degradation temperature is ≥20°C.

[0014] Preferably, the salinity of the substance to be degraded is ≤50g / L.

[0015] Preferably, the pH of the substance to be degraded is 5 to 8.

[0016] This invention provides a deep-sea Campylobacter sp. strain W7, deposited at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, with accession number CGMCC No. 27013. This Campylobacter sp. strain can secrete PHB depolymerase, effectively degrading PHB, with a PHB degradation rate as high as 79.70±0.52% after 5 days. This Campylobacter sp. strain W7 can be used for the bioremediation of PHB-contaminated products, reducing the PHB content in the environment, providing a new biological resource for the biodegradation of PHB products, and has significant value for environmental remediation, with objective economic and social benefits. Attached Figure Description

[0017] Figure 1 The colony morphology of Campylobacter strain W7 in solid culture medium and the cell morphology under a microscope are shown; where A is the colony morphology of strain W7, and B and C are the microscopic morphology of strain W7.

[0018] Figure 2 Physiological and biochemical results for deep-sea Campylobacter strain W7;

[0019] Figure 3 The phylogenetic tree results for Campylobacter strain W7 from the deep sea;

[0020] Figure 4 The DNAMAN alignment results of the gene sequence of Campylobacter strain W7 from the deep sea with the gene sequence of Campylobacter alkanivorans TMPB967 (OK489464) from the alkaline-eating seaborne Campylobacter.

[0021] Figure 5 The results show the pH variation trend of Campylobacter strain W7 under different culture conditions; where A represents the pH variation trend under different salinity culture conditions; B represents the pH variation trend under different initial pH culture conditions; and C represents the pH variation trend under different temperature culture conditions.

[0022] Figure 6 The results show the trends of relative enzyme activity and PHB degradation rate of deep-sea Campylobacter strain W7 under different salinity culture conditions; where A represents the trend of relative enzyme activity of strain W7 under different salinity culture conditions; and B represents the trend of degradation rate of strain W7 under different salinity culture conditions.

[0023] Figure 7 The results show the trends of relative enzyme activity and PHB degradation rate of deep-sea Campylobacter strain W7 under different initial pH conditions; where A represents the trend of relative enzyme activity of strain W7 under different initial pH conditions; and B represents the trend of degradation rate of strain W7 under different initial pH conditions.

[0024] Figure 8 The results show the trends of relative enzyme activity and PHB degradation rate of strain W7 from the deep-sea Campylobacter genus under different temperature culture conditions; where A represents the trend of relative enzyme activity of strain W7 under different temperature culture conditions; and B represents the trend of degradation rate of strain W7 under different temperature culture conditions.

[0025] Biological Preservation Instructions

[0026] Thalassolituus sp. strain W7 was deposited on April 3, 2023, at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, with accession number CGMCC No. 27013. Detailed Implementation

[0027] This invention provides a strain W7 of the genus Thalassolituus sp., with the deposit address at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, and the deposit number CGMCC No. 27013.

[0028] In this invention, the deep-sea Campylobacter strain W7 was isolated from the marine fish recirculating aquaculture system of Tianjin Haifa Zhenpin Industrial Co., Ltd.

[0029] The deep-sea Campylobacter sp. strain W7 described in this invention was isolated using the following method: PHB was added to the collected water sample, and the sample was enriched by aeration at 28°C for 7 days. The enriched culture was then cultured in a liquid medium with PHB as the sole carbon source for 5–6 days at 28°C and a rotation speed of 200 r / min to isolate and purify PHB-degrading bacteria. The above culture solution was diluted and spread onto a PHB solid medium for further culture for 4–6 days. Colonies producing a clear zone were picked from the PHB plates and named W7. Molecular biological identification was performed using 16S rRNA gene sequencing. DNAMAN results showed that the gene sequence of W7 was 98.75% identical to that of *Thalassolituus alkanivorans* TMPB967 (OK489464). Physiological and biochemical identification combined with molecular identification based on 16S rRNA gene sequencing confirmed that it was a deep-sea Campylobacter sp. strain, named W7.

[0030] The deep-sea Campylobacter strain W7 of this invention has the following biological characteristics: Gram-negative, non-flagellated, and aerobic. The bacterial cells are curved and morphologically diverse; during the logarithmic growth phase, cells are curved arcs or a few short rods. In PHB and LB semi-solid media, the bacteria only show obvious growth along the puncture line, with cell sizes ranging from 0.42–0.74 μm × 1.5–6.0 μm. In PHB solid medium, colonies are round, colorless, transparent, moist, with a raised center and neat edges, and a diameter of 2–3 mm.

[0031] In this invention, the liquid culture medium using PHB as the sole carbon source preferably contains 10.0 g / L of artificial sea salt, 0.2 g / L of NH4Cl, and 1.0 g / L of PHB. In this invention, the PHB solid culture medium is based on the liquid culture medium with the addition of 15.0 g / L of agar.

[0032] In this invention, the PHB semi-solid culture medium is based on liquid culture medium with the addition of 7.5 g / L agar; the LB semi-solid culture medium preferably contains 10 g / L yeast extract, 5 g / L tryptone, and 10 g / L agar.

[0033] In this invention, the 16S rRNA gene sequence of the deep-sea Campylobacter strain W7 is shown in SEQ ID No. 1, specifically as follows:

[0034] GCTGCATGGCGGAGCTTACCATGCAGTCGAGCGGTAGAAAGTAGCTTGCTACTTTTGAGAGCGGCGGACGGGTGAGTAATGCGTGGGAATCTACCTGGTAGTGGGGGACAACAGTTGGAAACGACTGCTAATACCGCATACGCCCTACGGGGGAAAGCGGGGGATCTTCGGACCTCGTGCTATCAGATGAGCCCGCGTGAGATTAGCTAGTTGGTGGGGTAAAGGCCTACCAAGGCGACGATCTCTAGCTGGTCTGAGAGGATGATCAGCCACACTGGGACTGAGACACGGCCCAGACTCCTACGGGAGGCAGCAGTGGGGAATATTGGACAATGGGCGCAAGCCTGATCCAGCCATGCCGCGTGTGTGAAGAAGGCCTTCGGGTTGTAAAGCACTTTCAGCGAGGAGGAAAGGTTGTAGCTTAATACGCTGCAGCTGTGACGTTACTCGCAGAAGAAGCACCGGCTAACTCCGTGCCAGCAGCCGCGGTAATACGGAGGGTGCAAGCGTTAATCGGAATTACTGGGCGTAAAGCGCGCGTAGGTTGTTTGTTAAGCGAGATGTGAAAGCCCCGGGCTCAACCTGGGAACTGCAtTTCGAACTGGCAAGCTAGAGTACAGTAGAGGGTGGCGGAATTTCCTGTGTAGCGGTGAAATGCGTAGAGATGGGAAGGAACATCAGTGGCGAAGGCGGCCACCTGGACTGATACTGACACTGAGGTGCGAAAGCGTGGGGAGCAAACAGGATTAGATACCCTGGTAGTCCACGCCGTAAACGATGTCTACTAGTTGTCGGGAGACTTGATCTCTTGGTAACGAAGCTAACGCGATAAGTAGACCGCCTGGGGAGTACGGCCGCAAGGTTAAAACTCAAATGAATTGACGGGGGCCCGCACAAGCGGTGGAGCATGTGGTTTAATTCGAAGCAACGCGAAGAACCTTACCTACTCTTGACATCCTGCGAACTTTCGAGAGATCGATTGGTGCCTTCGGGAACGCAGAGACAGGTGCTGCATGGCTGTCGTCAGCTCGTGTTGTGAAATGTTGGGTTAAGTCCCGTAACGAGCGCAACCCTTGTCCTTAGTTGCCATCATTTAGTTGGGGACTCTAAGGAGACTGCCGGTGACAAACCGGAGGAAGGCGGGGACGACGTCAAGTCATCATGGCCCTTACGAGTAGGGCTACACACGTGCTACAATGG CCGGTACAGAGGGTCGCGAAGCCGCGAGGTGGAGCTAATCTCACAAAGCCGGTCGTAGTCCGGATTGGAGTCTGCAACTCGACTCCATGAAGTCGGAATCGCTAGTAATCGTGAATCAGAATGTCACGGTGAATACGTTCCCGGGCCTTGTACACACCGCCCGTCACACCATGGGAGTGGGTTGCTCCAGAAGTAGATAGCCTAACCTTCGGGAGGGCGTTACCACGAGTATTAGGTGT.

[0035] The present invention also provides a culture of the deep-sea Campylobacter strain W7 described in the above-described scheme.

[0036] In this invention, the culture preferably comprises a culture supernatant free of Campylobacter spp. strain W7; the culture supernatant is obtained by centrifugation of the culture medium. In this invention, the culture preferably comprises extracellular products of Campylobacter spp. strain W7; the extracellular products include PHB depolymerase.

[0037] The present invention also provides a microbial agent comprising the deep-sea Campylobacter strain W7 described in the above-described scheme and / or the culture thereof.

[0038] In this invention, the effective viable count of Campylobacter spp. strain W7 in the bacterial agent is preferably 2 to 4 × 10⁻⁶. 7 cfu / mL, more preferably 3×10⁻⁶ 7 cfu / mL.

[0039] The present invention also provides the application of the deep-sea Campylobacter strain W7, the culture, or the agent described above in the degradation of poly-3-hydroxybutyrate.

[0040] In this invention, the degradation of poly-3-hydroxybutyrate preferably includes the following steps: mixing the deep-sea Campylobacter strain W7 or the culture or the bacterial agent described in the above scheme with the material to be degraded containing poly-3-hydroxybutyrate, and then performing degradation.

[0041] In this invention, the substance to be degraded preferably includes PHB product particles or PHB released into the environment; the environment preferably includes seawater; apart from PHB, no other carbon source needs to be added to the substance to be degraded.

[0042] In this invention, the salinity of the substance to be degraded is preferably ≤50g / L, more preferably 10-50g / L, and even more preferably 30g / L; the deep-sea Campylobacter strain of this invention reaches maximum relative enzyme activity under a salinity of 30g / L, and can effectively degrade PHB in seawater.

[0043] In this invention, the pH of the water in which the substance to be degraded is located is preferably 5 to 8.

[0044] In this invention, the degradation temperature is preferably ≥20℃, more preferably 20~37℃, and even more preferably 25~28℃.

[0045] The technical solutions of the present invention will be clearly and completely described below with reference to the embodiments thereof.

[0046] The following examples are provided to better understand the present invention, but are not intended to limit the invention. Unless otherwise specified, the experimental methods used in the following examples are conventional methods. Unless otherwise specified, the experimental materials used in the following examples were purchased from conventional biochemical reagent stores.

[0047] In an embodiment of the present invention, the liquid culture medium formulation using PHB as the sole carbon source is as follows: 10.0 g / L artificial sea salt, 0.2 g / L NH4Cl, and 1.0 g / L PHB. In this invention, the PHB solid culture medium is based on the liquid culture medium, with the addition of 15.0 g / L agar.

[0048] Example 1: Isolation, screening and identification of Campylobacter strain W7 from the deep sea

[0049] I. Isolation and Screening of W7 Strains

[0050] Sample collection: The samples came from the marine fish recirculating aquaculture system of Tianjin Haifa Zhenpin Industrial Co., Ltd.

[0051] Isolation and Screening: 0.03 g PHB was added to 200 mL of water sample and cultured under aeration at 28℃ for 7 days. 10 mL of the enriched culture was then incubated in 200 mL of liquid medium with PHB as the sole carbon source for 5–6 days under the following conditions: 28℃, 200 rpm, to isolate and purify the PHB-degrading bacteria. 0.1 mL of the diluted culture was then plated onto PHB solid medium and cultured for another 4–6 days. Colonies exhibiting a clear transparent zone were picked from the PHB plates and named W7.

[0052] II. Identification of W7 strain

[0053] 1. Morphological identification

[0054] The W7 strain was inoculated into PHB solid medium and cultured for 2 days using the point value method. Colony morphology was observed. The culture was continued until day 5, and the diameter of the degradation zone and the colony diameter were measured.

[0055] Take 10 μL of the strain in the logarithmic growth phase and place it on a glass slide. Add 2.5% glutaraldehyde for fixation. Wash off the external salts with 0.5 mol / L ammonium acetate. Rinse with 1×PBS to remove glutaraldehyde. Dehydrate with ethanol at different concentration gradients. Observe the cell morphology under a scanning electron microscope (FEIApreo, USA).

[0056] The colony morphology of strain W7 is as follows Figure 1 As shown in A, the microscopic morphology is as follows Figure 1 As shown in B and C. Among them, the colonies of strain W7 after 2 days of cultivation on PHB solid medium were round, colorless, transparent, moist, with a raised center, neat edges, and a diameter of 2–3 mm, and were Gram-negative. After 5 days of cultivation, the diameter of the degradation zone and the colony diameter were 1.28±0.08 cm and 0.72±0.08 cm, respectively, with a ratio of 1.98±0.16 (…). Figure 1 A). Under a scanning electron microscope, cells in the logarithmic growth phase appear as curved arcs or a few short rods (in the image). Figure 1 B in Figure 1 In the C section, no flagella were observed, and the bacteria only showed obvious growth along the puncture line in PHB and LB semi-solid media. The cell size was 0.42–0.74 μm × 1.5–6.0 μm (n = 30).

[0057] 2. Physiological and biochemical identification

[0058] Strains W7 were cultured in PHB solid medium to the logarithmic growth phase, then inoculated into liquid medium prepared by Biolog and cultured at 28°C for 3 days. Physiological and biochemical characteristics were analyzed using an automated microbial identification system (GENⅢMicroStation, USA) and GENⅢMicroPlate plates.

[0059] The colorimetric reaction and physiological and biochemical results of Biolog are as follows: Figure 2 As shown. Carbon source utilization tests indicated that strain W7 exhibited strong metabolic capacity for 3-hydroxy-D,L-butyric acid, L-lactic acid, methyl pyruvate, Tween 40, propionic acid, and acetic acid, and may have some metabolic capacity for glucuronide and acetoacetic acid. Chemical sensitivity tests showed that strain W7 could grow under pH 5–8 and 1%–8% NaCl conditions, and showed positive resistance results to sodium lactate, rifamycin SV, aztreonam, and sodium butyrate.

[0060] 3. Molecular biological identification

[0061] Strains of strain W7 in the logarithmic growth phase were collected by centrifugation, and total DNA was extracted using the Tiangen bacterial DNA extraction kit. PCR amplification was performed using the universal bacterial upstream primer 27F: 5'-AGAGTTTGATCCTGGCTCAG-3' (SEQ ID NO.2) and downstream primer 1492R: 5'-GGTTACCTTGTTACGACTT-3' (SEQ ID NO.3). The PCR reaction system (total volume 25 μL) consisted of: 12.5 μL Taq enzyme, 0.5 μL primer 1, 0.5 μL primer 2, and 11.5 μL ddH2O. The PCR reaction conditions were: 94℃ pre-denaturation for 5 min; 94℃ denaturation for 1 min, 56℃ annealing for 1 min, 72℃ extension for 2 min, 35 cycles; and 72℃ extension for 10 min. PCR products were sent to Beijing Liuhe BGI Genomics Co., Ltd. for sequencing. Sequences were aligned against the NCBI database using Mega X software and the Clustal W algorithm. A phylogenetic tree was constructed using the maximum likelihood method, with the values ​​on the branches representing the step size. The number of iterations was 1000. Sequence homology analysis was performed using DNAMAN software.

[0062] The 16S rRNA gene sequence of Campylobacter strain W7 is shown in SEQ ID No. 1.

[0063] Developmental tree results as follows Figure 3 As shown, through morphological, physiological and biochemical characteristics and 16S rDNA analysis, strain W7 was identified as belonging to the genus Campylobacter; DNAMAN results are as follows. Figure 4 As shown, the results indicate that the gene sequence of the deep-sea Campylobacter strain W7 has a homology similarity of 98.75% with that of the alkali-eating marine Campylobacter Thalassolituus alkanivorans TMPB967 (OK489464).

[0064] Example 2: Analysis of the degradation characteristics of PHB by Campylobacter W7 (deep-sea spirobacterium)

[0065] I. Effects of different initial pH, temperature, and salinity conditions on the degradation characteristics of PHB by Campylobacter W7 cultured in the deep sea.

[0066] Strain W7 was activated by inoculating it into PHB liquid medium, and then transferred to seed medium until the logarithmic growth phase to obtain seed culture. The seed culture was inoculated into PHB liquid medium at a 3% (v / v) inoculation rate and cultured for 5 days at different initial pH (pH 6, pH 7.2, and pH 8, with fixed temperatures and salinities of 28℃ and 30 g / L, respectively), different temperatures (20℃, 28℃, and 37℃, with fixed initial pH and salinities of 7.2 and 30 g / L, respectively), and different salinities (10 g / L, 30 g / L, and 50 g / L, with fixed initial pH and temperature of 7.2 and 28℃, respectively).

[0067] 1. Measurement of pH in the culture medium

[0068] Strain W7 was cultured under different salinity, initial pH and temperature conditions, and the pH value of the culture medium was measured on days 0.5, 1, 2, 3, 4 and 5 of culture.

[0069] The results are shown in Table 1 and Figure 5 As shown, strain W7 was cultured under different salinity, initial pH and temperature conditions. The pH of all bacterial solutions decreased rapidly within 12 hours, reached its lowest point in about 1 day, and the overall pH tended to stabilize (pH 5.5-6.0) from 1 to 5 days.

[0070] Table 1. Results of pH variation trends of Campylobacter strain W7 under different culture conditions.

[0071]

[0072] 2. Determination of PHB depolymerase activity and PHB degradation rate

[0073] The culture medium was centrifuged at 4℃ and 12000r / min for 20min, and the supernatant crude enzyme solution was collected.

[0074] Dissolve 0.1g PHB particles in 10mL chloroform and mix with 0.5g / L sodium dodecyl sulfate. Make up to 100mL with distilled water. After sonication for 30min, prepare PHB emulsion. Heat and stir at 75℃ for 90min to remove chloroform and obtain PHB emulsion.

[0075] Using PHB emulsion as a substrate, 3 mL of PHB emulsion was placed in a constant temperature water bath. Once the desired culture temperature was reached, 3 mL of the corresponding crude enzyme solution was added. In the control group, other conditions remained unchanged, but the supernatant from the liquid culture medium was added. After incubation for 40 min, the absorbance (OD value) of the mixture was measured at 650 nm using a spectrophotometer (China, UV-3100, Meptop). The PHB depolymerase activity was calculated based on the OD value. One unit of enzyme activity was defined as the amount of enzyme required to cause a 0.001 unit decrease in absorbance per minute.

[0076] The mixture was centrifuged to collect the precipitate. The precipitate was dried in a 60℃ oven, and 2 mL of 98% concentrated sulfuric acid was added. The mixture was then boiled in a water bath for 1 hour and rapidly cooled to room temperature. The precipitate was diluted 50 to 200 times with 5 mM dilute sulfuric acid. The content of PHB in the precipitate was determined by calculating the PHB content using an Aminex HPX-87H ion exclusion organic acid column (300×7.8 mm) equipped with an HPLC system. The PHB degradation rate was then calculated by subtraction. HPLC conditions: 5 mM dilute sulfuric acid as eluent, flow rate of 0.7 mL / min, sample loading of 20 μL, column temperature of 65℃, and detection at 210 nm. The standard curve is shown in equation (1).

[0077] y = 39416x - 365754, R 2 =0.9944 Equation (1).

[0078] The results under different salinity culture conditions are shown in Table 2 and Figure 6 As shown, the maximum depolymerase activity was reached at salinities of 30 g / L and 10 g / L on day 3, while the maximum relative enzyme activity appeared at salinity of 50 g / L on day 4. With continued extension of the culture time, the enzyme activity of the strain showed a continuous decreasing trend. Figure 6 (A) The PHB degradation rate increased rapidly from 0 to 0.5 days, and increased slowly from 0.5 to 2 days. After 2 days, the PHB degradation rate gradually increased. The PHB degradation rate was highest at the end of the treatment under a salinity of 30 g / L. Figure 6 (B)

[0079] Table 2. Trends in relative enzyme activity and PHB degradation rate of Campylobacter strain W7 under different salinity conditions.

[0080]

[0081] The results under different initial pH culture conditions are shown in Table 3 and Figure 7 As shown, there was no significant difference in PHB depolymerase activity. Figure 7 (A) The PHB degradation rate is high from 0 to 0.5 days, and low from 0.5 to 2 days, with a slow increase in PHB degradation rate. After 2 days, the PHB degradation rate gradually increases. Figure 7(B in the text). The degradation rate was highest at an initial pH of 7.2 and lowest at pH 6.

[0082] Table 3. Trends in relative enzyme activity and PHB degradation rate of Campylobacter strain W7 under different initial pH conditions.

[0083]

[0084] The results under different temperature cultivation conditions are shown in Table 4 and Figure 8 As shown, the relative enzyme activity at 37℃ reached its highest level on day 3 and its lowest level at 20℃. Figure 8 (A) The PHB degradation rate increased rapidly from 0 to 0.5 days, increased slowly from 0.5 to 1 day, gradually increased after 2 days, and reached its maximum degradation rate at 4 days. The degradation rate was higher at 37℃ and 28℃, and lower at 20℃, but there was no significant difference in degradation rates between the two at 37℃ and 28℃. Figure 8 (B in the text). The degradation rate decreased on day 5, possibly due to the strain resynthesizing PHB under certain conditions. The degradation rate decreased on day 5, and the PHB depolymerase activity and degradation rate varied among different batches of experimental strains.

[0085] Table 4. Trends in relative enzyme activity and PHB degradation rate of Campylobacter strain W7 under different temperature conditions.

[0086]

[0087] Conclusion: When cultured at 28℃ and 30 g / L, the PHB depolymerase secreted by Campylobacter W7 reached its maximum relative enzyme activity, and the maximum PHB degradation rate reached 79.70 ± 0.52%.

[0088] Although the above embodiments have provided a detailed description of the present invention, they are only some embodiments of the present invention, and not all embodiments. People can obtain other embodiments based on these embodiments without creative effort, and these embodiments all fall within the protection scope of the present invention.

Claims

1. A strain W7 of the genus *Thalassolituus*, characterized in that... The address for the collection is No. 3, No. 1 Courtyard, Beichen West Road, Chaoyang District, Beijing, and the collection number is CGMCC No. 27013.

2. A microbial agent, characterized in that, Includes the deep-sea Campylobacter strain W7 as described in claim 1.

3. The microbial agent according to claim 2, characterized in that, The effective viable count of Campylobacter spp. strain W7 in the bacterial agent is 2-4 × 10⁻⁶. 7 cfu / mL.

4. The application of the deep-sea Campylobacter strain W7 as described in claim 1 or the bacterial agent as described in claim 2 or 3 in the degradation of poly-3-hydroxybutyrate.

5. The application according to claim 4, characterized in that, The degradation of poly-3-hydroxybutyrate includes the following steps: The deep-sea Campylobacter strain W7 as described in claim 1, or the bacterial agent as described in claim 2 or 3, is mixed with the material to be degraded containing poly-3-hydroxybutyrate, and then degraded.

6. The application according to claim 5, characterized in that, The degradation temperature is ≥20℃.

7. The application according to claim 5, characterized in that, The salinity of the substance to be degraded is ≤50g / L.

8. The application according to claim 5, characterized in that, The pH of the substance to be degraded is 5-8.

Images