Thermophilic strain with high efficiency of producing polyhydroxyalkanoate and application thereof

Abstract

This invention provides a thermophilic bacterial strain with high efficiency in producing polyhydroxyalkanoates and its applications. The thermophilic strain with high efficiency in producing polyhydroxyalkanoates is *Thermomonas thermophila* (…). Caldimonas hydrothermalis The strain identified is WH-YN, with accession number GDMCC No. 68346. This thermophilic bacterium, WH-YN, possesses advantages such as thermophilicity, strong adaptability to various culture media, high tolerance for inoculum size errors, stable PHA synthesis ability, and a wide range of carbon source utilization. It is well-suited for the industrial production of polyhydroxyalkanoates and can simultaneously facilitate the resource utilization of industrial wastewater.

Description

Technical Field

[0001] This invention belongs to the field of microbial fermentation technology and biodegradable materials, specifically involving a thermophilic strain with the ability to produce polyhydroxyalkanoates (PHA) with high efficiency, and the application of this strain in the industrial production of PHA, especially suitable for scenarios where PHA is produced by high-temperature fermentation using inexpensive carbon sources (such as industrial wastewater, agricultural waste hydrolysate, etc.). Background Technology

[0002] Polyhydroxyalkanoates (PHAs) are intracellular energy storage polymers synthesized by microorganisms under nutrient-limited conditions such as excess carbon and nitrogen. They possess excellent biocompatibility, biodegradability, and mechanical processing properties, and can replace traditional petroleum-based plastics (such as polyethylene and polypropylene) in applications such as packaging materials, medical implants, and agricultural films. As one of the key materials for solving white pollution, PHAs have extremely high environmental and economic value.

[0003] Currently, the PHA-producing strains reported both domestically and internationally mainly include mesophilic strains and a few thermophilic strains. Among them, mesophilic strains (optimal growth temperature 25~37℃) are the mainstream in research and application, with *R. euoxygenans* being a typical example. Ralstonia eutropha (Now renamed) Cupriavidus necator ), copper necrophorum ( Alcaligenes latus ), Pseudomonas spp. Pseudomonas These are mesophilic strains, etc. The mechanism of action of these strains is as follows: under suitable mesophilic conditions, utilizing high-quality carbon sources such as glucose and sucrose, in a nitrogen-limited fermentation system, they synthesize PHA through intracellular metabolic pathways and accumulate it within the cells; after fermentation, the PHA product is obtained through cell disruption, extraction, and purification.

[0004] With the increasing demand for industrial production of PHA, the inherent defects of mesophilic strains have gradually become apparent: First, the fermentation process requires strict temperature control between 25 and 37°C. Especially in the high-temperature environment of summer, a large amount of energy is required for cooling, resulting in high energy consumption and increased costs. Second, the mesophilic fermentation system is susceptible to contamination by other microorganisms. To maintain a sterile environment, additional sterilization costs are required, which further limits its large-scale application.

[0005] To address the aforementioned issues, the production of PHA by thermophilic strains (optimal growth temperature ≥45℃) has become a research hotspot. Existing technologies have reported some thermophilic PHA-producing strains; for example, patent CN109868259A discloses a thermophilic *Bacillus steatis* strain (…). Geobacillus stearothermophilusIts optimal growth temperature is 50-55℃, and the yield of PHA produced by glucose fermentation is 0.8-1.2 g / L, with PHA accounting for 35%-42% of the cell dry weight; another study reported a strain of Thermomonas genus ( Caldimonas thermodepolymerans Its optimal temperature is 60℃, but the yield of PHA is only 0.5g / L, and it can only utilize a few pure carbon sources such as glucose and xylose, which cannot be adapted to cheap industrial waste carbon sources.

[0006] In summary, existing thermophilic PHA-producing strains generally suffer from the following shortcomings: ① Low PHA yield and intracellular accumulation rate, making it difficult to meet the production requirements of industrial-scale production; ② Narrow carbon source adaptability, unable to efficiently utilize inexpensive carbon sources such as starch wastewater and agricultural straw hydrolysate, resulting in high raw material costs; ③ Some strains have a narrow optimal temperature range (e.g., only 50~52℃), and temperature fluctuations easily affect fermentation stability, increasing the difficulty of industrial-scale control. Therefore, screening a thermophilic PHA-producing strain with high yield, broad carbon source adaptability, and strong fermentation stability is a pressing technical problem to be solved in the current field of PHA industrial production. Summary of the Invention

[0007] To address the technical bottlenecks of existing thermophilic PHA-producing strains, such as low yield, narrow carbon source adaptability, narrow fermentation temperature range, and difficulty in adapting to industrial production scenarios (e.g., using industrial hot wastewater), there is an urgent need for a thermophilic strain and supporting application scheme that originates from a natural high-temperature environment, can withstand high temperatures of 60-70℃, is compatible with various culture media and inexpensive carbon sources, can be directly grown using industrial hot wastewater and produce PHA efficiently, and has a simple and controllable screening and fermentation process.

[0008] To achieve the above objectives, the technical solution adopted by the present invention is as follows: This invention provides a thermophilic strain with high efficiency in producing polyhydroxyalkanoates, wherein the thermophilic strain with high efficiency in producing polyhydroxyalkanoates is *Thermomonas thermophila* (…). Caldimonas hydrothermalis The strain was identified as WH-YN, with accession number GDMCC No. 68346.

[0009] Furthermore, the nucleotide sequence of the thermomonas hydrophila WH-YN is shown in SEQ ID NO. 1.

[0010] Furthermore, the thermonovial thermomonas WH-YN is a Gram-positive bacterium, which is short rod-shaped, with a size of 0.5~0.8μm×2.0~3.5μm, and includes elliptical spores.

[0011] Furthermore, the thermonovial bacterium WH-YN can grow stably at temperatures of 50~70℃ and can synthesize polyhydroxy fatty acid esters using pure carbon sources or inexpensive carbon sources (28%~43%) as raw materials.

[0012] This invention also provides a method for screening thermophilic strains with high efficiency in producing polyhydroxyalkanoates, the screening method comprising: The pure bacterial strain obtained from the sample was inoculated into a nitrogen-limiting medium and cultured at 60℃ and 180rpm for 48-72h to obtain the bacterial solution. The Nile blue fluorescence staining method was used for qualitative screening: a smear of bacterial culture was prepared, 1-2 mL of staining solution was added and incubated for 30 min, and then observed under a fluorescence microscope. Strains that showed bright red fluorescent particles in their cells were potential PHA-producing strains, and the strains obtained in the initial screening were obtained. The nitrogen-limiting culture medium is formulated as follows: glucose 20 g / L, (NH4)2SO4 0.5 g / L, K2HPO4 3H2O3g / L, KH2PO41.5g / L, MgSO4 7H2O 0.5g / L, CaCl2 2H₂O 0.1 g / L, pH 7.0; the excitation light of the fluorescence microscope is 365 nm; The strains obtained from the initial screening were inoculated into the nitrogen-limiting medium and cultured at 60°C and 180 rpm for 70 h to obtain the thermomonas hydrophila WH-YN. The yield of PHA was determined by gas chromatography-mass spectrometry (GC-MS).

[0013] The present invention also provides a method for culturing the above-mentioned thermophilic strain with high efficiency in producing polyhydroxy fatty acid esters. The method includes: inoculating the thermophilic bacterium WH-YN into tryptone soybean broth (TSB), yeast extract-starch-glucose (YSG), LB broth, and R2A bacterial culture medium, respectively; after inoculation, placing all culture media in a constant temperature shaking incubator at 60°C and 180 rpm for shaking culture.

[0014] Furthermore, the culture conditions for the thermomonas hydrophila WH-YN are TSB medium and an inoculum size of 15% (v / v).

[0015] The present invention further provides the application of the above-mentioned thermophilic strain with high efficiency in producing polyhydroxyalkanoates in the fields of industrial production of polyhydroxyalkanoates and resource utilization of industrial wastewater.

[0016] Furthermore, the thermomonas hydrophila WH-YN directly utilizes industrial hot wastewater at 50~70℃ to produce polyhydroxy fatty acid esters without the need for temperature regulation or nutrient supplementation.

[0017] Furthermore, under industrial hot wastewater conditions at 50°C, the proportion of strain PHA in the dry weight of *Thermomonas hydrophila* WH-YN was 48.75%; under industrial hot wastewater conditions at 70°C, the proportion of strain PHA in the dry weight of *Thermomonas hydrophila* WH-YN was 43.65%, the dry weight of the cells was 7.0 g / L, and the PHA yield was 3.2 g / L.

[0018] Compared with the prior art, the technical solution provided by the present invention has at least the following advantages: This invention provides a highly efficient thermophilic Bacillus strain for PHA production, along with its screening and application methods. This strain was screened from high-temperature hot spring sediments and, after morphological, physiological, and molecular biological identification, confirmed its advantages in high-temperature growth (stable growth at 50-70℃), broad-spectrum carbon source utilization, and adaptability to industrial hot wastewater. Furthermore, through optimized screening and fermentation processes, it achieves highly efficient PHA synthesis, effectively overcoming existing technological limitations. This provides low-cost, highly stable strain resources and technical support for the industrial production of PHA, demonstrating significant industrial application value and promising prospects for wider application. Attached Figure Description

[0019] Figure 1 illustrates the sampling and screening process of the strains in an embodiment of the present invention; Figure 2 is a flowchart of the strain screening process in an embodiment of the present invention; Figure 3 shows the growth curves of the strain of the present invention in different culture media; Figure 4 shows the growth curves of the strain of the present invention at different inoculum amounts in different culture media; Figure 5 shows the morphological observation of the strain of the present invention; A: Transmission electron microscope (TEM) image; B: Transmission optical microscope image; C: Nile blue stained fluorescence image; Figure 6 shows the yield of PHA strain of the present invention; Figure 7 shows the quantitative detection results of PHA production by the strain of the present invention using different carbon sources; Figure 8 shows the quantitative detection results of PHA production by the strain of the present invention in industrial hot wastewater.

[0020] Deposit description The thermomonas hydrophila of this invention is classified and named as *Thermomonas hydrophila* WH-YN. Caldimonas hydrothermalis It is deposited at the Guangdong Provincial Center for Microbial Culture Collection, located at 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, Guangdong Academy of Sciences, with accession number GDMCC No. 68346, deposited on May 26, 2026. Detailed Implementation

[0021] To enable those skilled in the art to better understand the present invention, the embodiments of the present invention will be described below with reference to the accompanying drawings in the embodiments of the present invention. The embodiments described are only some of the embodiments of the present invention, and not all of them, and are not intended to limit the present invention.

[0022] This invention proposes a highly efficient PHA-producing strain of Thermomonas hydrophila (THH). Caldimonas hydrothermalis The strain is WH-YN, and its screening, validation, and application methods are described in the following flowchart. Figure 1 , Figure 2 As shown, the core steps include: I. Strain Screening and Identification This step aims to screen for target strain WH-YN, which possesses thermophilic properties and PHA synthesis capabilities, from high-temperature hot spring environments.

[0023] Sampling: Sediments from the Lotus High-Temperature Hot Spring (water temperature 70~75℃, pH 7.0~7.5) in Tengchong City, Yunnan Province were selected as the sample source. 10g of sediment samples were collected using sterile sampling tubes and transported at low temperature in an ice box to avoid damage to the activity of thermophilic microorganisms in the samples.

[0024] Enrichment culture: Add 90 mL of sterile physiological saline to the sediment sample and shake for 30 min to prepare a bacterial suspension; take 10 mL of the bacterial suspension and inoculate it into 100 mL of enrichment medium (formula: glucose 20 g / L, peptone 5 g / L, yeast extract 3 g / L, NaCl 5 g / L, K2HPO4). 3H2O 2g / L, KH2PO41g / L, MgSO4 (7H2O 0.5g / L, pH 7.0) was placed in a constant temperature shaking incubator at 60℃ and 180rpm for 48h to enrich thermophilic microorganisms.

[0025] Isolation and purification: The enriched bacterial culture was serially diluted (10⁻⁶ ppm). -4 ~10 -6 Take 0.1 mL of the diluted solution and spread it on TSB, YSG and R2A agar plates respectively. Incubate at 60℃ for 24 h. Pick single colonies with different morphologies and streak them three times on the corresponding medium to obtain pure strains.

[0026] Initial screening (PHA qualitative detection): The pure strain was inoculated into a nitrogen-limiting medium (formulation: glucose 20 g / L, (NH4)2SO4 0.5 g / L, K2HPO4). 3H2O 3g / L, KH2PO41.5g / L, MgSO4 7H2O 0.5g / L, CaCl2 Incubate with 0.1 g / L Nile blue 2H2O (pH 7.0) at 60℃ and 180 rpm for 48–72 h; qualitative screening is performed using Nile blue fluorescence staining: take a smear of bacterial culture, add 1–2 mL of staining solution, incubate for 30 min, and observe under a fluorescence microscope (365 nm excitation light). Strains showing bright red fluorescent granules inside the cells are potential PHA-producing strains (typical results can be found in [link to relevant documentation]). Figure 5 (C in the middle).

[0027] Secondary screening (PHA quantitative detection): The strains obtained from the primary screening were inoculated into the secondary screening medium (the same nitrogen-limiting medium as the primary screening medium) and cultured at 60℃ and 180rpm for 70 h (12 h, 36 h and 70 h were selected); the PHA yield was determined by GC-MS.

[0028] Identification of strains of Thermomonas hydrophila WH-YN: Morphological identification: Strains in the logarithmic growth phase were observed under optical microscopy and TEM (procedure: bacterial fixation → gradient dehydration → negative staining → electron microscopy imaging). The morphology, size, spore characteristics, and intracellular structure of the strains were recorded (typical results are shown in...). Figure 5 A in Figure 5 (B in the middle) Molecular biological identification: Genomic DNA was extracted from the strain, and the 16S rRNA gene was amplified (primers: 27F / 1492R), the sequence of which is shown in SEQ ID NO.1: GTCGAACGGCAGCGGGTCCTTCGGGATGCCGGCGAGTGGCGAACGGGTGA GTAATGCATCGGAACGTACCCAGTCGTGGGGGATAACTACTCGAAAGAGT AGCTAATACCGCATACGACCTGAGGGTGAAAGCGGGGGACCGCAAGGCCT CGCGCGATTGGAGCGGCCGATGTCGGATTAGCTAGTTGGTGGGGTAAAGG CTTACCAAGGCGACGATCCGTAGCTGGTCTGAGAGGACGACCAGCCACAC TGGGACTGAGACACGGCCCAGACTCCTACGGGAGGCAGCAGTGGGGAATT TTGGACAATGGGCGCAAGCCTGATCCAGCCATGCCGCGTGCGGGAAGAAG GCCTTCGGGTTGTAAACCGCTTTTGTCAGGGAAGAAAAGCTCTGGGCTAA TACCTCGGAGTGATGACGGTACCTGAAGAATAAGCACCGGCTAACTACGT GCCAGCAGCCGCGGTAATACGTAGGGTGCGAGCGTTAATCGGAATTACTG GGCGTAAAGCGTGCGCAGGCGGTTGTGCAAGACAGATGTGAAATCCCCGG GCTTAACCTGGGAACTGCATTTGTGACTGCACGGCTAGAGTGTGGCAGAG GGGGATGGAATTCCGCGTGTAGCAGTGAAATGCGTAGATATGCGGAGGAA CACCGATGGCGAAGGCAATCCCCTGGGCCTGCACTGACGCTCATGCACGA AAGCGTGGGGAGCAAACAGGATTAGATACCCTGGTAGTCCACGCCCTAAA CGATGTCAACTGGTTGTTGGGGATTCATTTCCTCAGTAACGAAGCTAACG CGTGAAGTTGACCGCCTGGGGAGTACGGCCGCAAGGTTGAAACTCAAAGG AATTGACGGGGACCCGCACAAGCGGTGGATGATGTGGTTTAATTCGATGC AACGCGAAAAACCTTACCTACCCTTGACATGCCAGGAACCCTGCAGAGAT GTGGGGGTGCTCGAAAGAGAGCCTGGACACAGGTGCTGCATGGCCGTCGT CAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCT TGCCATTAGTTGCTACGAAAGGGCACTCTAATGGGACTGCCGGTGACAAA CCGGAGGAAGGTGGGGATGACGTCAGGTCCTCATGGCCCTTATGGGTAGG GCTACACACGTCATACAATGGCCGGTACAGAGGGCTGCCAACCCGCGAGG GGGAGCCAATCCCAGAAAACCGGTCGTAGTCCGGATCGCAGTCTGCAACT CGACTGCGTGAAGTCGGAATCGCTAGTAATCGCGGATCAGCATGTCGCGG TGAATACGTTCCCGGGTCTTGTACACACCGCCCGTCACACCATGGGAGCG GGTTCTGCCAGAAGTGGGTAGCCTAACCGCAAG.

[0029] After sequencing, the sequences were uploaded to GenBank, and their taxonomic status was analyzed by BLAST alignment. Final identification results: This strain is a Gram-positive bacterium, short rod-shaped (0.5~0.8μm × 2.0~3.5μm), with elliptical spores, and... Caldimonas hydrothermalis With a homology of 99.2%, it was finally identified as a hot spring thermomonas strain.

[0030] II. Verification of strain growth characteristics This step aims to clarify the growth capacity of the strain under different conditions, providing a basis for subsequent fermentation applications.

[0031] Multi-culture medium adaptability test: to identify the target strain Caldimonas hydrothermalis The compatibility of different conventional culture media was investigated, and the optimal culture system was screened to provide support for subsequent PHA synthesis and related physiological metabolism experiments, thereby activating PHA to the logarithmic phase. Caldimonas hydrothermalis The bacterial strains were inoculated into four commonly used bacterial culture media: TSB, YSG, LB broth, and R2A, with an inoculation amount of 2% (v / v) to ensure consistent initial bacterial count. After inoculation, all media were placed in a constant temperature shaking incubator at 60℃ and 180 rpm for shaking culture, maintaining uniform and stable culture conditions throughout the process to avoid interference from environmental factors on the growth of the strains.

[0032] During the cultivation process, samples were collected at selected time points (0, 4, 8, 12, 24, and 36 hours). Three replicates were taken from each culture medium each time, and the OD of the bacterial suspension was measured using a UV spectrophotometer at a wavelength of 600 nm. 600 Value (OD) 600The absorbance value is positively correlated with the bacterial concentration, indirectly reflecting the growth and reproduction of the strain. A blank control group without inoculation of the corresponding culture medium was also set up to subtract the absorbance value of the culture medium itself, ensuring the accuracy and reliability of the measurement data. Based on the measured OD... 600 Values, with incubation time as the x-axis and OD... 600 Plot the average value on the y-axis. Caldimonas hydrothermalis Growth curves in four different culture media (typical results are shown in...) Figure 3 ).

[0033] Growth curve and OD 600 The results of the dynamic changes in values ​​show that Caldimonas hydrothermalis The strain grew well in all four culture media: TSB, YSG, LB, and R2A, without any growth inhibition. Only slight differences were observed in growth rate, duration of the logarithmic phase, and peak OD value during the stationary phase. Among these, the strain showed the fastest growth initiation and the shortest lag phase (approximately 2-4 hours) in YSG medium, with the highest OD value after entering the logarithmic phase. 600 The value rises rapidly; the stationary phase peak is higher in YSG and TSB media, indicating that the carbon and nitrogen source combination in these media is more conducive to the accumulation of bacterial biomass; in LB and R2A media, the growth trend of the strain is stable, the logarithmic phase lasts longer, and the OD value in the stationary phase is higher. 600 The values ​​remained at a high level, indicating that the strain tolerated both culture media well. Overall, the target strain... It exhibits strong adaptability to all four test media, and the appropriate media can be selected for culture according to the needs of subsequent experiments.

[0034] Different inoculum amounts were tested: Based on the previous culture medium adaptability test results, the target strain grew well in four media: TSB, YSG, LB, and R2A. Therefore, these four media were selected to simultaneously conduct tests with different inoculum amounts to explore the effect of inoculum amount on growth. Caldimonas hydrothermalis The effects of different culture media on growth rate, biomass accumulation, and growth cycle were investigated to determine the optimal inoculum size parameters suitable for different culture media, providing comprehensive support for subsequent experiments. Three inoculum size gradients were set up: 5%, 10%, and 15% (v / v). Each culture medium corresponded to three inoculum size gradients, and each group had three parallel replicates. A blank control without inoculation of the corresponding culture medium was also included to eliminate interference from the absorbance of the culture medium itself and microbial contamination on the experimental results.

[0035] The inoculation procedure strictly followed aseptic techniques, and the activated cells were brought to the logarithmic phase. Caldimonas hydrothermalis CaldimonasThe bacterial suspension was inoculated into Erlenmeyer flasks containing equal volumes of four types of culture media: TSB, YSG, LB, and R2A, according to the corresponding inoculation amounts. After inoculation, all experimental groups were placed under the same culture conditions (60℃, 180rpm constant temperature shaking) as the culture medium adaptability test to ensure the uniformity of the culture environment and reduce the interference of irrelevant variables on the growth of strains in different culture media.

[0036] During the cultivation process, the sampling time and detection methods remained consistent with those described above. Sampling was conducted at selected time points (0, 4, 8, 12, 24, and 36 hours) to measure the OD values ​​of the bacterial suspensions under four different culture media and three inoculum size gradients. 600 The absorbance values ​​of each group of parallel samples were recorded and the average value was calculated. After subtracting the absorbance value of the corresponding blank control group, the values ​​were plotted with the incubation time as the x-axis and the OD value as the y-axis. 600 Plot the average values ​​on the ordinate for different inoculum amounts in the four culture media. hydrothermalis The growth curve (which can be labeled accordingly) Caldimonas hydrothermalis The results (presented in different culture media) were used to visually analyze the differences in the effect of inoculum size on the growth of the strain in different culture media. Among them, a 15% inoculum size showed the best performance in TSB medium, exhibiting the shortest lag phase, the fastest logarithmic growth rate, and the highest biomass accumulation (OD) during the stationary phase. 600 The peak value was significantly higher than that of the 5% and 10% inoculum groups in the same medium, indicating that the 15% inoculum is suitable for TSB medium. Figure 4 The optimal inoculum size for growth.

[0037] During the experiment, it is important to ensure precise control of the inoculum size and maintain consistent inoculation procedures for each culture medium to avoid discrepancies between the actual inoculum size and the set value caused by residual bacterial solution. During shaking culture, ensure consistent aeration rates in all Erlenmeyer flasks to prevent insufficient aeration from affecting the aerobic growth of the strains. Ensure uniform culture conditions for all four culture media and three inoculum size gradients. Strict aseptic technique must be maintained during sampling to avoid cross-contamination between different culture media and inoculum size groups, ensuring the authenticity and reproducibility of each set of measurement data. This will provide reliable experimental evidence for subsequent optimization of inoculum sizes under different culture media and efficient culture of the strains.

[0038] III. Qualitative Testing of PHA To verify the present invention Caldimonas hydrothermalis The strain's PHA synthesis capacity was assessed, and qualitative PHA testing was conducted simultaneously. Morphological observation of the strain was combined with the testing process to visually analyze PHA accumulation within the strain. Morphological images of the strain of this invention were used (A: TEM image; B: optical microscope image; C: Nile blue stained fluorescence image). Among them, the TEM image (… Caldimonas hydrothermalisA) in the image can visually present the internal structure of the strain's cells, clearly showing the distribution, morphology, and size of PHA particles within the cells. By observing the presence of characteristic PHA particles (usually round or oval particles with high electron density) within the cells, a preliminary qualitative assessment of PHA synthesis can be achieved; optical microscope images ( Figure 5 (B) allows for clear observation of the overall morphology, size, and aggregation state of the strain, providing a basis for preliminary determination of the location of PHA accumulation; Nile blue stained fluorescence images ( Figure 5 C) can further corroborate the existence of PHA, because Nile blue dye can specifically bind to PHA in cells and emit characteristic fluorescence under excitation light. By measuring the fluorescence intensity and distribution, the accumulation and distribution characteristics of PHA in the strain can be preliminarily determined. Combined with the observation results of the first two microscopic images, the qualitative verification of the PHA synthesis ability of the strain of this invention is completed, providing an intuitive morphological basis for subsequent quantitative testing of PHA and optimization of the synthesis process.

[0039] IV. PHA Yield Measurement and Carbon Source Optimization This step aims to optimize Figure 5 The carbon source conditions for PHA synthesis by the strain were determined, its optimal carbon source for PHA production was identified, and the strain's ability to utilize a wide range of carbon sources was verified, providing a theoretical basis and data support for the efficient synthesis of PHA from complex substrates (such as lignocellulose hydrolysate).

[0040] (a) PHA production test To investigate the effect of different culture times on the accumulation of PHA by the strain, samples were taken and yields were measured at three key culture time points: 12 h, 36 h, and 72 h. The results showed that throughout the culture cycle, PHA production generally increased with increasing culture time. The strain experienced rapid growth and reproduction in the early stage, gradually initiated intracellular polymer synthesis in the middle stage, and entered a stable accumulation phase in the later stage. The PHA production peaked at 70 h, reaching a maximum of 27.89%, indicating that this time point was more conducive to the strain's efficient utilization of carbon sources and maximized PHA synthesis. Caldimonas hydrothermalis ).

[0041] (II) PHA production test of different pure carbon sources A nitrogen-limited synthetic medium was used as the basal medium, and the nitrogen concentration in the system was strictly controlled to induce a large accumulation of PHA. The carbon source was the only variable. Four different types of pure carbon sources were selected for the experiment: monosaccharides (mannose, fructose), disaccharides (maltose), and polysaccharides (inulin). Each carbon source was added to the basal medium separately, and the final carbon source concentration was uniformly adjusted to 20 g / L to ensure a consistent carbon source supply level.

[0042] The inoculation process uses the culture parameters optimized and determined above, and the cells activated to the logarithmic phase are inoculated.Figure 6 Caldimonas The bacterial culture was inoculated at a rate of 15% (v / v) into nitrogen-limiting media containing different pure carbon sources. Three biological replicates were set up for each group, and an uninoculated blank medium was set up as a control. After inoculation, all experimental groups were placed in a constant temperature shaking incubator at 60℃ and 180 rpm and cultured in the dark for 24 h to 36 h.

[0043] After cultivation, fermentation broth from all experimental groups was collected, centrifuged, and washed to remove supernatant and residual culture medium components, yielding cell precipitate. The cells were then freeze-dried to constant weight, precisely weighed, and the cell dry weight (CDW) was calculated. GC-MS was used for qualitative and quantitative analysis of PHA in the freeze-dried cells. After derivatization, the chromatographic retention time and mass spectrometric characteristic peaks were compared with those of PHA standards to determine the PHA composition. PHA yield and content (the percentage of PHA in cell dry weight) were calculated using peak area normalization, comprehensively evaluating the impact of different pure carbon sources on the strain's ability to synthesize PHA. PHA production tests using different pure carbon sources: Based on a nitrogen-limited medium, galactose, fructose, maltose, and inulin were used as carbon sources (all with a carbon concentration of 20 g / L). After inoculation, the strains were cultured at 60°C and 180 rpm for 70 h. GC-MS was used to determine PHA yield and cell dry weight. The results showed that all carbon sources supported PHA synthesis, with inulin yielding the highest yield (89.00%) (typical results are shown in...). hydrothermalis ).

[0044] V. Industrial Hot Wastewater Adaptability Test This step aims to verify the application potential of the strain in industrial settings and evaluate its ability to produce PHA from industrial hot wastewater.

[0045] Sample preparation: Industrial hot wastewater discharged from a municipal solid waste processing plant (temperatures of 50℃ and 70℃, pH 7.0, carbon content of approximately 15g / L, without any added nutrients) was collected, filtered through a 0.22μm filter membrane for sterilization, and used as the fermentation substrate.

[0046] Fermentation test: The seed culture of the strain (OD) was used to test the fermentation solution. 600 =1.2) Inoculate with industrial hot wastewater at a rate of 5% (v / v) and culture at 65℃ and 180rpm for 70 h with shaking; after fermentation, measure cell dry weight and PHA yield.

[0047] Results Analysis: The strain could grow stably in industrial hot wastewater at both 50℃ and 70℃, demonstrating good high-temperature tolerance and substrate utilization ability. At 50℃, PHA accounted for 48.75% of the cell dry weight; at 70℃, the PHA proportion was 43.65%, corresponding to a stationary phase cell dry weight of 7.0 g / L and a PHA yield of 3.2 g / L (typical results can be found in...).Figure 7 The above results indicate that this strain can be directly grown and synthesized using industrial hot wastewater as raw material without the need for additional carbon sources or other nutrients, which can significantly reduce the raw material cost of industrial PHA production and has good potential for industrial application.

[0048] VI. Summary of Verification Results Morphological and qualitative verification: The morphological characteristics of the strain were confirmed by optical microscopy and TEM observation, and Nile blue staining proved that it had the ability to synthesize PHA. Figure 8 ); Growth characteristic verification: The strain can grow in various culture media and at high temperatures of 60~70℃, and its growth kinetic parameters are suitable for industrial fermentation. Figure 5 ); PHA production capability verification: PHA can be produced efficiently from pure carbon sources, inexpensive carbon sources, and industrial hot wastewater, with yields significantly superior to existing thermophilic strains. Figure 3 and Figure 6 ); Industrial compatibility verification: PHA can be directly produced from industrial hot wastewater at 50~70℃ without temperature regulation or nutrient supplementation, demonstrating strong potential for industrial applications. Figure 7 ).

[0049] VII. Conclusion This invention revolves around Figure 8 A series of experiments were conducted to optimize the culture conditions of the strain, verify its PHA synthesis ability, and test its carbon source compatibility. Based on the combined results of all experiments, the following core conclusions were drawn: First, Caldimonas hydrothermalis Caldimonas The strain exhibits strong culture medium adaptability, growing well in four commonly used bacterial media: TSB, YSG, LB, and R2A, with only slight differences in growth rate and biomass accumulation. The appropriate culture system can be flexibly selected based on different experimental needs. Secondly, the inoculum size significantly affects the growth of the strain in the four media. A 15% (v / v) inoculum size in TSB medium is the optimal parameter, exhibiting the shortest lag phase, the fastest logarithmic growth rate, and the highest biomass accumulation in the stationary phase, providing reliable parameter support for subsequent efficient culture of the strain. Thirdly, PHA qualitative testing confirms… hydrothermalis The strain possesses the ability to synthesize PHA. Observation using optical microscopy, TEM, and Nile blue-stained fluorescence microscopy clearly revealed the presence of characteristic PHA particles within the strain's cells, clarifying the distribution and morphological characteristics of PHA within the cells and laying a morphological foundation for quantitative PHA research. Fourth, PHA production tests using different pure carbon sources showed... Caldimonas hydrothermalisThe strain exhibits broad-spectrum carbon source utilization capabilities, synthesizing PHA from monosaccharides, disaccharides, and polysaccharides. Different carbon sources significantly affect the strain's PHA yield and cell dry weight (the optimal carbon source and corresponding yield are based on GC-MS measured data). Fifth, the PHA production test results from industrial thermal wastewater show that this strain possesses excellent adaptability to high-temperature wastewater and substrate utilization potential. It can grow stably in industrial thermal wastewater at two different temperatures, 50℃ and 70℃, without the need for additional carbon or nitrogen sources; it can efficiently synthesize PHA using only the substrates present in the wastewater itself.

[0050] In conclusion, Caldimonas hydrothermalis Caldimonas hydrothermalis The strain exhibits advantages such as thermophilicity, strong culture medium adaptability, high inoculum size tolerance, stable PHA synthesis ability, and a wide range of carbon source utilization, especially suitable for culture conditions with a 15% inoculum size in TSB medium. More importantly, this strain possesses excellent high-temperature tolerance and adaptability to industrial wastewater, allowing it to directly utilize industrial hot wastewater as a substrate for growth and PHA production without the need for additional nutrients. It can stably grow and efficiently synthesize PHA in industrial wastewater at 50℃~70℃, significantly reducing raw material costs. This not only makes it well-suited for the industrial production of polyhydroxyalkanoates but also enables the resource utilization of industrial wastewater. These characteristics provide solid experimental data and theoretical support for subsequent optimization of the PHA synthesis process of this strain and the development of low-cost PHA production technologies, and also lay the foundation for further promotion and application of this strain in the fields of industrial wastewater resource treatment and PHA biomanufacturing.

Claims

1. A thermophilic bacterial strain with a high efficiency in producing polyhydroxyalkanoates, characterized in that, The thermophilic strain with high efficiency in producing polyhydroxy fatty acid esters is *Thermomonas hydrophila* (…). Caldimonas hydrothermalis The strain was identified as WH-YN, with accession number GDMCC No. 68346.

2. The thermophilic strain with high efficiency in producing polyhydroxy fatty acid esters according to claim 1, characterized in that, The nucleotide sequence of the thermomonas hydrophila WH-YN is shown in SEQ ID NO.

1.

3. The thermophilic strain with high efficiency in producing polyhydroxy fatty acid esters according to claim 1, characterized in that, The thermomonas hydrophila WH-YN is a Gram-positive bacterium, which is short rod-shaped, with a size of 0.5~0.8μm×2.0~3.5μm, and includes elliptical spores.

4. The thermophilic strain with high efficiency in producing polyhydroxy fatty acid esters according to claim 1, characterized in that, The thermomonas hydrophila WH-YN can grow stably at temperatures of 50~70℃ and can synthesize polyhydroxy fatty acid esters using pure carbon sources or inexpensive carbon sources as raw materials.

5. A method for screening thermophilic strains with high efficiency in producing polyhydroxyalkanoates according to any one of claims 1 to 4, characterized in that, The screening method includes: The pure bacterial strain obtained from the sample was inoculated into a nitrogen-limiting medium and cultured at 60℃ and 180rpm for 48-72h to obtain the bacterial solution. The Nile blue fluorescence staining method was used for qualitative screening: a smear of bacterial culture was prepared, 1-2 mL of staining solution was added and incubated for 30 min, and then observed under a fluorescence microscope. Strains that showed bright red fluorescent particles in their cells were potential PHA-producing strains, and the strains obtained in the initial screening were obtained. The nitrogen-limiting culture medium is formulated as follows: glucose 20 g / L, (NH4)2SO4 0.5 g / L, K2HPO4 3H2O 3g / L, KH2PO4 1.5g / L, MgSO4 7H2O 0.5g / L, CaCl2 2H₂O 0.1 g / L, pH 7.0; the excitation light of the fluorescence microscope is 365 nm; The strains obtained from the initial screening were inoculated into the nitrogen-limiting medium and cultured at 60°C and 180 rpm for 70 h to obtain the thermomonas hydrophila WH-YN. The yield of PHA was determined by gas chromatography-mass spectrometry.

6. A method for culturing a thermophilic strain with high efficiency in producing polyhydroxyalkanoates according to any one of claims 1 to 4, characterized in that, The culture method includes: inoculating the thermomonas hydrophila WH-YN into tryptone soybean broth, yeast extract-starch-glucose, LB broth, and R2A bacterial culture medium, respectively; after inoculation, placing all culture media in a constant temperature shaking incubator at 60℃ and 180 rpm for shaking culture.

7. The method for cultivating a thermophilic strain with high efficiency in producing polyhydroxyalkanoates according to claim 6, characterized in that, The culture conditions for the thermomonas hydrophila WH-YN were TSB medium and an inoculum size of 15% (v / v).

8. The application of a thermophilic strain with high efficiency in producing polyhydroxyalkanoates according to any one of claims 1 to 3 in the field of industrial production of polyhydroxyalkanoates and resource utilization of industrial wastewater.

9. The application according to claim 8, characterized in that, The thermomonas hydrophila described above directly utilizes industrial hot wastewater at 50~70℃ to produce polyhydroxy fatty acid esters without the need for temperature regulation or nutrient supplementation.

10. The application according to claim 9, characterized in that, Thermomonas hydrophila WH-YN strain, under industrial hot wastewater conditions at 50°C, had a PHA content of 48.75% of the cell dry weight. Under industrial hot wastewater conditions at 70℃, the strain of Thermomonas hydrophila WH-YN had a PHA content of 43.65% of the cell dry weight, a cell dry weight of 7.0 g / L, and a PHA yield of 3.2 g / L.

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