Delftia tsuruhatanensis and application thereof
By using Delft bacterium WC2 and its compound bacterial agent, the problem of malodorous gas treatment in pig farming has been solved, achieving efficient conversion of sulfides into odorless substances, thus improving the health of pigs and the quality of the environment.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- CHENGDU INSTITUTE OF BIOLOGY CHINESE ACADEMY OF SCIENCES
- Filing Date
- 2023-02-20
- Publication Date
- 2026-06-05
AI Technical Summary
Malodorous gases in pig farming have a serious impact on the health of pigs and the environment, and existing technologies are insufficient to treat them efficiently and at low cost.
The deodorizing compound microbial agent is formed by using Delftia tsuruhatensis WC2 and its compound microbial agent, which oxidizes sulfides into elemental sulfur and further oxidizes them into sulfates, and combines them with plant extracts to inhibit the production of odor substances.
It significantly improved the conversion efficiency of malodorous gases, reduced the emission of malodorous substances, and improved the environmental quality of pig farming.
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Abstract
Description
Technical Field
[0001] This invention belongs to the field of microbiology, specifically relating to a strain of Delftobacterium tsurugi and its applications. Background Technology
[0002] While intensive and large-scale pig farming has improved production efficiency, it has also brought about serious environmental pollution problems.
[0003] Foul-smelling gases are composed of various harmful volatile compounds, with feces and urine being the main sources of odor in pig farming. High concentrations of foul-smelling gases in pig pens are not only harmful to the health of pigs, easily inhibiting their growth and reducing their production performance, but also have a serious impact on the surrounding atmospheric environment. Long-term inhalation of foul-smelling gases can cause serious harm to people's physical and mental health.
[0004] Therefore, developing efficient and low-cost odor reduction technologies and improving the odor treatment efficiency of pig farms is of great practical significance for promoting the green and sustainable development of the pig farming industry. Summary of the Invention
[0005] The purpose of this invention is to provide a strain of Delftobacterium tsurugi and its applications.
[0006] To achieve the above-mentioned objectives, the technical solution adopted in this invention is as follows: a strain of Delftia tsuruhatensis WC2 was deposited at the Guangdong Provincial Center for Microbial Culture Collection on December 12, 2022, with the accession number GDMCC NO: 62522.
[0007] Accordingly, the application of Delftobacterium tsurugi in deodorization.
[0008] Accordingly, the application of the Delftobacterium tsurugi in desulfurization and / or denitrification.
[0009] Accordingly, the bacterial agent contains the aforementioned Delftus tsurugi.
[0010] Preferably, the bacterial agent also includes denitrifying paracocci.
[0011] Preferably, the microbial agent further includes plant components, which are components that have deodorizing effects and do not inhibit the growth of microorganisms in the microbial agent.
[0012] Preferably, the plant ingredient is derived from one or more of green tea, ginger, or yucca.
[0013] Accordingly, the application of the bacterial agent in deodorization.
[0014] Accordingly, the application of the microbial agent in desulfurization and / or denitrification.
[0015] Accordingly, the method of applying the microbial agent in deodorization, desulfurization or denitrification involves preparing the microbial agent into a solution and spraying it onto the object or area to be deodorized, or mixing it with the substance to be deodorized.
[0016] This invention has the following beneficial effects: For the first time, this invention domesticated and screened a strain of *Delftibacterium tsurugi* with heterotrophic desulfurization effects, which can rapidly oxidize sulfides to elemental sulfur, and then further oxidize it to sulfate. Therefore, this microorganism has a good deodorization effect on malodorous gases and substances mainly composed of sulfides (hydrogen sulfide). Based on this *Delftibacterium tsurugi*, this invention further provides a microbial composition and deodorizing compound bacterial agent with better deodorization effects. The various microorganisms in the composition work synergistically, significantly improving the desulfurization and denitrification effects, and can convert malodorous substances into odorless substances before the malodorous substances in feces volatilize. The compound bacterial agent uses plant extracts to enhance microbial deodorization: active substances such as yucca saponins can inhibit the urease activity of odor-producing microorganisms, thereby inhibiting the production of ammonia; green tea polyphenols and turmeric can inhibit the growth and reproduction of pathogens, thereby inhibiting their metabolism of residual nutrients in feces. Attached Figure Description
[0017] Figure 1 This is a schematic diagram illustrating the changes in desulfurization during the domestication of microorganisms;
[0018] Figure 2 A schematic diagram illustrating the changes in nitrogen removal by denitrifying microorganisms;
[0019] Figure 3 This is a schematic diagram illustrating the changes in desulfurization by desulfurizing microorganisms;
[0020] Figure 4 A schematic diagram showing the growth of Delftobacterium tsurugi WC2;
[0021] Figure 5 This is a schematic diagram showing the growth of Paracoccus denitrifyingis WN8.
[0022] Figure 6 This is a schematic diagram illustrating the changing trend of WC2 odor gas removal rate;
[0023] Figure 7 This is a schematic diagram showing the trend of WN8 odor gas removal rate. Detailed Implementation
[0024] This invention provides a novel *Delftia tsuruhatensis* WC2 strain, which was deposited at the Guangdong Provincial Microbial Culture Collection Center on December 12, 2022, with accession number GDMCC NO: 62522. Its 16S rDNA sequence is shown in SEQ ID NO: 1.
[0025] The described Delftobacterium tsurugi exhibits high desulfurization efficiency, rapidly oxidizing sulfides to elemental sulfur, and further oxidizing it to sulfate. Therefore, it demonstrates excellent deodorization effects on malodorous gases and substances primarily composed of sulfides (hydrogen sulfide). Similar effects or mechanisms have not been observed in other Delftobacterium tsurugi strains.
[0026] Based on the aforementioned *Delftia tsuruhata*, the present invention further provides a composite deodorizing microbial composition. The microbial composition includes *Delftia tsuruhata* and nitrifying bacteria. The nitrifying bacteria are preferably *Paracoccus denitrifyingus*. In the microbial composition, the ratio of *Delftia tsuruhata* to nitrifying bacteria is 1:1 to 2:3, with a viable count ≥10-1. 9 CFU / mL.
[0027] A more preferred method is to add a plant extract with deodorizing effect and no inhibitory effect on microbial growth to the microbial composition to prepare a compound microbial agent before use. The plant extract is preferably any one or a mixture of several of green tea extract, ginger extract, or yucca extract. The concentration of the plant extract is 5% (w / w). The microbial composition is diluted with water to 5% (v / v) and then mixed with the plant extract at a volume ratio of (1-2):(1-2). In use, the compound microbial agent is sprayed onto the object or area to be deodorized, or mixed with the substance to be deodorized.
[0028] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Unless otherwise specified, the technical means used in the embodiments are conventional means well known to those skilled in the art. All obtained data are average values obtained after at least three repetitions, and each repetition yields valid data.
[0029] Example 1: Domestication, Screening and Identification of Microorganisms
[0030] 1. Acclimation. Pig manure samples were collected from the pig farm and placed in a laboratory acclimation tank to acclimate heterotrophic nitrifying bacteria and heterotrophic sulfur oxidizing bacteria. The acclimation culture conditions were as follows: at room temperature, a small air pump was connected to a 20L glass tank, and aeration was carried out continuously at a rate of 10L / min.
[0031] Acclimation medium (g / L): Na2S·9H2O 2.58 (per gram of drug S) 2- (Content approximately 0.15g), anhydrous glucose 2, NH4Cl 0.6g, KH2PO4 1, K2HPO4 1, MgCl2·6H2O 0.4, NaHCO3 0.4, trace element solution 10mL / L, pH=7.0.
[0032] Trace element solution (g / L): Disodium EDTA 50, ZnSO4·7H2O 22, CaCl2 5.54, MnCl2·4H2O 5.06, FeSO4·7H2O 4.99, (NH4)6MoO 24 ·4H2O 1.10, CaSO4·5H2O 1.57, CoCl2·6H2O 1.61, pH=7.0.
[0033] After two months of continuous acclimatization, samples were taken periodically in the later stages (every 12 hours for the first 5 days, and every 6 hours for the next 5 days) to determine the sulfur ion and ammonia nitrogen content in the acclimatization solution. After each stage of sulfur ion depletion (when the sulfur ion concentration reached 0), the substrate was allowed to settle, the water was changed, and new acclimatization medium was added. Simultaneously, the sulfur and nitrogen loads were gradually increased by 1, 1.5, 2, 2.5, and 3 times, progressively increasing the content of Na₂S·9H₂O and NH₄Cl in the medium, while increasing the anhydrous glucose content to maintain a constant C / N ratio of 15. Results are as follows: Figure 1 As shown, at the end of the acclimatization period, after the sulfur ion removal rate reached more than 85%, the acclimatization was stopped, and the acclimatization culture medium was obtained.
[0034] (2) Strain isolation. Take the acclimatization culture medium and dilute it with sterile water to a concentration of 10:1. -4 10 -5 10 -6 10 -7 Four different gradient dilutions were spread onto selective media for heterotrophic nitrifying bacteria and selective media for heterotrophic sulfur oxidizing bacteria, respectively. After incubation at 30°C for 48 hours, streak separation was performed, and the samples were purified repeatedly.
[0035] Selective culture medium for heterotrophic nitrifying bacteria (g / L): glucose 5, NaCl 1, (NH4)2SO4 0.6, K2HPO4 1.0, MgSO4·7H2O 0.5, FeSO4·7H2O 0.05, agar 20, pH=7.0, sterilized at 115℃ for 30 min.
[0036] Selective culture medium for heterotrophic sulfur oxidizing bacteria (g / L); peptone 10, beef extract 2, sodium citrate buffer 1000 mL, agar 20, pH = 7.0, sterilized at 121℃ for 20 min. Before use, add sodium sulfide solution through a sterile filter membrane on a sterile operating table to adjust the sulfur content in the culture medium. 2- The concentration is 0.2 mol / L.
[0037] Eight denitrifying bacteria strains (WN1–WN8) and five desulfurizing bacteria strains (WC1–WC5) were obtained. Each isolated strain underwent denitrification and desulfurization screening, and seed cultures of each strain were prepared using beef extract peptone liquid medium. The seed cultures were inoculated at 5% (v / v) into the corresponding screening medium (denitrifying bacteria into denitrifying bacteria screening medium, and desulfurizing bacteria into desulfurizing bacteria screening medium), and cultured on a constant-temperature shaking incubator (180 rpm, 30℃). A blank control group was also established (inoculated with an equal volume of sterilized beef extract peptone liquid medium). Sulfide ion and ammonia nitrogen concentrations in each group were measured periodically, and the removal rate was calculated based on the blank control.
[0038] Beef extract peptone liquid medium (g / L): beef extract 3, peptone 10, NaCl 5, pH 7.0~7.2.
[0039] Denitrifying bacteria selection medium (g / L): glucose 5, NaCl 1, (NH4)2SO4 0.6, K2HPO4 1.0, MgSO4·7H2O 0.5, FeSO4·7H2O 0.05, pH=7.0, sterilized at 115℃ for 30 min.
[0040] Sulfur-removing bacteria screening medium (g / L); peptone 10, beef extract 2, sodium citrate buffer 1000mL, pH=7.0, sterilized at 121℃ for 20min. Before use, add sodium sulfide solution through a sterile filter membrane on a sterile operating table to reduce the sulfur content in the medium. 2- The concentration is 0.2 mol / L.
[0041] The denitrification and desulfurization effects of each group of microorganisms are as follows: Figure 2 , 3 As shown in the figure, denitrifying strains were screened using a selection medium with an initial ammonia nitrogen concentration of 200 mg / L. Among them, WN8 achieved the highest ammonia nitrogen removal rate of 100% at 24 h. Desulfurizing strains were screened using a selection medium with an initial sulfide ion concentration of 600 mg / L. Among them, WC2 achieved the highest ammonia nitrogen removal rate of 99.45% at 24 h. Therefore, the strains with the best denitrification and desulfurization effects were selected as WN8 and WC2, respectively.
[0042] (3) Strains identification. The target strains WN8 and WC2 were identified based on their morphological and physiological-biochemical characteristics. Genomic DNA was extracted and amplified using the universal primers 27F / 1492R for bacterial 16S rRNA gene amplification. The PCR products were then sent to Shanghai Sangon Biotech Co., Ltd. for sequencing. Finally, their taxonomic status was verified based on the NCBI comparison results.
[0043] WC2 was identified as Delftobacterium tsurugi, and its growth in the culture dish is as follows: Figure 4As shown; WN8 was identified as *Paracoccus denitrificans*, and its growth in the culture dish is as follows. Figure 5 As shown.
[0044] WC2 cells are Gram-negative, spherical, and non-spore-forming; colonies are light pink with regular edges and a relatively smooth and moist surface. WN8 cells are Gram-negative, rod-shaped, and non-spore-forming; colonies are white with regular edges and a relatively smooth and moist surface. The physiological and biochemical characteristics of both are shown in Table 1. In Table 1, "+" represents positive results or the presence of the corresponding characteristic, and "-" represents negative results or the absence of the corresponding characteristic. For example, "+, -" indicates that the microorganism produces acid but not gas when fermenting the corresponding sugar.
[0045] Table 1. Physiological and Biochemical Characteristics of Microorganisms
[0046]
[0047] Example 2: Deodorization effect demonstration
[0048] 1. Demonstration of the deodorizing effect of a single microorganism.
[0049] WC2 was inoculated into beef extract peptone liquid medium and cultured at 30°C for 48 hours to obtain WC2 culture medium; WN8 was inoculated into beef extract peptone liquid medium and cultured at 30°C for 48 hours to obtain WN8 culture medium. The viable count of both cultures reached 102. 9 CFU / mL.
[0050] Fresh pig manure was collected from a pig farm in Pengzhou City. The experimental setup consisted of a 5L sealed container filled with fresh pig manure and water at a mass ratio of 3:7 to simulate the water-soaked state of a manure ditch.
[0051] Two experimental groups and one control group were set up. In each group, 200g of water-soaked feces was placed in the experimental apparatus, spread evenly, and mixed thoroughly. 6mL of WC2 culture medium (experimental group 1), 6mL of WN8 culture medium (experimental group 2), and 6mL of sterile beef extract peptone liquid culture medium (control group) were sprayed onto the respective water-soaked feces. The odor concentration was measured at 9:00 AM daily on days 1, 2, 3, 4, and 5. Each group was divided into three replicates.
[0052] (4) Referring to the control group, the average values were used to calculate the removal rates of ammonia, hydrogen sulfide, total volatile organic compounds, and overall odor concentration, and the deodorization effects of WC2 and WN8 were calculated. The calculation formula is: Odor removal rate of a certain test gas = (Control group data measured on the same day - Test group data measured on the same day) / Control group data measured on the same day × 100%.
[0053] The removal rates of odorous gases in each group are shown in Table 2. The trend of the removal rate of WC2 odorous gases is as follows: Figure 6 As shown, the trend of WN8 odor gas removal rate is as follows: Figure 7 As shown.
[0054] Table 2 Comparison of Odor Gas Removal Rate
[0055]
[0056] The results showed that during the treatment of fresh pig manure with WC2, the hydrogen sulfide removal rate reached 60.53% on the first day, then slowly decreased and remained at around 16%. This demonstrates that WC2 can quickly colonize and function immediately upon addition of manure. In the later stages of treatment, as the amount of hydrogen sulfide produced by anaerobic fermentation gradually decreases, the deodorization effect becomes relatively less significant. The removal rates of ammonia and total odor concentration (OU) remained above 20%. The removal rate of total volatile organic compounds (VOCs) by WC2 was not very stable, possibly because bacterial interactions and changes in the microecological environment can also generate some VOCs, leading to a lower removal rate.
[0057] During the treatment of fresh pig manure, WN8 showed a high and stable ammonia removal rate, reaching a maximum of 52.45% on day 5, indicating that WN8 had formed a dominant community in the pig manure at this time. Since WN8 did not demonstrate a significant advantage over other denitrifying paracocci in odor removal, subsequent experiments focused primarily on WC2.
[0058] 2. Demonstration of the deodorizing effect of the microbial composition.
[0059] WC2 was inoculated into beef extract peptone liquid medium and cultured at 30°C for 48 h to obtain WC2 culture medium; WN8 was inoculated into beef extract peptone liquid medium and cultured at 30°C for 2 days to obtain WN8 culture medium, with a viable count of 10⁻⁶ for both. 9 CFU / mL. Simultaneously, viable bacterial counts of 10⁻⁶ were obtained by culturing using the same method. 9 CFU / mL of Delftobacterium tsurugi 1 culture medium (CCTCC NO: M2017664), Delftobacterium tsurugi 2 culture medium (CFCC 81205), Paracoccus denitrificans 1 culture medium (ATCC13543), and Paracoccus denitrificans 2 culture medium (CGMCC 1.7287).
[0060] Fresh pig manure was collected from Emperor Hank's pig farm. The experimental setup consisted of a 5L sealed container filled with fresh pig manure and water at a mass ratio of 3:7 to simulate the water-filled state of a manure ditch.
[0061] The experimental groups were set up as shown in Table 3. 200g of water-soaked feces was placed in each experimental device, spread evenly, and mixed thoroughly. 6mL of culture medium from each group was sprayed onto the water-soaked feces. The odor gas content was measured at 9:00 AM daily on days 1, 2, 3, 4, and 5. A control group was also set up, using an equal volume of sterile beef extract peptone medium instead of the culture medium. Each group had three replicates. The proportions in Table 3 are volume ratios. The inventors also conducted many other combination experiments (including other ratios of WC2 and WN8, and substitution combinations between different subspecies of the same species or different species of the same genus). Due to space limitations, only a few representative combinations are presented below; other combinations are not listed here.
[0062] Table 3 Comparison of Microbial Compositions in Each Group
[0063] Group Delftus tsurugi denitrifying paracocci Delftobacterium tsuruhata: Denitrifying paracoccus Group 1 WC2 WN8 1:1 Group 2 WC2 WN8 1:2 Group 3 WC2 WN8 2:1 Group 4 WC2 WN8 2:3 Group 5 WC2 WN8 3:2 Group 6 Delftella tsurugi 1 WN8 2:1 Group 7 Delftella tsurugi 2 WN8 2:1 Group 8 WC2 Denitrifying paracoccus 1 2:1 Group 9 WC2 Denitrifying paracoccus 2 2:1 Group 10 Delftella tsurugi 1 WN8 2:3 Group 11 Delftella tsurugi 2 WN8 2:3 Group 12 WC2 Denitrifying paracoccus 1 2:3 Group 13 WC2 Denitrifying paracoccus 2 2:3
[0064] The removal rates of odorous gases in each group are shown in Table 4.
[0065] Table 4 Comparison of Odor Gas Removal Rate
[0066]
[0067]
[0068]
[0069] Table 4 shows that the microbial composition in group 4 exhibited the best removal efficiency for ammonia, overall odor concentration, and volatile organic compounds (VOCs). The highest removal rates were achieved on day 5, with ammonia removal rates of 78.89%, overall odor concentration removal rates of 87.34%, and VOC removal rates of 72.87%. Hydrogen sulfide removal reached its highest rate of 99.5% on day 4. This demonstrates a synergistic effect between the two microorganisms, superior to single-bacterial solutions in ammonia and desulfurization, with a better ammonia removal efficiency when WC2:WN8 = 2:3. Replacing WN8 with other denitrifying paracocci did not significantly alter the results.
[0070] Group 3's microbial composition showed the best performance in hydrogen sulfide removal, achieving 100% removal on day 5. At this point, the ratio of WC2:WN8 was 2:1, indicating that a higher proportion of sulfur-oxidizing bacteria resulted in higher conversion efficiency for sulfur-containing odorous substances, thus reducing the amount of hydrogen sulfide released. Replacing WC2 with other strains of Delftobacterium truncatum resulted in no desulfurization effect, and the ammonia removal effect also decreased significantly. This proves that only the specific strain of Delftobacterium truncatum, WC2, possesses heterotrophic nitrification desulfurization effects and a synergistic effect with denitrifying paracocci.
[0071] 3. Demonstration of the deodorizing effect of compound microbial agent on pigsty floor.
[0072] The microbial composition was prepared according to Group 4 of Table 3. Yucca powder, turmeric powder, and tea polyphenol powder were then prepared into 5% (w / v) aqueous solutions of plant extracts. The microbial composition was then diluted with sterile water to prepare a 5% microbial culture medium. The aqueous solutions of each plant extract were mixed with equal volumes of the microbial culture medium to obtain three groups of compound microbial agents.
[0073] Four pigsties belonging to pig farmers in Shuangliu District, Chengdu, each containing the same number (3 pigs) of pigs, were used as experimental subjects. Compound microbial agents were loaded into agricultural sprayers and sprayed onto the floors of the three pigsties every morning for three consecutive days until the floors were moist. The concentrations of NH3, H2S, VOCs, and OU in the air were measured before spraying and at 6, 12, and 24 hours after spraying. One pigsty was set up as a blank control group, sprayed with the same amount of tap water every morning, with all other conditions remaining the same. The results are shown in Table 5. The concentration units in Table 5 are in ppm.
[0074] Table 5. Odor removal effect of pigsty floor
[0075]
[0076] The results showed that the trends of NH3, H2S, VOC, and OU were basically consistent during the three-day microbial agent spraying deodorization experiment. The concentration of malodorous gases in the control group pigsty showed almost no decrease. Among the groups sprayed with compound microbial agents, the deodorization efficiency, from highest to lowest, was: tea polyphenol-microbial agent group > yucca-microbial agent group > turmeric-microbial agent group.
[0077] 4. Demonstration of the deodorization effect of compound microbial agent on pigsty manure ditches.
[0078] A compound microbial agent was prepared based on a tea polyphenol-microbial agent combination. Three pig houses in a pig farm in Pengzhou City, each containing the same number and age of pigs, were used as experimental subjects. After emptying the manure ditches in the pig houses, based on the volume of the manure ditches, 1% of the compound microbial agent was poured into the manure ditch of the first pig house, and 1% of a commercially available deodorizing microbial agent was poured into the manure ditch of the second pig house. The third pig house was left untreated as a control group. The concentrations of NH3, H2S, VOCs, and OU were measured for seven consecutive days. The results are shown in Table 6. The concentration units in Table 6 are in ppm.
[0079] Table 6. Deodorization Effect of Pigsty Manure Ditches
[0080]
[0081]
[0082] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Any modifications, alterations, substitutions, or variations made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention shall fall within the protection scope defined by the claims of the present invention.
Claims
1. A strain of Delftobacterium tsurugi ( Delftia tsuruhatensis WC2, characterized in that: It was deposited at the Guangdong Provincial Center for Microbial Culture Collection on December 12, 2022, with accession number GDMCC NO: 62522.
2. The application of Delftobacterium tsurugi WC2 as described in claim 1 in deodorization.
3. The application of Delftobacterium tsurugi WC2 as described in claim 1 in desulfurization and / or denitrification.
4. An inoculum containing the Delftobacterium tsurugi WC2 as described in claim 1.
5. The microbial agent according to claim 4, characterized in that: The bacterial agent also includes denitrifying paracocci.
6. The microbial agent according to claim 4 or 5, characterized in that: The microbial agent also includes plant components, which are components that have deodorizing effects and do not inhibit the growth of microorganisms in the microbial agent.
7. The microbial agent according to claim 6, characterized in that: The plant ingredients are derived from one or more of green tea, ginger, or yucca.
8. The use of the microbial agent according to any one of claims 4 to 7 in deodorization.
9. The use of the microbial agent according to any one of claims 4 to 7 in desulfurization and / or denitrification.
10. The method of applying the microbial agent according to any one of claims 4 to 7 in deodorization, desulfurization, or denitrification, characterized in that: Prepare the microbial agent into a solution and spray it on the object or area to be deodorized, or mix it with the substance to be deodorized.