High-temperature-resistant Gordonia and application thereof in sewage treatment

Abstract

The invention relates to the technical field of microbiological sewage treatment, in particular to a high-temperature-resistant Gordonia sp. HD01 strain and application thereof in sewage treatment, the classification name of the Gordonia sp. HD01 strain is Gordonia sp. HD01, and the preservation number of the Gordonia sp. HD01 strain is CCTCC NO: M 20242968. The Gordonia disclosed by the invention can grow under the aerobic condition of 40 DEG C, can tolerate high-grease and high-COD (Chemical Oxygen Demand) environments in restaurant sewage, can effectively remove grease in the sewage under medium and high temperature conditions, and has a wide application prospect.

Description

Technical Field

[0001] The invention relates to the technical field of microbial sewage treatment, in particular to a high-temperature resistant Gordonia and its application in sewage treatment. Background Art

[0002] With steady economic growth and continued improvements in people's quality of life, the catering industry has flourished, leading to an increasingly serious problem of oily wastewater discharge. This wastewater is rich in complex components, particularly high levels of animal and vegetable oils and fats. It is also accompanied by high concentrations of organic matter, high turbidity, high salinity, and excessive chemical oxygen demand (COD) and ammonia nitrogen concentrations, placing significant pressure on municipal wastewater treatment systems. While traditional physical and chemical methods can remove oil, they are prone to secondary pollution and are costly. In contrast, biological methods, leveraging microbial degradation of oils, offer advantages that are both environmentally friendly and economical. This technology leverages the ability of microorganisms to break down complex oil compounds into simpler, harmless substances through their metabolic activity, offering advantages such as low cost, high efficiency, and environmental friendliness. The fundamental principle of oil removal is that microbial degradation of oil pollutants relies primarily on enzymes produced by the microorganisms. These enzymes break down oil into smaller molecules, making them more accessible to microbial absorption and further degradation. For example, certain bacteria and fungi secrete lipases and hydrocarbon oxidases, which break down complex oil compounds into simpler molecules such as fatty acids, glycerol, and carbon dioxide.

[0003] Currently, a large number of studies have been reported on the microbial degradation of petroleum or crude oil, mainly focusing on the screening and isolation of microbial strains and lipases with high oil degradation efficiency from the environment. Although many reports have been published on the isolation and identification of highly efficient oil-degrading microorganisms, most of them are for petroleum, lubricating oil, and crude oil. There are few reports on pure strains for removing edible oil and animal oil from catering wastewater. In addition, the high oil and COD content in catering wastewater also limits the practical application of some microorganisms. In view of this, there is an urgent need to obtain a microorganism that can tolerate the catering wastewater environment and has a good ability to remove oil from wastewater. Summary of the Invention

[0004] In light of this, the present invention provides a thermostable strain of Gordonia and its application method in wastewater. This strain can tolerate high oil and COD content found in restaurant wastewater and effectively remove oil at medium to high temperatures, aiming to address the high oil content in restaurant wastewater.

[0005] The technical solution of the present invention is achieved as follows: In one aspect, the present invention provides a heat-resistant Gordonia strain, preferably, the Gordonia strain was deposited in the China Center for Type Culture Collection (Address: Wuhan University, Wuhan, China) on December 31, 2024, and its classification is named Gordonia sp.HD01, the accession number is CCTCC NO: M 20242968.

[0006]

[0007] Based on the above scheme, preferably, the culture temperature of Gordonia is 25-40°C, and further preferably, the culture temperature of Gordonia is 35°C.

[0008] Based on the above solution, preferably, the Gordonia can remove grease at 30-40°C.

[0009] Based on the above solution, preferably, the oil includes one or more of edible oil, animal oil and lubricating oil.

[0010] Based on the above scheme, preferably, the concentration of the oil is 600~29125 mg / L.

[0011] Based on the above scheme, it is further preferred that the Gordonia can effectively remove edible oil and animal oil from sewage at 30°C, with a removal rate of 40.8% for edible oil with a concentration of 600 mg / L, 59.28% for edible oil with a concentration of 29125 mg / L, and 43.96% for animal oil with a concentration of 15771 mg / L; the removal rate of lubricating oil with a concentration of 600 mg / L at 40°C is 29.7%.

[0012] In a second aspect, a microbial agent is provided, preferably containing the thermostable Gordonia as described above.

[0013] In a third aspect, a use of the thermostable Gordonia as described above in sewage treatment is provided.

[0014] The thermostable Gordonia of the present invention and its application in sewage have the following beneficial effects compared with the prior art: The Gordonia of the present invention can grow at 40°C under aerobic conditions, while the reported growth temperature of pure cultured Gordonia is no higher than 37°C; The Gordonia of the present invention can remove various oils and fats from wastewater at 30°C, achieving efficient removal at both low and high concentrations, which is suitable for practical application conditions. It can also effectively degrade lubricating oils at 40°C, showing great application potential in some high-temperature restaurant wastewater. The Gordonia of the present invention can tolerate high-oil, high-COD environments in catering wastewater, and does not require additional pretreatment of the wastewater in practical applications, and has broad application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments or the description of the prior art. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0016] Figure 1 This is a graph showing the growth of Gordonia HD01 at 25-40°C; Figure 2 This is a diagram showing the removal effect of Gordonia HD01 on low-concentration edible oil wastewater at 30°C; Figure 3 This is a diagram showing the removal effect of Gordonia HD01 on high-concentration edible oil wastewater and animal oil wastewater at 30°C; Figure 4 This is a diagram showing the removal effect of Gordonia HD01 on low-concentration lubricating oil wastewater at 40°C. DETAILED DESCRIPTION

[0017] The following will be combined with the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the embodiments described are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts are within the scope of protection of the present invention.

[0018] The culture medium used in the present invention is prepared as follows. All culture media are sterilized at high temperature and high pressure (121°C, 20 min) before use: Activation medium: tryptone 10 g / L, yeast extract 5 g / L, sodium chloride 10 g / L.

[0019] Basal culture medium: FeSO4·7H2O 0.4 g / L, MgSO4·7H2O 0.05 g / L, K2HPO4 1 g / L, NaHCO3 1.5 g / L, CaCl2·2H2O 1.5 g / L, NaCl 2.0 g / L, KNO3 1.4 g / L, fructose 4 g / L, trace element solution 1 mL / L, pH 7.5.

[0020] Trace element solution: EDTA 0.5 g / L, CuSO4·5H2O 0.075 g / L, ZnSO4·7H2O 0.3 g / L, CoCl2·6H2O 0.375 g / L, MnCl2·2H2O 0.3 g / L, H3BO4 0.014 g / L, NaMoO4·2H2O 0.22 g / L.

[0021] The water quality index detection involved in this invention all adopts the national standard method, among which the oil content detection method adopts the infrared spectrophotometry method for the determination of petroleum and animal and vegetable oils in water (HJ 637-2018).

[0022] Specific growth rate was calculated based on OD 600 The specific calculation formula is as follows: M (specific growth rate) =

[0023] OD1 and OD2 are the OD at time t1 and t2 respectively. 600 Value, t1, t2 unit: h.

[0024] The grease removal rate is calculated based on the oil content at the beginning and end. The specific calculation formula is as follows: Removal rate =

[0025] Where C1 is the initial oil concentration, mg / L; C2 is the final oil concentration, mg / L.

[0026] Example 1: Screening and identification of bacterial strains Activated sludge from a sewage treatment facility at a tunnel construction site in Haidong City, Qinghai Province, was inoculated into 100 mL of basal culture medium at a 5% ratio and concentrated in a shaker at 30°C and 140 rpm until turbidity became apparent. The enriched bacterial solution was streaked onto a solid culture plate and incubated at 30°C until a clear streak mark appeared. The terminal colony was selected and streaked three times until a single colony appeared. The single colony was transferred to 100 mL of basal culture medium using an inoculation needle and incubated in a shaker at 30°C and 140 rpm. The purified culture was PCR-amplified for its 16s rDNA gene fragment, and molecular biological identification was performed by sequencing and comparison. The primer sequences used for amplification are as follows: 16s rDNA upstream primer 27F: 5′-AGAGTTTGATCMTGGCTCAG-3′ (SEQ ID NO. 2); 16S rDNA downstream primer 1492R: 5′-GGTTACCTTGTTACGACTT-3′ (SEQ ID NO. 3).

[0027] According to the sequencing results of the PCR product completed by Shanghai Sangon Biotechnology Co., Ltd. (shown as SEQ ID NO. 1), the isolated strain was identified as Gordonia by comparing the 16S rDNA sequence in the NCBI database ( Gordonia alkanivorans The strain was deposited in China Center for Type Culture Collection (Address: Wuhan University, Wuhan, China) on December 31, 2024, and its classification was named Gordonia sp. HD01, the deposit number is CCTCC NO: M20242968.

[0028] Example 2: Growth of Gordonia HD01 at 25-40°C Preparation of inoculum: Inoculate a single colony from the plate into 100 mL of activation medium and culture at 30°C and 140 rpm. 600 When the concentration is ≥2.0, it is used as inoculum.

[0029] The above inoculum was inoculated into the basal culture medium at a volume ratio of 1%, and cultured at 25, 30, 35 and 40 °C, with shaking at 140 r / min. Each group was set up in triplicate, and samples were taken every day to measure the OD 600 , the experimental results are as follows Figure 1 shown.

[0030] according to Figure 1 Gordonia HD01 can grow in 25~40℃ culture medium, and OD 600 can reach 2.0, indicating that it can adapt to medium and high temperatures without being significantly inhibited; according to OD 600 From the results, Gordonia HD01 grew best at 35℃, and the OD 600 It reached 2.14±0.13, and its fastest specific growth rate reached 0.13±0.001; at the same time, the maximum specific growth rate of Gordonia HD01 at 40℃ was 0.123±0.003, which was higher than the maximum specific growth rates at 30℃ and 25℃, indicating that Gordonia HD01 still had a faster growth rate under these conditions.

[0031] Example 3: Removal effect of Gordonia HD01 in low-concentration edible oil wastewater Preparation of inoculum: 1 mL of Gordonia HD01 bacterial solution was inoculated into 100 mL of basal medium and cultured at 30°C and 200 rpm until OD 600 When the concentration is ≥2.0, it is used as inoculum.

[0032] The above HD01 inoculum was inoculated into the edible oil-containing catering wastewater at a volume ratio of 1%, with an initial oil concentration of 600 mg / L and a shaking speed of 140 r / min at 30°C. Two parallel cultures were set up for each group. The oil concentration was tested at the beginning and on the 6th day. The results were as follows: Figure 2 shown.

[0033] according to Figure 2 After 6 days of treatment at 30℃, the oil concentration of each experimental group decreased significantly compared with the blank group. The initial edible oil concentration was 611.57±22.82 mg / L. After 6 days, the blank control group had almost no decrease, and the removal rate was less than 1%. The edible oil concentration of the HD01 group dropped to 243.67±21.96 mg / L, and the removal rate was 40.8%.

[0034] Example 4: Removal effect of Gordonia HD01 on high-concentration edible oil wastewater and animal oil wastewater at 30°C Preparation of inoculum solution: refer to the inoculum solution preparation method of Example 3.

[0035] Wastewater from a restaurant in Wuhan, Hubei Province, was spiked with high-concentration edible oil (29125 mg / L) and high-concentration animal oil (15771 mg / L). 1 mL of the Gordonia HD01 inoculum was added to 100 mL of the edible oil wastewater and animal oil wastewater, respectively. The blank control group was left uninoculated. Two replicates were set up for each group. The incubation was carried out in a shaker at 30°C and 140 rpm. Samples were taken every 12 days to measure the oil concentration. The results are shown in Table 1. Figure 3 shown.

[0036] according to Figure 3 Figures (a) and (b): After 12 days of treatment at 30°C, the oil removal rate in each test group was significantly higher than that in the blank group. At 30°C, HD01 achieved an edible oil removal efficiency of 59.28±4.23% in edible oil wastewater and an animal oil removal efficiency of 43.96±1.96% in animal oil wastewater, demonstrating its ability to treat both edible and animal oil wastewater at moderate temperatures. According to the report "Research on Optimization of Coagulation Process for Catering Wastewater Using Response Surface Methodology" by Jia Yanping et al., catering wastewater contains high levels of COD, BOD, and animal and plant oils. Specifically, animal and plant oils form an oil film on the water surface, hindering oxygen dissolution and causing wastewater hypoxia, which is detrimental to microbial growth. COD, which contains organic components such as detergents, can damage the integrity of microbial cell membranes, interfere with microbial physiological functions, and even directly kill them.

[0037] Example 5: Removal effect of Gordonia HD01 in low-concentration lubricating oil wastewater Preparation of inoculum solution: refer to the inoculum solution preparation method of Example 3.

[0038] The above HD01 inoculum was inoculated into lubricating oil-containing wastewater at a volume ratio of 1%, with an initial oil concentration of 600 mg / L and a shaking speed of 140 r / min at 40°C. Two replicates were set up for each group. The oil concentration was tested by sampling at the beginning and on the 6th day. The results were as follows: Figure 4 shown.

[0039] according to Figure 4 After treatment at 40℃ for 6 days, the oil concentration of each experimental group decreased significantly compared with the blank group. The initial lubricating oil concentration was 658.13±128.87 mg / L. After 6 days, the blank control group had almost no decrease, and the removal rate was less than 1%. The lubricating oil concentration of the HD01 group was 462.63±32.35 mg / L, and the removal rate was 29.7%.

[0040] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included in the scope of protection of the present invention.

Claims

1. A thermostable strain of Gordonia, characterized by: The taxonomic designation of the Gordonia Gordonia sp. HD01, the accession number is CCTCC NO: M 20242968.

2. The thermostable Gordonia according to claim 1, characterized in that The 16s rDNA sequence of Gordonia is shown as SEQ ID NO.

1.

3. The thermostable Gordonia according to claim 1, characterized in that The culture temperature of the Gordonia is 25-40°C.

4. The thermostable Gordonia according to claim 1, characterized in that The Gordonia bacteria can remove grease at 30-40°C.

5. The thermostable Gordonia according to claim 4, characterized in that The oil includes one or more of edible oil, animal oil and lubricating oil.

6. The thermostable Gordonia according to claim 4, characterized in that The concentration of the oil is 600~29125 mg / L.

7. A microbial agent, characterized in that: Contains the heat-resistant Gordonia as claimed in claim 1.

8. Use of the thermostable Gordonia according to claim 1 in sewage treatment.

Images