Ochrobactrum and application thereof in anaerobic degradation of halogenated organic matters

Abstract

The ochrobactrum is named as SC-Y-1 and is preserved in the China Center for Type Culture Collection on September 5, 2024, the preservation address is No. 299 on eight road, Wuchang District, Wuhan City, Hubei Province, and the preservation number is CCTCC NO: M 20241914. The Ochrobactrumsp. SC-Y-1 disclosed by the invention can take halogenated organic matters as a carbon source and an energy source, degrade the halogenated organic matters and degrade the halogenated organic matters through co-metabolism, can keep relatively high activity in a poor-nutrient polluted site and a combined polluted site, is suitable for being applied to the fields of wastewater, drinking water, soil remediation and the like, and has a wide application prospect. And a new breakthrough is expected to be made in the field of engineering application of aryl chloride biodegradation.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and particularly relates to an Ochrobacterium and application thereof in anaerobic degradation of halogenated organic matter. Background Art

[0002] Halogenated organics are common organohalogen compounds widely used in industrial manufacturing, but their emissions and leaks can cause serious harm to the environment as they are considered a type of persistent organic pollutants (POPs). The presence of POPs in soil and groundwater can cause ecosystem pollution and harm human health, so their effective degradation and removal become crucial.

[0003] The genera and microorganisms that degrade halogenated organics have great potential for treating environments contaminated by halogenated organics. These microorganisms usually have efficient enzyme systems that degrade halogenated organics and can decompose halogenated organics into safer metabolites, such as ethylene and harmless inorganic substances. The biodegradation of halogenated organics is a complex process that usually requires multiple microbial strains to work together to degrade organochlorine compounds such as halogenated organics into harmless products. The anaerobic biodegradation of halogenated organics has significant advantages and plays an important role in environmental governance and waste treatment. Organochlorine compounds are usually difficult to completely degrade by physical or chemical methods, but anaerobic biodegradation can degrade them into more environmentally friendly products. Secondly, anaerobic biodegradation does not require the presence of oxygen and is therefore suitable for anoxic or microaerobic environments, such as deep soils, sediments, and groundwater saturated zones, which makes anaerobic biodegradation the only feasible degradation method in some cases. In addition, the degradation process usually reduces halogenated organics to safer products, such as chlorides and ethylene, which are relatively stable and not easily converted into toxic substances again. Anaerobic biodegradation is also applicable to a variety of organochlorine compounds, including not only halogenated organics but also other similar compounds such as chlorinated ethylenes, 1,2-dihalogenated organics, and tetrahalogenated organics, which increases its application prospects in treating a variety of pollutants.

[0004] There are no reports on the use of Ochrobacterium for the degradation of halogenated organic matter. Among the functional strains reported so far, only a single form of co-metabolism or aerobic dechlorination can be used to degrade halogenated organic matter. There is little research on anaerobic degradation, which, to a certain extent, limits the application of anaerobic degradation of halogenated organic matter in engineering. Summary of the invention

[0005] The technical problem to be solved by the present invention is to provide an Ochrobactrum and its application in anaerobic degradation of halogenated organic matter, so as to solve the technical problem that halogenated organic matter cannot be degraded by engineering anaerobically.

[0006] To achieve the above-mentioned purpose, the technical solution adopted by the present invention is: to provide an Ochrobactrum sp., named SC-Y-1, which was deposited in the China Center for Type Culture Collection on September 5, 2024, with the storage address of No. 299, Bayi Road, Wuchang District, Wuhan City, Hubei Province, and the storage number is CCTCC NO: M 20241914.

[0007] The invention also discloses the application of the Ochrobactrum SC-Y-1 in anaerobic degradation of halogenated organic matter.

[0008] On the basis of the above technical solution, the present invention can also be improved as follows:

[0009] Furthermore, Ochrobacterium SC-Y-1 uses halogenated organic matter as the sole carbon source in the anaerobic degradation of halogenated organic matter.

[0010] Furthermore, the halogenated organic compound is a brominated flame retardant or a pesticide.

[0011] The invention also discloses a biocatalyst comprising Ochrobacterium SC-Y-1.

[0012] The beneficial effects of the present invention are as follows: the Ochrobacterium SC-Y-1 disclosed in the present invention can degrade halogenated organic matter as a carbon source and energy source, and can also degrade it through cometabolism; therefore, the complete cells of Ochrobacterium SC-Y-1 can be used as a raw material for a biocatalyst, and the liquid culture density OD 600 It can reach 1.4, and can maintain high activity even out of the environmental system. The culture medium required for expanded culture is simple in composition and low in cost. It is particularly suitable for the degradation of halogenated organic pollutants in wastewater and soil biological modification. It can be widely promoted. It is the only Ochrobacterium with halogenated organic degradation function discovered so far, which solves the problem that anaerobic and facultative anaerobic biodegradation of chlorinated hydrocarbon pollutants cannot be engineered in the existing technology. BRIEF DESCRIPTION OF THE DRAWINGS

[0013] Figure 1 is the phylogenetic tree of Ochrobacterium SC-Y-1;

[0014] Figure 2 This is a curve diagram of the degradation effect of Ochrobacterium SC-Y-1 on different types of halogenated organic matter;

[0015] Figure 3 is the OD of Ochrobacterium SC-Y-1 600 Change graph;

[0016] Figure 4 is the concentration variation diagram of the remaining dichloroethylene;

[0017] Figure 5This is a curve diagram of the degradation effect of Ochrobacterium SC-Y-1 on halogenated organic matter of different concentrations;

[0018] Figure 6 This is a curve diagram of the degradation effect of Ochrobacterium SC-Y-1 on halogenated organic matter under different salinity conditions. DETAILED DESCRIPTION

[0019] The specific embodiments of the present invention are described below to facilitate the understanding of the present invention by those skilled in the art. If the specific conditions are not specified in the examples, the methods are carried out according to conventional conditions or conditions recommended by the manufacturer. If the manufacturers are not specified in the reagents or instruments used, they are all conventional products that can be purchased commercially. However, it should be clear that the present invention is not limited to the scope of the specific embodiments. For those skilled in the art, as long as various changes are within the spirit and scope of the present invention defined and determined by the attached claims, these changes are obvious, and all inventions and creations using the concept of the present invention are protected.

[0020] The composition of the MSN liquid culture medium used in the present invention is as follows: CaCl2 0.1g, MgSO4·7H2O 0.2g, NH4SO42.5g, KH2PO4 4.5g, NaCl 0.2g, halogenated organic matter (60mg / L, 100mg / L, 150mg / L, 200mg / L, 300mg / L, 350mg / L) and distilled water 1L.

[0021] The composition of LB liquid medium is as follows: 5.0 g beef extract, 10 g peptone, 10 g NaCl and 1 L distilled water.

[0022] The MSN solid medium and the LB solid medium can be prepared by adding 20 g / L agar to the liquid medium as described above.

[0023] Example 1 Enrichment and Optimization of Ochrobactrum SC-Y-1

[0024] 1. Enrichment culture

[0025] A total of 10 g of contaminated particles under the 4 mm sieve and above the 2 mm sieve in the contaminated soil cover layer were taken from the domestic waste landfill in Rongchang District, Chongqing, and 100 mL of deionized water was added. The mixture was then placed in a shaker for 3 h. After the shaking, 10 mL of the supernatant was taken and inoculated into a 250 mL conical flask containing 100 mL of LB liquid culture medium. The mixture was placed in a shaker for 72 h to obtain an enriched bacterial solution. The storage temperature of the enriched bacterial solution was 4 °C, and the temperature of the shaker was 30 °C and the rotation speed was 160 r / min during shaking.

[0026] 2. Pure bacteria isolation

[0027] The enriched bacterial solution was diluted 10-fold in series with sterile distilled water to make a dilution of 10 -1 and 10 -2 dilution; the MSN solid medium was cultured by pouring method, the MSN solid medium was placed in a biochemical incubator, cultured at 30°C for 3 days, and finally the strains with excellent growth and single colony formation were subcultured for multiple times to purify the pure bacteria.

[0028] 3. Strain optimization

[0029] The bacterial suspension prepared using pure strains was added to a saline bottle containing 100 mL of culture medium in an anaerobic environment, and three halogenated organic substances (ethylene dichloride, decabromodiphenyl ether, chlordane) were added at concentrations of 60 mg / L, 130 mg / L and 200 mg / L, respectively. The culture medium was then placed in a shaker and cultured under shaking conditions at a temperature of 30°C and a rotation speed of 160 r / min, and the bacterial solution concentration and chlorobenzene concentration were tested every 24 hours.

[0030] The OD value of the bacterial solution concentration was detected by UV2000 spectrophotometer with a detection wavelength of 600nm; the viable cell concentration was determined by plate colony counting method; the dry weight of the bacteria was obtained by drying 10mL of bacterial solution at 80℃ to constant weight and weighing it with a precision electronic balance. At least 2 sets of parallel tests were performed for each experiment to ensure that the RSD was less than 5%.

[0031] Halogenated organic compounds were detected by gas chromatography under the following chromatographic conditions: a DB-23 elastic quartz capillary column (30 m × 0.32 mm × 0.25 μm), an injection port temperature of 240 ° C, a detector temperature of 260 ° C, a column temperature of 90 ° C (1.0 min), a heating rate of 10 ° C / min to 140 ° C, and then a heating rate of 20 ° C / min to 200 ° C (1.0 min); the carrier gas was nitrogen (99.999%), the carrier gas flow rate in the column was 2.0 mL / min, split injection, and the split ratio was 10: 1; the ECD current was 0.5 nA; the tail gas flow rate was 40 mL / min.

[0032] Decabromodiphenyl ether was detected by gas chromatography under the following chromatographic conditions: HP5 capillary column (15m×0.53mm×1.5μm), carrier gas was nitrogen (>99.99%), column head pressure was 120kPa, column flow control mode was constant pressure, non-divided injection was 1.00μL, diverter valve opening time was 0.75min, injection port temperature was 270℃, detector temperature was 300℃. The column heating program was as follows: initial temperature was set at 110℃, and the temperature was increased at 8℃·min -1 The temperature was raised to 300°C at a rate of 1.5°C and maintained for 40 min.

[0033] Chlordane detection was performed using gas chromatography, and the chromatographic conditions were as follows: DB-1701 quartz capillary column (30 m × 0.32 mm × 0.25 m), 1.0 mL of sample solution or standard solution was automatically injected in splitless mode, the injection port temperature was 2500 °C, the detector temperature was 3000 °C, the carrier gas and tail gas were both high-purity nitrogen with a purity of > 99.999%, the constant pressure was 50 kPa, and the constant flow was 60 mL·min -1 Column oven temperature program: maintain at 90℃ for 1 min, then increase the temperature at 40℃·min -1 The temperature was raised to 170℃ at a rate of 3℃·min and then maintained at 3℃·min -1 The temperature was raised to 230℃ and maintained for 3min, and finally at 25℃·min -1 The temperature was raised to 280°C and maintained for 2 min.

[0034] Example 2 Identification of Ochrobactrum SC-Y-1

[0035] The strain was cultured and then the colony morphology was observed. The colony diameter was 0.5-0.8 μm, the length was 0.9-2.0 μm, the end was round, and it usually moved with flagella around the peritrichous. It was anaerobic and most of the colonies were opaque and milky white. According to the identification of the chemical properties, the bacteria belonged to Gram-negative bacteria.

[0036] Then the strain was molecularly identified. The specific process was as follows: 1-5 mL of pure bacterial culture solution was used to extract genomic DNA using a bacterial genomic DNA extraction kit, and then 16SrRNA broad-spectrum amplification primers 27F: AGAGTTTGATCMTGGCTCAG (SEQ ID NO.1) and 1492R: GGTTACCTTGTTACGACTT (SEQ ID NO.2) were used. QIAquick Genomic DNA Buffer Set was used to PCR amplify the target fragment. 5 μL was taken for 3% agarose gel electrophoresis, and the target fragment was recovered by gel excision for DNA sequencing. The DNA sequencing was commissioned to Shanghai Sangon Biotech Co., Ltd., and DNA sequencing was performed using Seq Forward, Seq Reverse, and SeqInternal as primers.

[0037] The sequencing results of 16S rDNA of Ochrobactrum sp. SC-Y-1 showed that the base length was 1384 bp. The base sequence was compared with the GenBank nucleic acid sequence database and found that the homology with the strain 7002-134 of Ochrobactrum sp. was 100%. Figure 1) showed that Ochrobactrum sp. and Pseudochrobactrum sp. were closely related.

[0038] The nucleotide sequence of Ochrobactrum sp. SC-Y-1 (SEQ ID NO.3) is as follows:

[0039]

[0040] Example 3 Degradation effect of Ochrobacterium SC-Y-1 on different types of halogenated organic matter

[0041] Take equal amounts of three halogenated organic compounds (ethylene dichloride, decabromodiphenyl ether, chlordane) and add them to a 250 mL saline bottle containing 100 mL MSN liquid medium, with a concentration of 0.5 mg / L. Add 5 mL of Ochrobacterium SC-Y-1 bacterial solution (OD 600 1.4), cover with rubber stopper and shake well, put into shaker and set at 30℃, 160r / min anaerobic conditions for 168 hours, determine the content of dichloroethylene, decabromodiphenyl ether and chlordane by gas chromatography, and determine the degradation rate of dichloroethylene, decabromodiphenyl ether and chlordane ( Figure 2 ) and the OD of the strain 600 ( Figure 3 ).like Figure 2 As shown in the figure, when the degradation object is dichloroethylene, the degradation effect is good, and it is basically completely degraded on the third day, with a degradation rate of 88.25%. Due to the toxic effect that inhibits the activity of the bacteria, the degradation rate remains basically unchanged. When the degradation object is decabromodiphenyl ether, the degradation effect is significantly lower than that of dichloroethylene, and the maximum degradation rate is 71.04%. When the degradation object is chlordane, the degradation effect is basically the same as that of decabromodiphenyl ether, and the maximum degradation rate can reach 54.84%. Figure 3 As shown, from 0 to 12 hours, Ochrobactrum SC-Y-1 was in the growth stage, and the concentration of the bacterial solution gradually increased; from 12 to 120 hours, the concentration of the bacterial solution did not change much and was in a stable stage; under the inhibition of halogenated organic matter, the concentration of the bacterial solution decreased after 120 hours.

[0042] Example 4 Degradation effect of Ochrobacterium SC-Y-1 on halogenated organic matter of different concentrations

[0043] Take a certain amount of halogenated organic matter (ethylene dichloride) and add it to a 250mL saline bottle containing 100mL MSN liquid culture medium. The concentrations of ethylene dichloride are 60mg / L, 100mg / L, 150mg / L, 200mg / L, 300mg / L and 350mg / L. Add 5mL of Ochrobacterium SC-Y-1 bacterial solution (OD 600 1.4), cover with rubber stopper and shake well, put into shaker and set at 30℃, 160r / min for 168 hours, determine the content of halogenated organic matter by gas chromatography, and determine the degradation effect of halogenated organic matter. The concentration change of remaining dichloroethylene after degradation of different concentrations of dichloroethylene is as follows: Figure 4 As shown, the degradation rate is Figure 5The results show that when the concentration of dichloroethylene is 60 mg / L, the degradation effect is good, and dichloroethylene can be degraded by more than 80% on the seventh day; when the concentration of chlorobenzene is 100 mg / L, the final degradation rate is 71.66%; when the concentration of dichloroethylene is 200 mg / L, due to the toxic effect of inhibiting the activity of bacteria, the degradation rate of dichloroethylene can reach more than 58..22%; when the concentration of dichloroethylene is 300 mg / L, the degradation rate is 57.32%; when the concentration of chlorobenzene is 350 mg / L, the strong inhibition of dichloroethylene leads to a degradation effect of 50.32% of dichloroethylene.

[0044] Example 5 Degradation effect of Ochrobacterium SC-Y-1 on halogenated organic matter under different salinities

[0045] Take 200 mg / L of dichloroethylene and add it to a 250 mL saline bottle containing 100 mL of MSN liquid culture medium. The salinity is 2%, 3%, 4% and 5% respectively. Add 0.5 mL of Ochrobacterium spp. culture solution (OD 600 1.4), cover the rubber stopper and shake well, put it in a shaker and set it at 30℃ and 160r / min for 146 hours, determine the chlorobenzene content by gas chromatography, and determine the degradation effect of halogenated organic matter (ethylene dichloride). Figure 6 As shown, when the salinity is 2%, Ochrobacterium SC-Y-1 degrades the concentration of dichloroethylene from 2 mg / L to 0.4 mg / L within 7 days; when the salinity is 3%, the concentration of chlorobenzene is degraded from 2 mg / L to 0.6 mg / L; when the salinity is 4%, the salinity degradation rate is more than 70%; when the salinity is 5%, the concentration of chlorobenzene is degraded from 2 mg / L to 0.8 mg / L, and the degradation rate is more than 60%.

[0046] In summary, it can be seen that the Ochrobacterium SC-Y-1 screened from the contaminated soil of the cover soil of the domestic waste landfill in Rongchang District, Chongqing City in the present invention not only has high tolerance to chloride ions and halogenated organic matter, but can also degrade halogenated organic matter as a carbon source and energy source, and can also degrade it through cometabolism, and can maintain high activity in both oligotrophic and complex contaminated sites. The unique degradation characteristics of Ochrobacterium SC-Y-1 are expected to make new breakthroughs in the engineering application field of chlorinated hydrocarbon biodegradation.

Claims

1. An Ochrobactrum sp., named SC-Y-1, was deposited in the China Center for Type Culture Collection on September 5, 2024, with the deposit number CCTCC NO: M 20241914.

2. Use of the Ochrobactrum SC-Y-1 described in claim 1 in anaerobic degradation of halogenated organic matter.

3. The use according to claim 2, characterized in that: The Ochrobacterium SC-Y-1 uses halogenated organic matter as the sole carbon source in anaerobic degradation of halogenated organic matter.

4. The use according to claim 2 or 3, characterized in that: The halogenated organic matter is a brominated flame retardant or a pesticide.

5. A biocatalyst, characterized in that: It includes the Ochrobactrum SC-Y-1 described in claim 1.

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