Achromobacter and its application in anaerobic degradation of halogenated organic compounds

Abstract

The application discloses an ochrobactrum and application of the ochrobactrum in anaerobic degradation of halogenated organic matters, 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, Bayi Road, Wuchang District, Wuhan City, Hubei Province, and the preservation number is CCTCC NO: M 20241914.The ochrobactrum (Ochrobactrum sp.) SC-Y-1 disclosed by the application can take halogenated organic matters as carbon source and energy, and can degrade the halogenated organic matters, and can also degrade the halogenated organic matters through co-metabolism, can keep high activity in oligotrophic contaminated sites and compound contaminated sites, is suitable for application in the fields of wastewater, drinking water and soil remediation and the like, and is expected to make a new breakthrough in the engineering application field of chlorinated aromatic hydrocarbon biodegradation.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of biotechnology, and particularly relates to a Pseudomonas putida and its application in anaerobic degradation of halogenated organic compounds. BACKGROUND

[0002] Halogenated organic compounds are a common type of organic halides, 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 becomes crucial.

[0003] Bacterial genera and microorganisms that degrade halogenated organic compounds have great potential for treating environments contaminated with halogenated organic compounds, as these microorganisms often have efficient halogenated organic compound degradation enzyme systems that can break down halogenated organic compounds into safer metabolic products, such as ethylene and harmless inorganic substances. The biological degradation of halogenated organic compounds is a complex process that often requires multiple microbial strains to work together to degrade organic chlorine compounds such as halogenated organic compounds into harmless products. Anaerobic biodegradation of halogenated organic compounds has significant advantages and plays an important role in environmental management and waste treatment. Organic chlorine 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, so it is suitable for anoxic or micro-aerobic environments such as deep soil, sediments, and groundwater saturated zones, making anaerobic biodegradation the only viable degradation method in certain situations. In addition, this degradation process usually reduces halogenated organic compounds to relatively safe products such as chlorides and ethylene, which are relatively stable and not easily converted back into toxic substances. Anaerobic biodegradation is also suitable for a variety of organic chlorine compounds, including not only halogenated organic compounds but also other similar compounds such as chlorinated ethylene, 1,2-dihalogenated organic compounds, and tetrahalogenated organic compounds, which increases its application prospects in treating a variety of pollutants.

[0004] There is currently no report on the use of Pseudomonas putida for halogenated organic compound degradation research. Among the currently reported functional strains, halogenated organic compound degradation can only be achieved in the form of co-metabolism or aerobic dechlorination, and there is little research on anaerobic degradation, which to some extent limits the application of halogenated organic compound anaerobic degradation in engineering. SUMMARY

[0005] The technical problem to be solved by the present application is to provide a Pseudomonas putida and its application in anaerobic degradation of halogenated organic compounds, to solve the technical problem that halogenated organic compounds cannot be engineered to be degraded by anaerobic methods.

[0006] To achieve the above object, the technical scheme adopted by the present application is to provide a pale strain (Ochrobactrum sp. Ochrobactrum , named SC-Y-1, which was preserved in the China Center of Typical Culture Collection on September 5, 2024, and the address of the preservation center is No. 299, Bayi Road, Wuchang District, Wuhan City, Hubei Province, and the preservation number is CCTCC NO: M 20241914.

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

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

[0009] Further, the Ochrobactrum SC-Y-1 degrades halogenated organic matter as the sole carbon source in anaerobic degradation of halogenated organic matter.

[0010] Further, the halogenated organic matter is a brominated flame retardant or pesticide.

[0011] The present application also discloses a biological catalyst comprising the Ochrobactrum SC-Y-1.

[0012] The present application has the following beneficial effects: the Ochrobactrum SC-Y-1 disclosed in the present application can degrade halogenated organic matter as a carbon source and energy source, and can also degrade it through co-metabolism; therefore, the whole cell of the Ochrobactrum SC-Y-1 can be used as a raw material of a biological catalyst, and the liquid culture density OD 600 can reach 1.4, and can still maintain high activity in the environment system. The culture medium required for scale-up culture is simple in composition and low in cost, and is particularly suitable for degradation of halogenated organic matter pollutants in wastewater and soil biological modification, and can be widely promoted. It is the Ochrobactrum with halogenated organic matter degradation function found at present, and solves the problem that anaerobic and facultative anaerobic biological degradation of chlorinated hydrocarbon pollutants cannot be engineered in the prior art. BRIEF DESCRIPTION OF DRAWINGS

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

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

[0015] Figure 3 is an OD 600 variation graph of the Ochrobactrum SC-Y-1;

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

[0017] Figure 5The degradation effect curve of the Ochrobactrum sp. SC-Y-1 on different concentrations of halogenated organic compounds is shown in the figure;

[0018] Figure 6 The degradation effect curve of the Ochrobactrum sp. SC-Y-1 on halogenated organic compounds under different salinity conditions is shown in the figure. DETAILED DESCRIPTION

[0019] The specific embodiments of the present application are described below to facilitate the understanding of the present application by those skilled in the art. The specific conditions not mentioned in the examples are carried out according to the conventional conditions or the conditions recommended by the manufacturer. The reagents or instruments not mentioned by the manufacturer are conventional products that can be purchased on the market. However, it should be clear that the present application is not limited to the scope of the specific embodiments. For those skilled in the art, it is obvious that various changes are within the spirit and scope of the present application as defined and determined by the appended claims, and all the inventions utilizing the concept of the present application are within the scope of protection.

[0020] The composition of the MSN liquid medium used in the present application is as follows: CaCl20.1g, MgSO4·7H2O 0.2g, NH4SO42.5g, KH2PO44.5g, NaCl 0.2g, halogenated organic compound (60mg / L, 100mg / L, 150mg / L, 200mg / L, 300mg / L, 350mg / L) and distilled water 1L.

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

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

[0023] Example 1 Enrichment and optimization of Ochrobactrum sp. SC-Y-1

[0024] 1. Enrichment culture

[0025] 10g of contaminated particles of 4mm sieve and 2mm sieve from the contaminated soil cover layer of the household garbage landfill in Rongchang District, Chongqing City were taken, 100mL of deionized water was added, and then placed in a shaking bed for 3h. After the shaking was completed, 10mL of supernatant was taken and inoculated into a 250mL conical flask containing 100mL of LB liquid medium. After being placed in a shaking bed for 72h, an enriched bacteria solution was obtained. The storage temperature of the enriched bacteria solution was 4℃, and the temperature of the shaking bed was 30℃ and the rotation speed was 160r / min during shaking.

[0026] 2. Pure bacteria separation

[0027] The enriched bacteria liquid was diluted 10 times by sterile distilled water to prepare dilutions with dilution degrees of 10 -1 and 10 -2 . The MSN solid culture medium was placed in a biochemical incubator and cultured at 30°C for 3 days. Finally, the bacteria strains with excellent growth and single colony formation were subcultured multiple times to obtain pure bacteria.

[0028] 3. Strain optimization

[0029] The bacterial suspension prepared by using the pure strain was added to a salt water bottle containing 100 mL of culture medium in an anaerobic environment, and three kinds of halogenated organic compounds (dichloroethylene, decabromobiphenyl ether, chlordane) with concentrations of 60 mg / L, 130 mg / L and 200 mg / L were added, respectively, and then the culture medium was placed in a shaker and cultured at a temperature of 30°C and a speed of 160 r / min, and the concentration of the bacterial liquid and the concentration of chlorobenzene were detected every 24 h.

[0030] The OD value of the bacterial liquid concentration was detected by UV2000 spectrophotometer with a detection wavelength of 600 nm; the viable cell concentration was determined by plate colony counting method; and the dry weight of the bacterial body was obtained by drying 10 mL of bacterial liquid at 80°C to a constant weight and weighing with a precision electronic balance. Each experiment was performed at least in two parallel groups to ensure that the RSD was less than 5%.

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

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

[0033] Chlordane detection was performed using gas chromatography under the following conditions: DB-1701 quartz capillary column (30m × 0.32mm × 0.25m); 1.0 mL of sample solution or standard solution was automatically injected using a splitless injection method; the injection port temperature was 2500°C; the detector temperature was 3000°C; both the carrier gas and the make-up gas were high-purity nitrogen with a purity >99.999%; the constant pressure was 50 kPa; and the constant flow rate was 60 mL / min. -1 The column oven heating program is as follows: hold at 90°C for 1 minute, then at 40°C for 1 minute. -1 The temperature was increased to 170°C and maintained for 3 minutes, then increased to 3°C / min. -1 The temperature was increased to 230°C and maintained for 3 minutes, then finally increased to 25°C / min. -1 The temperature was increased to 280°C and then maintained for 2 minutes.

[0034] Example 2: Identification of *Bacillus anthracis* SC-Y-1

[0035] The strain was cultured, and colony morphology was observed. Colonies were 0.5–0.8 μm in diameter and 0.9–2.0 μm in length, with rounded ends. They typically exhibit peritrichous motility, are anaerobic, and are mostly opaque and milky white. Physicochemical analysis confirmed that this bacterium is Gram-negative.

[0036] Next, molecular identification of the strain was performed. The specific procedure was as follows: Genomic DNA was extracted from 1-5 mL of pure bacterial culture using a bacterial genomic DNA extraction kit. Then, 16S rRNA broad-spectrum amplification primers 27F: AGAGTTTGATCMTGGCTCAG (SEQ ID NO.1) and 1492R: GGTTACCTTGTTACGACTT (SEQ ID NO.2) were used. PCR amplification of the target fragment was performed using QIAquick Genomic DNA Buffer Set. 5 μL of the amplified fragment was subjected to 3% agarose gel electrophoresis. The target fragment was recovered by gel excision and DNA sequencing was performed. The DNA sequencing was commissioned to Shanghai Sangon Biotech Co., Ltd., using Seq Forward, Seq Reverse, and Seq Internal primers.

[0037] Paleobacterium ( Ochrobactrum Sequencing results of the 16S rDNA of sp. SC-Y-1 showed a base length of 1384 bp. Comparison of the base sequence with the GenBank nucleic acid sequence database revealed 100% homology with the strain Ochrobactrum sp. strain 7002-134. A phylogenetic tree of Ochrobactrum sp. SC-Y-1 was constructed. Figure 1It can be known that Ochrobactrum sp. is closely related to Pseudochrobactrum sp.

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

[0039]

[0040] Example 3: Degradation effect of Bacillus angulans SC-Y-1 on different types of halogenated organic compounds

[0041] Equal amounts of three halogenated organic compounds (dichloroethylene, decabromodiphenyl ether, and chlordane) were added to 250 mL saline bottles containing 100 mL of MSN liquid culture medium, each at a concentration of 0.5 mg / L. 5 mL of *Bacillus cereus* SC-Y-1 bacterial suspension (OD200) was then added. 600 (1.4), seal with rubber stopper, shake well, place in a shaker and incubate under anaerobic conditions of 30℃ and 160r / min for 168 hours. Determine the contents of dichloroethylene, decabromodiphenyl ether, and chlordane using gas chromatography, and determine the degradation rates of dichloroethylene, decabromodiphenyl ether, and chlordane. Figure 2 ) and OD of bacterial strains 600 ( Figure 3 ).like Figure 2 As shown, when the target being degraded is chlordane, the degradation effect is good, with almost complete degradation by the 3rd day, reaching a degradation rate of 88.25%. Due to the toxic effect inhibiting bacterial activity, the degradation rate remains essentially unchanged. When the target being degraded is decabromodiphenyl ether, the degradation effect is significantly less than that of chlordane, with a maximum degradation rate of 71.04%. When the target being degraded is dichloroethylene, the degradation effect is basically the same as that of decabromodiphenyl ether, with a maximum degradation rate reaching 54.84%. Figure 3 As shown, from 0 to 12 hours, Bacillus SC-Y-1 was in the growth stage, and the bacterial concentration gradually increased; from 12 to 120 hours, the bacterial concentration did not change much and remained in a stable stage; under the inhibition of halogenated organic matter, the bacterial concentration decreased after 120 hours.

[0042] Example 4: Degradation effect of Bacillus anthracis SC-Y-1 on different concentrations of halogenated organic compounds

[0043] A certain amount of the halogenated organic compound (dichloroethylene) was taken and added to a 250 mL saline bottle containing 100 mL of MSN liquid culture medium, with dichloroethylene concentrations of 60 mg / L, 100 mg / L, 150 mg / L, 200 mg / L, 300 mg / L, and 350 mg / L, respectively. 5 mL of *Bacillus cereus* SC-Y-1 bacterial suspension (OD200) was added. 600 (1.4), seal with rubber stopper, shake well, place in a shaker and incubate at 30℃ and 160r / min for 168 hours. Determine the content of halogenated organic compounds using gas chromatography and determine the degradation effect of halogenated organic compounds. The changes in the concentration of residual dichloroethylene after degradation of different concentrations of dichloroethylene are shown in the figure. Figure 4 As shown, the degradation rate is as follows Figure 5The results show that when the concentration of dichloroethylene is 60 mg / L, the degradation effect is good, and the 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 the bacterial body, 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%; and when the concentration of chlorobenzene is 350 mg / L, the strong inhibitory effect of dichloroethylene leads to a dichloroethylene degradation effect of 50.32%.

[0044] Example 5 Degradation effect of paleobacter SC-Y-1 on halogenated organic matter under different salinity conditions

[0045] 200 mg / L of dichloroethylene is added to a 250 mL salt water bottle containing 100 mL of an MSM liquid medium, and the salinity is 2%, 3%, 4% and 5%, respectively. 0.5 mL of paleobacter bacterial liquid (OD 600 1.4) is added, the rubber plug is covered, and the mixture is shaken and placed in a shaking bed under the condition of 30°C and 160 r / min for 146 hours. The content of chlorobenzene is determined by gas chromatography, and the degradation effect of halogenated organic matter (dichloroethylene) is determined. As Figure 6 shown, when the salinity is 2%, the paleobacter 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 degradation rate is more than 70%; and 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] As can be seen from the above, the paleobacter SC-Y-1 screened from the contaminated soil of the covering soil of the living garbage landfill in Rongchang District of Chongqing has not only high tolerance to chlorine ions and halogenated organic matter, but also can degrade halogenated organic matter as a carbon source and energy source, and can degrade halogenated organic matter through co-metabolism. The paleobacter SC-Y-1 can maintain high activity in oligotrophic contaminated sites and complex contaminated sites, and the unique degradation characteristics of the paleobacter SC-Y-1 are expected to make new breakthroughs in the engineering application field of chlorinated hydrocarbon biodegradation.

Claims

1. A type of pale bacillus ( Ochrobactrum The application of sp.)SC-Y-1 in the degradation of halogenated organic compounds under anaerobic and high-salt conditions is characterized by: The Ochrobactrum SC-Y-1 degrades halogenated organic matter under anaerobic conditions with halogenated organic matter as the sole carbon source; the halogenated organic matter is dichloroethylene, decabromobiphenyl ether or chlordane; the Ochrobactrum SC-Y-1 is preserved in the China Center for Type Culture Collection on September 5, 2024, and the preservation number is CCTCC NO: M 20241914.

2. A biocatalyst characterized in that: The Ochrobactrum SC-Y-1 of claim 1. The Ochrobactrum SC-Y-1 of claim 1.

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