Salt-resistant and high-temperature-resistant halomonas pinnata LH-B.0008 and application thereof

Abstract

The invention relates to Halomonas pinnata LH-B.0008, which is classified and named as Halomonas arcis, and the preservation number of the Halomonas pinnata LH-B.0008 is CGMCC (China General Microbiological Culture Collection Center) NO: 16316. The strain LH-B.0008 disclosed by the invention can be used for synchronously removing nitrogen and degrading COD (Chemical Oxygen Demand) at a high temperature of 38-45 DEG C and in a high-salt range of 1-30% in salinity, including treatment of refractory COD in carbon fiber production wastewater containing refractory acrylonitrile and high-concentration DMSO (Dimethylsulfoxide), papermaking wastewater, tannery wastewater, new energy lithium battery disassembly and recovery and other wastewater, and has a better effect in a low-salt environment of less than 1%. The strain is used for biological denitrification and COD degradation in low-salt, high-salt and high-temperature environments, can effectively solve the problems of biological denitrification and COD degradation in the high-salt and high-temperature environments, reduces the treatment cost, and has good application prospects and popularization values in high-salt biochemical systems, nitrogen-containing and refractory COD wastewater, and particularly in sewage treatment plants with covered biochemical systems in summer.

Description

Technical Field

[0001] This invention relates to the field of environmental remediation microorganisms, and in particular to a salt-tolerant and heat-resistant Haloxylon ammodendron (Haloxylon esculenta). Halomonas arcis LH-B.0008, the present invention also relates to the use of this strain. Background Technology

[0002] High-salinity wastewater originates widely from various industries, including chemical, pharmaceutical, electroplating, leather tanning, and the dismantling and recycling of new energy lithium batteries. It is characterized by high salinity, persistent COD, and high nitrogen content. With increasingly stringent environmental regulations requiring covered wastewater treatment systems for centralized waste gas collection and treatment, the temperature of biochemical systems can reach 38–45°C in summer. Traditional biological treatment methods become inefficient or even fail to operate normally under these high-salinity and high-temperature conditions.

[0003] Traditional biological nitrogen removal and COD degradation methods face numerous challenges when treating high-salt and high-temperature wastewater. High salinity dehydrates microbial cells, leading to reduced or even inactivated enzyme activity, thus affecting normal metabolism and growth. High temperatures can damage the protein and nucleic acid structures within microorganisms, similarly hindering their degradation of pollutants in wastewater. Under these extreme conditions of high salinity and high temperature, conventional microbial communities struggle to survive and function, resulting in extremely low efficiency in biological nitrogen removal and COD degradation.

[0004] Currently, the main methods for treating high-salinity, high-temperature wastewater include physicochemical and biological methods. Physicochemical methods, such as evaporation concentration, ion exchange, and advanced oxidation, can remove pollutants to a certain extent, but they suffer from high costs, high energy consumption, and the potential for secondary pollution. For example, evaporation concentration requires a large amount of heat energy, resulting in high equipment investment and operating costs; single advanced oxidation methods, such as Fenton oxidation, ozone oxidation, and electrochemical oxidation, are energy-intensive and costly, and some may even cause secondary pollution.

[0005] Biological methods have attracted widespread attention due to their advantages such as low cost and environmental friendliness. However, as mentioned earlier, conventional microorganisms are not very effective in treating wastewater under high-salt and high-temperature environments. Developing microbial strains and treatment technologies that can adapt to high-salt and high-temperature environments and efficiently carry out biological denitrification and COD degradation has become a key issue that urgently needs to be addressed in the field of wastewater treatment.

[0006] Regarding the challenges of denitrification under high salinity and high temperature conditions and the degradation of difficult-to-treat COD using existing technologies, several relevant patents have been published: Chinese patent publication CN115851516A discloses a thermo- and salt-tolerant aerobic denitrifying bacterium, its cultivation method, and its applications. This strain exhibits high temperature and salt tolerance (50–60℃) and high salt tolerance (4%–6%), demonstrating high aerobic denitrification capacity. Chinese patent publication CN104830710A discloses a thermo-tolerant aerobic denitrifying bacterium for denitrification in eutrophic lakes, and also for treating high-temperature wastewater after wet desulfurization processes. Chinese patent publication CN109486699A discloses a salt-tolerant aerobic denitrifying bacterium that can efficiently remove nitrate nitrogen from high-salt wastewater. Chinese patent publication CN110564642B discloses a salt-tolerant heterotrophic nitrifying aerobic denitrifying bacterium and its applications. It exhibits excellent heterotrophic nitrification and aerobic denitrification capabilities in high-salt environments. The degradation rate of ammonia nitrogen in a medium with a salt concentration of 10 wt% and an initial ammonia nitrogen concentration of 200 mg / L is as high as 99% after 45 hours. The bacteria still grow well when the salt concentration is increased to 15 wt%. All of the above technologies are solutions to the problem of salt-tolerant or high-temperature-tolerant denitrification.

[0007] Existing technologies have provided some technical experience in solving high-salt denitrification, high-temperature denitrification, or high-salt and high-temperature denitrification. However, there are few reports on simultaneous denitrification and COD degradation under high-salt and high-temperature conditions, especially for recalcitrant COD. Summary of the Invention

[0008] One technical problem this invention aims to solve is to address the shortcomings of existing technologies by providing a novel *Haloxylon ammodendron* species capable of high salt and high temperature resistance, and simultaneous denitrification. Halomonas arcis LH-B.0008 strain.

[0009] Another technical problem to be solved by the present invention is to provide the aforementioned Haloxylon ammodendron ( Halomonas arcis Applications of strain LH-B.0008: It can solve the problems of biological denitrification and COD degradation in wastewater under high salinity and / or high temperature conditions, especially the treatment of wastewater containing high salinity, high temperature, and recalcitrant COD.

[0010] To achieve the above-mentioned objectives, the present invention is implemented through the following technical solution: This invention relates to Haloxylon ammodendron (Haloxylon esculenta). Halomonas arcis The characteristics of strain LH-B.0008 are: the preservation number of strain LH-B.0008 is: CGMCC NO:16316.

[0011] The Haloxylon ammodendron disclosed in this invention ( Halomonas arcisLH-B.0008, hereinafter referred to as *Haloxylon ammodendron* LH-B.0008 or LH-B.0008. The inventors used 27F and 1492R as primers to determine the 16S rRNA gene sequence. Through comparison and phylogenetic analysis with known model organisms (http: / / www.ezbiocloud.net / eztaxon / ), it was identified as... Halomonas arcis The category is named Halomonas arcis LH-B.0008. This strain was deposited on August 20, 2018, at the China General Microbiological Culture Collection Center (CGMCC), with accession number CGMCC NO:16316. The 16S rRNA sequence of the *Haloxylon ammodendron* LH-B.0008 of this invention is 1433 bp in length.

[0012] The *Haloxylon ammodendron* LH-B.0008 of the present invention can grow in a range of salt concentrations of 0% to 30%, pH of 6 to 10, and temperatures of 8 to 50°C. The optimal growth salinity is 0% to 15%, the optimal growth pH is 7.0 to 9.0, and the optimal growth temperature is 25 to 40°C.

[0013] The *Haloxylon ammodendron* LH-B.0008 of this invention was isolated from sediments of the Qarhan Salt Lake in Qinghai Province. It grows using methanol, ethylene glycol, glycerol, methylamine, phenol, toluene, etc. as carbon sources, and ammonia nitrogen, nitrate nitrogen, nitrite nitrogen, urea, and organic amines as nitrogen sources.

[0014] This invention also discloses *Haloxylon ammodendron* (… Halomonas arcis LH-B.0008 is used in the treatment of high-salinity wastewater with a salinity of 1% to 30% and / or high-temperature wastewater at 38 to 45°C.

[0015] The preferred use of the present invention is: the use is for *Haloxylon ammodendron* (… Halomonas arcis Application of LH-B.0008 in biological denitrification and COD degradation of high-salinity wastewater (1%–30%) and / or high-temperature wastewater (38–45℃). Simultaneous denitrification and COD degradation can be carried out under anoxic or aerobic conditions.

[0016] The preferred use of the present invention is: to use the aforementioned *Haloxylon ammodendron* (… Halomonas arcis LH-B.0008 is used for low-cost treatment of COD in recalcitrant wastewater by coupling a biochemical unit with an advanced oxidation process; the advanced oxidation process includes ozone, Fenton, photocatalysis, electrochemistry and other advanced oxidation processes that can modify recalcitrant organic substances; the recalcitrant wastewater is selected from papermaking wastewater, leather tanning wastewater or wastewater from the dismantling and recycling of new energy lithium batteries.

[0017] The present invention also discloses the aforementioned Haloxylon ammodendron ( Halomonas arcisThe use of LH-B.0008 in the treatment of bottom salt wastewater with a salinity of less than 1%, the wastewater including nitrogen-containing and COD-containing wastewater, especially nitrogen-containing and COD-containing wastewater in a high-temperature environment of 38-45℃.

[0018] Preferably, the wastewater described in this invention comes from carbon fiber production wastewater containing recalcitrant acrylonitrile and high concentrations of DMSO, papermaking wastewater, leather tanning wastewater, and wastewater from the dismantling and recycling of new energy lithium batteries.

[0019] When using this invention, it is preferable to use *Haloxylon ammodendron* (… Halomonas arcis The solid microbial agent prepared by drying the fermentation broth of LH-B.0008 or its fermentation broth loading medium is then used for wastewater treatment.

[0020] When using the *Haloxylon ammodendron* LH-B.0008 of this invention, the bacteria can be fermented in a scale-up environment using tryptone, yeast extract, and various carbon sources such as methanol, ethylene glycol, glycerol, methylamine, phenol, and toluene to obtain a fermentation broth. Alternatively, the fermentation broth can be further mixed with a carrier, dried, and prepared into a solid inoculum for treating high-salt, nitrogenous, and COD-containing wastewater with a salt concentration of 1%–30%. It can also be used to treat nitrogenous and COD-containing wastewater with a salinity <1%, especially for biological denitrification and COD degradation under high-temperature conditions of 38–45℃. Simultaneous denitrification can be carried out under anoxic / aerobic conditions, achieving both effective COD degradation and co-metabolic removal of some recalcitrant organic matter.

[0021] When applying the *Haloxylon ammodendron* LH-B.0008 strain of this invention to low-cost treatment of high-salt, recalcitrant COD, the fermentation broth of *Haloxylon ammodendron* LH-B.0008 can be inoculated into an aerobic biological system at an inoculation ratio of 0.5‰ to 5‰, or solid bacterial agent at 0.1‰ to 2‰. This inoculates the strain with the system sludge to form a composite sludge floc, preferably with a biofilm carrier to form a biofilm system. Wastewater modified by ozone oxidation or Fenton oxidation is then inoculated into the composite sludge floc or biofilm system to promote the treatment of recalcitrant wastewater to meet standards. Compared to ozone or Fenton oxidation alone, this method saves more than 30% in costs and has a strong bio-enhancing effect. It can be coupled with ozone or Fenton oxidation for low-cost treatment of recalcitrant COD in wastewater.

[0022] The preferred strain of this invention is *Haloxylon ammodendron* (…). Halomonas arcis The preparation method of LH-B.0008 fermentation broth is as follows: Add 0.5% sodium chloride salt to culture medium 1 by weight / volume percentage as fermentation culture medium. The formula of culture medium 1 is as follows: 10 g / L tryptone, 5 g / L yeast extract, 0.2 ml / L methanol, 0.2 ml / L ethylene glycol, 0.1 ml / L glycerol, 0.1 ml / L methylamine, 0.2 g / L phenol, 0.1 ml / L toluene, 1 ml / L trace elements, and pH 7.0–8.0. Primary fermentation: *Haloxylon ammodendron* (… Halomonas arcis The slant culture of LH-B.0008 was transferred to the fermentation broth and cultured on a shaker at 37℃ and 120r / min for 48h to obtain the primary fermentation broth. Secondary scale-up fermentation: 10% of the primary fermentation broth was transferred into 2L of fermentation culture medium and cultured on a shaker at 37℃ and 160r / min for 48h to obtain the secondary fermentation broth; Three-stage scale-up fermentation: 2L of secondary fermentation broth was inoculated into a 20L aerobic fermenter for further scale-up fermentation. A fed-batch culture was used, with a feeding time of 36 hours and a total culture time of 48 hours. The culture temperature was 37℃, the stirring speed was 160 rpm, and the dissolved oxygen was controlled at 2.0–6.0 mg / L. After 48 hours, *Haloxylon ammodendron* was obtained. Halomonas arcis LH-B.0008 fermentation broth.

[0023] The preferred strain of this invention is *Haloxylon ammodendron* (…). Halomonas arcis The preparation method of LH-B.0008 solid microbial agent is as follows: The above-preferred method was used to prepare *Haloxylon ammodendron* ( Halomonas arcis The fermentation broth of LH-B.0008 was concentrated by sedimentation to obtain a concentrated bacterial solution, which was then mixed with a carrier to prepare a solid bacterial agent. The concentrated bacterial solution (75-85 parts) and the carrier (15-25 parts) were stirred until homogeneous, and then dried in a fluidized bed at 45°C to obtain the solid bacterial agent. The carrier was any one or more of plant straw powder, rice husk powder, natural mineral carriers, and polysaccharide carriers.

[0024] The application of the *Haloxylon ammodendron* LH-B.0008 of the present invention is also reflected in actual wastewater treatment: it can be used for wastewater from carbon fiber production, electroplating, papermaking, leather tanning, and the dismantling and recycling of new energy lithium batteries, especially for the treatment of the above-mentioned wastewater in high-temperature environments during summer.

[0025] Compared with the prior art, the present invention has the following beneficial effects: 1. The *Haloxylon ammodendron* LH-B.0008 of this invention is tolerant to high salt and high temperature, with a wide salt tolerance range of 0-30%. It can denitrify and degrade COD within a high salt range of 1-30%. It still has high efficiency in denitrification and COD degradation at high temperatures of 38-45°C, which can improve denitrification efficiency by more than 30% and COD treatment efficiency by 5-15%. The higher the temperature, the more significant the enhancement effect on the system. It is suitable for addition to covered biochemical systems when operating in high-temperature environments in summer to enhance the treatment capacity of biochemical systems under high-temperature environments.

[0026] 2. The *Haloxylon ammodendron* LH-B.0008 of this invention, in the low-cost treatment of high-salt, recalcitrant COD, is used in the coupled treatment of biochemical units and advanced oxidation processes. Compared to the control group without the strain, it improves COD treatment efficiency by more than 30%. Furthermore, compared to simple advanced oxidation processes such as ozone and Fenton oxidation, it saves more than 30% in costs, exhibiting a strong bio-enhancing effect. It can be coupled with advanced oxidation processes for low-cost treatment of recalcitrant COD in wastewater. *Haloxylon ammodendron* LH-B.0008 demonstrates significant treatment effects in actual wastewater treatment systems without altering existing structures, effectively enhancing the treatment capacity of biochemical systems. It shows promising application prospects in the treatment of wastewater from carbon fiber production, electroplating, papermaking, leather tanning, and the dismantling and recycling of new energy lithium batteries, especially in high-temperature environments during summer. Attached Figure Description

[0027] Figure 1 It is Haloxylon ammodendron ( Halomonas arcis Phylogenetic tree diagram of LH-B.0008 and known model bacteria constructed using the maximum parsimony method based on 16S rRNA; Haloxylon ammodendron ( Halomonas arcis The biomaterial preservation information for LH-B.0008 is as follows: LH-B.0008, classified and named... Halomonas arcis, The deposit date is August 20, 2018. The depositary institution is the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences. The accession number is CGMCC NO:16316. Detailed Implementation

[0028] The present invention will be further described below with reference to specific embodiments, but the present invention is not limited to the following embodiments. Unless otherwise specified, the following embodiments are all conventional methods, and the materials and reagents used can be purchased from biological or chemical reagent companies.

[0029] The following are some of the culture media involved in the examples: Culture medium 1: Supplementing the culture medium based on tryptone and yeast extract with multiple carbon sources and toxic organic matter enhances the strain's adaptability to multiple carbon sources and tolerance to toxic environments. The formula is: 10 g / L tryptone, 5 g / L yeast extract, 0.2 ml / L methanol, 0.2 ml / L ethylene glycol, 0.1 ml / L glycerol, 0.1 ml / L methylamine, 0.2 g / L phenol, 0.1 ml / L toluene, 1 ml / L trace elements, and pH 7.0–8.0.

[0030] Culture medium 2: glucose 0.5 g / L, methanol 0.5 ml / L, ethylene glycol 0.2 ml / L, glycerol 2 mL / L, phenol 0.1 g / L, aniline 0.05 ml / L, peptone 1 g / L, dipotassium hydrogen phosphate 0.3 g / L, potassium dihydrogen phosphate 0.1 g / L, ammonium chloride 0.5 g / L, MgSO4 0.5 g / L, NaCl 250 g / L; trace elements 1 mL / L, pH 7.0–8.0; agar 2%.

[0031] Trace elements (1000mL): H3BO3 0.5 g, MnSO4·7H2O 0.5 g, ZnSO4·7H2O 0.5 g CuSO4·5H2O 0.025 g, Na2MoO4·2H2O 0.025 g, CoCl2·6H2O 0.025g, NiCl2·6H2O 0.025 g, FeCl3·6H2O 2.0 g, Na2EDTA 5.0 g Example 1: Isolation, identification, and preservation of *Haloxylon ammodendron* LH-B.0008 Haloxylon ammodendron LH-B.0008 was isolated from sediments of the Qarhan Salt Lake in Qinghai Province.

[0032] Enrichment and screening of bacterial strains: 25% sodium chloride was added to culture medium 1, and the sediment samples of Qinghai Chaka Salt Lake were enriched and cultured under the conditions of 37℃ and 120r / min shaking. Gram staining and microscopic examination were performed regularly until the bacterial solution was enriched to a certain concentration and abundance of bacteria, thus obtaining a high-salt bacterial community enrichment solution.

[0033] Strain isolation: Solid isolation medium was prepared by adding 2% agar to medium 2 for later use.

[0034] The streak plating method was used for separation. One loop of the enriched solution was streaked onto a solid separation medium, sealed with sealing film, and incubated upside down in a constant temperature incubator at 37°C for 3 days. For plates with incomplete separation, streak plating was repeated until a large number of single colonies were obtained. Strain purification: When the colonies on the plate have grown to a point where their appearance characteristics are easily identifiable, select colonies of different shapes and colors and streak them on fresh culture medium for purification until single colonies with distinct characteristics and vigorous growth are obtained.

[0035] Strain identification and preservation: Single colony pure cultures were sent to the China Agricultural Microbial Culture Collection Center for identification. A 1433 bp gene fragment was obtained based on the 16S gene sequence amplified by 27F / 1492R. The fragment was compared with known type bacteria via http: / / www.ezbiocloud.net / eztaxon / . The comparison results and phylogenetic analysis are attached. Figure 1 It shares 99.36% similarity with the known species *Halomonas arcis* and is classified as *Halomonas arcis* LH-B.0008. It was deposited on August 20, 2018, at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC NO.16316.

[0036] Example 2: Preparation of fermentation broth and solid inoculum of *Haloxylon ammodendron* LH-B.0008 (1) Preliminary exploration of the salt tolerance of the strain and its growth rate under different salinity levels Sodium chloride was added to culture medium 1 at concentrations of 0 g / L, 10 g / L, 50 g / L, 100 g / L, 150 g / L, 200 g / L, and 250 g / L, respectively, to prepare culture solutions with sodium chloride salinities of 0%, 1%, 5%, 10%, 15%, 20%, and 25%. These solutions were dispensed into 500 ml Erlenmeyer flasks and sterilized. Well-developed bacterial colonies of *Haloxylon ammodendron* LH-B.0008 were inoculated into physiological saline to prepare high-concentration bacterial suspensions. These suspensions were then inoculated into Erlenmeyer flasks containing different salinities (10 ml / flask) and cultured at 37°C on a shaker at 120 rpm. Water was replenished during culture to prevent changes in salt concentration. After 48 hours of culture, the OD600 values ​​were measured and found to be 2.34, 2.23, 2.05, 1.99, 1.65, 1.88, and 1.46, respectively. The OD600 trend shows that this strain can grow in a salinity range of 0-25%, making it a typical salt-tolerant bacterium. Its growth rate is particularly faster in salt-free and low-salt environments, consistent with the growth characteristics of salt-tolerant bacteria. The optimal salinity for fermentation culture was 0-1%.

[0037] (2) Preparation of fermentation broth of Haloxylon ammodendron LH-B.0008 0.5% sodium chloride was added to culture medium 1 as the fermentation broth. Primary fermentation: The slant culture of *Haloxylon ammodendron* was transferred to the fermentation broth and cultured at 37℃ and 120 rpm for 48 h on a shaker to obtain a primary fermentation broth with an OD600 value of 2.3. Secondary scale-up fermentation: 10% of the primary fermentation broth was transferred to 2 L of fermentation broth and cultured at 37℃ and 160 rpm for 48 h on a shaker to obtain a secondary fermentation broth with an OD600 value of 2.58. Tertiary scale-up fermentation: 2 L of the secondary fermentation broth was inoculated into a 20 L aerobic fermenter for further scale-up fermentation. A fed-batch culture was used with a feeding time of 36 h, a culture time of 48 h, a culture temperature of 37℃, a stirring speed of 160 rpm, and dissolved oxygen controlled at 2.0–6.0 mg / L. After 48 h, a fermentation broth with an OD600 value of 2.73 was obtained, completing the preparation of the *Haloxylon ammodendron* fermentation broth and obtaining a mature fermentation broth.

[0038] (3) Preparation of solid inoculum of Haloxylon ammodendron LH-B.0008 The above fermentation broth was sedimented and concentrated to obtain concentrated bacterial solution. Using straw powder as a carrier, 80 parts of concentrated bacterial solution and 20 parts of straw powder were stirred and mixed evenly, and then dried in a fluidized bed at 45°C to complete the preparation of solid bacterial agent.

[0039] During the cultivation process, samples can be taken daily using a microscope to check for the presence of contaminating microorganisms; at the same time, the growth and morphological changes of the strain can be observed. The experimental results show that strain LH-B.0008 can be rapidly fermented and scaled up, indicating that LH-B.0008 has the potential for large-scale production and engineering applications.

[0040] Example 3: Salt tolerance test of Haloxylon ammodendron LH-B.0008 (1) Culture media 1 with sodium chloride was used to prepare culture solutions with salinity gradients of 0%, 0.5%, 1%, 3%, 5%, 10%, 15%, 20%, 25%, 28%, 30%, 31%, and 32%. Blank culture medium was used as a control. (2) Inoculation and culture: The bacterial suspension of Haloxylon ammodendron LH-B.0008 was prepared according to the method in Example 2 and inoculated into culture media of various salinities, 1 ml / bottle. The initial OD600 of the experimental group after inoculation was measured to be 0.1-0.15. The culture was carried out at 37℃ and 160 r / min for 48 h. The OD600 value after culture was measured to judge the growth of the bacterial strain.

[0041] (3) Experimental results: The salt tolerance range of LH-B.0008 strain is 0%–30%. The blank control medium was clear and transparent. The turbidity of LH-B.0008 strain tended to increase after 48 h of culture in medium with a salinity of 0%–30% (see Table 1), but no significant change was observed in medium with a salinity of 31% and 32% (near saturation). This indicates that LH-B.0008 can grow rapidly in the salt concentration range of 0%–30%, and LH-B.0008 is a salt-tolerant bacterium with a salt tolerance range of 0–30%.

[0042] Table 1. OD600 values ​​of LH-B.0008 after 48 h of culture in different salt concentrations.

[0043] Example 4: Growth Temperature Test of Haloxylon ammodendron LH-B.0008 (1) Using culture medium formula 1, the temperature gradient was set at 0℃, 5℃, 8℃, 10℃, 15℃, 20℃, 25℃, 30℃, 37℃, 40℃, 45℃, 48℃, 50℃, and 52℃, with a blank culture medium as a control. (2) Inoculation and culture: The bacterial suspension of *Haloxylon ammodendron* LH-B.0008 was prepared according to the method in Example 2 and inoculated into the test culture medium at various temperatures, 1 ml / bottle. The initial OD600 of the test group after inoculation was measured to be 0.1-0.15. The temperature was controlled by a 0℃ refrigerator at 0℃, and the culture was statically incubated with intermittent shaking. The temperature was controlled by a constant temperature shaker at 5℃, 8℃, 10℃, 15℃, 20℃, 25℃, 30℃, 37℃, 40℃, 42℃, 45℃, 48℃, 50℃, and 52℃. The culture was carried out at 120-160 r / min for 24 h. The OD600 value after culture was measured to judge the growth of the bacterial strain.

[0044] (3) Experimental results: The obvious growth temperature range of LH-B.0008 strain is 8–50℃. The blank culture medium is clear and transparent. As a blank sample, the turbidity of LH-B.0008 strain tends to increase after 24 hours of culture at 8–50℃ (see Table 2), but there is no obvious change when cultured at temperatures above 50℃. This indicates that LH-B.0008 can proliferate and grow within the temperature range of 8–50℃, and its growth outside this temperature range is unclear.

[0045] Table 2. OD600 values ​​of LH-B.0008 after 24 hours of incubation at different temperatures.

[0046] Example 5: Salt-tolerant simultaneous denitrification and COD degradation test of *Haloxylon ammodendron* LH-B.0008 (1) Culture medium: The dosage of the drug is calculated based on COD. The dosage is 300 mg / L of glucose, 200 mg / L of methanol, 200 mg / L of ethylene glycol, 100 mg / L of glycerol, and 200 mg / L of phenol. The nitrogen sources are ammonium chloride, potassium nitrate, sodium nitrite and urea. Ammonia nitrogen, nitrate nitrogen, nitrite nitrogen and organic nitrogen are added respectively to make the total nitrogen concentration reach 50 mg / L. The salinity is set to 0% to 30%. 10 ml of trace elements are added and the pH is adjusted to 7.2.

[0047] (2) Experimental design: The experiment was conducted in 500mL Erlenmeyer flasks, with 500mL of culture medium added to each flask. The salinity gradients were set as 0%, 1%, 3%, 5%, 10%, 15%, 20%, 25%, and 30% (no additional salt was added at 0% salinity).

[0048] (3) Salt-tolerant simultaneous denitrification and COD removal test: After sterilization and cooling, all samples were inoculated with approximately 30 mL of LH-B.0008 culture medium and tested on a constant-temperature magnetic stirrer at 30℃ and 120 r / min. Samples were taken at 24 h to determine the concentrations of TN and COD in the Erlenmeyer flasks. The test results are shown in Table 3 below. Table 3. Test results of simultaneous denitrification and COD removal of LH-B.0008 at different salt concentrations (Unit: mg / L)

[0049] The experimental results show that *Haloxylon ammodendron* LH-B.0008 has the effect of simultaneous denitrification and COD degradation in high salinity environments of 1–30%, and the effect is better in salinity treatment of <1%. The nitrogen source for denitrification can be ammonia nitrogen, nitrate nitrogen, nitrite nitrogen, or organic nitrogen.

[0050] Example 6: Experiment on the effect of *Haloxylon ammodendron* LH-B.0008 on simultaneous denitrification and COD degradation in an enhanced anoxic / aerobic system under high temperature and high salinity conditions of 38–45℃. The propylene oxide production project generates high-salinity wastewater with a salinity of 5%. The top of the biological system tank is sealed for waste gas collection. During the summer, the on-site temperature reaches a maximum of 45℃. The system's performance gradually deteriorates between 38 and 45℃, failing to consistently meet standards. The on-site process involves hydrolysis + anoxic + aerobic + sedimentation + deep treatment. The anoxic tank has a retention time of 4–8 hours due to fluctuations in influent flow, while the aerobic tank has a retention time of 8–16 hours. The *Haloxylon ammodendron* LH-B.0008 fermentation broth is added to both the anoxic and aerobic tanks at a dosage of 2‰ of the tank volume. The average effluent quality of the production lines with and without the added broth during different high-temperature periods (based on the tank water temperature) is shown in Table 4 below. Table 4. Results of simultaneous denitrification and COD removal tests of LH-B.0008 at different salt concentrations (Unit: mg / L)

[0051] The results showed that the addition of *Haloxylon ammodendron* LH-B.0008 could improve the simultaneous denitrification and COD degradation of the high-temperature, high-salt anoxic / aerobic system. The denitrification effect was significantly improved, by more than 30%, and the COD treatment effect was improved by 5-15%. The higher the temperature, the more significant the enhancement effect of the thermotolerant bacterium LH-B.0008 on the system.

[0052] Example 7: Experimental use of *Haloxylon ammodendron* LH-B.0008 in carbon fiber production wastewater containing recalcitrant acrylonitrile and high concentrations of DMSO. The application of *Haloxylon ammodendron* LH-B.0008 in carbon fiber production wastewater containing recalcitrant acrylonitrile and high concentrations of DMSO, the water quality indicators of which are shown in Table 5 below: Table 5. Wastewater Quality in Carbon Fiber Production

[0053] The treatment process consisted of pre-aerobic + anoxic + aerobic + sedimentation tank. The pre-aerobic retention time was 20 hours, the anoxic retention time was 16 hours, and the aerobic retention time was 32 hours. The system was inoculated with sludge from the on-site biological treatment tank. The fermentation broth of *Haloxylon ammodendron* LH-B.0008 was added to the pre-aerobic, anoxic, and aerobic tanks at a dosage of 1‰ of the tank volume. The control group (within the same sequence) did not receive any bacterial agent. After 30 days of stable treatment, the data are shown in Table 6 below. Table 6. Effect of *Haloxylon ammodendron* LH-B.0008 on wastewater treatment in carbon fiber production.

[0054] The results showed that using *Haloxylon ammodendron* LH-B.0008 to treat carbon fiber production wastewater containing recalcitrant acrylonitrile and high concentrations of DMSO yielded significant COD removal, along with substantial reductions in acrylonitrile and DMSO. *Haloxylon ammodendron* utilized readily degradable substances in the fermentation broth and wastewater to co-metabolize the recalcitrant acrylonitrile, simultaneously removing most of the recalcitrant acrylonitrile while degrading readily degradable COD. Compared to the control group, *Haloxylon ammodendron* LH-B.0008 demonstrated significant degradation effects on acrylonitrile and DMSO in the enhanced application of carbon fiber production wastewater containing recalcitrant acrylonitrile and high concentrations of DMSO, achieving COD removal rates exceeding 90% and total nitrogen removal rates exceeding 90%.

[0055] Example 8: Experimental use of *Haloxylon ammodendron* LH-B.0008 in high-salt nitrogen-containing electroplating wastewater Application of *Haloxylon ammodendron* LH-B.0008 in high-salt, nitrogen-containing electroplating wastewater. This wastewater exhibits fluctuating water quality, with water quality indicators ranging from 1% to 5% salinity, 100 to 1000 mg / L COD, and NH3... -N was 40–200 mg / L, NO2-N was 100–300 mg / L, and TN was 200–500 mg / L. The wastewater biological treatment process was: primary anoxic / aerobic + secondary sedimentation tank ① + secondary anoxic / aerobic + secondary sedimentation tank ②. The retention times in the primary anoxic / aerobic tanks were 20 h / 25 h and 10 h / 20 h, respectively. The system was inoculated with sludge from the on-site biological treatment tank. The *Haloxylon ammodendron* LH-B.0008 solid bacterial agent was added to the anoxic and aerobic tanks at dosages of 0.5‰, 1‰, 0.5‰, and 1‰ of the tank volume, respectively. The control group did not receive any bacterial agent. Other process parameters were the same. Multiple batches of water samples were run for 30 days. The average influent and effluent data for the groups with and without LH-B.0008 bacterial agent are shown in Table 7 below. Table 7. Effect of *Haloxylon ammodendron* LH-B.0008 on high-salt, nitrogen-containing electroplating wastewater.

[0056] The results showed that the addition of Haloxylon ammodendron LH-B.0008 could enhance the treatment effect of the biochemical system for electroplating wastewater under high salinity conditions, increasing denitrification capacity by more than 40% and COD degradation capacity by more than 20%.

[0057] Example 9: Experimental use of *Haloxylon ammodendron* LH-B.0008 in papermaking wastewater containing recalcitrant COD. A paper mill in a chemical industrial park uses waste paper pulp and finished wood pulp as its main raw materials. This type of wastewater is characterized by high organic load, high suspended solids concentration, large fluctuations in water quality, and high hardness. The wastewater discharged into the downstream sewage treatment plant is particularly difficult to biodegrade. The downstream sewage treatment plant's process is: hydrolysis acidification tank + A / O + secondary sedimentation tank + Fenton treatment. The waste paper mill's influent has low biodegradability, with a COD of 220 mg / L. Other indicators meet standards, but the effluent is required to have a COD < 50 mg / L. On-site biochemical COD removal rate is only about 10%. Fenton treatment is needed to meet the standards, but the cost of Fenton treatment is high, reaching 20 yuan per ton of water treated.

[0058] The fermentation broth of *Haloxylon ammodendron* LH-B.0008 obtained in Example 2 was introduced into the A / O tank at dosages of 1‰ and 2‰, respectively. Fenton oxidation was used to reduce the dosage, and UV254 was used to control the modification effect, keeping the cost of Fenton reagent below 3 yuan. The effluent was then recycled back to tank A for Fenton and *Haloxylon ammodendron* bio-enhanced coupled treatment. The control group only added Fenton oxidation effluent recycling without adding LH-B.0008 fermentation broth. Results showed that the system effluent COD was around 150 mg / L in the group with only Fenton effluent recycling and no LH-B.0008 fermentation broth, failing to meet the standard. After the Fenton and *Haloxylon ammodendron* bio-enhanced coupled group stabilized, the effluent COD could be consistently <50 mg / L, consistently meeting the standard. The treatment cost, calculated based on the costs of chemicals, electricity, and sludge, was more than 50% lower than that of Fenton treatment alone, while ensuring stable effluent COD compliance.

[0059] The feasibility of using ozone-biochemical coupling treatment for the above-mentioned wastewater was investigated. The fermentation broth of *Haloxylon ammodendron* LH-B.0008 obtained in Example 2 was introduced into the A / O tank at dosages of 1‰ and 2‰, respectively. Ozone or ozone catalytic oxidation was used for treatment, with UV254 controlling the modification effect and keeping the ozone process cost below 3 yuan. The ozone effluent was recycled back to tank A for further coupling treatment with *Haloxylon ammodendron* bio-enhanced treatment. A control group was set up with only ozone effluent recycling and no *Haloxylon ammodendron* fermentation broth added. Results showed that in the group with only ozone effluent recycling and no *Haloxylon ammodendron* fermentation broth, the system effluent COD was around 120 mg / L, which did not meet the standards. After the ozone and *Haloxylon ammodendron* bio-enhanced coupling group stabilized, the effluent COD could be stably maintained below 50 mg / L, achieving stable treatment compliance.

[0060] Example 10: Application of *Haloxylon ammodendron* LH-B.0008 in the treatment of recalcitrant effluent from high-salt tanning wastewater to meet standards. *Haloxylon ammodendron* LH-B.0008 is used for the treatment of high-salinity tanning wastewater that is difficult to degrade, achieving compliance standards. This high-salinity tanning wastewater exhibits fluctuating inflow water quality, with effluent quality indicators ranging from salinity 2-3%, COD 200-400 mg / L, high alkalinity, high hardness, and high color. After upstream treatment, the inflow water has low biodegradability. The on-site treatment process involves hydrolysis + A / O + A / O + MBR + flotation + ozone, achieving a biochemical COD removal rate ≤30%, with the effluent COD around 250 mg / L. Further ozone treatment only removes approximately 20 mg / L of COD, and the O / C ratio is >4, resulting in low ozone utilization and failure to meet standards. Increasing the ozone dosage can achieve compliance, but the treatment cost is over 6 yuan.

[0061] The fermentation broth of *Haloxylon ammodendron* LH-B.0008 obtained in Example 2 was introduced into the A / O tank. The dosage of both the pre-A / O and post-A / O tanks was 1‰. Ozone oxidation was controlled with UV254 to control the modification effect. The effluent was returned to the pre-A / O tank. After stable operation, the effluent COD was consistently <50mg / L, meeting the standard. The overall O / C ratio was ≤2, and the cost per ton of water treatment was ≤4 yuan. The enhanced treatment of high-salt tanning wastewater and the coupled ozone treatment with *Haloxylon ammodendron* LH-B.0008 can stably meet the standards, saving more than 30% in cost per ton of water treatment.

[0062] Example 11: Experimental use of *Haloxylon ammodendron* LH-B.0008 in 6%–12% high-salt wastewater from the dismantling and recycling of new energy lithium batteries. The fermentation broth of *Haloxylon ammodendron* LH-B.0008 obtained in Example 2 is used for the treatment of wastewater from the dismantling and recycling of high-salt new energy lithium batteries. The wastewater from the dismantling and recycling of new energy lithium batteries produces various types of wastewater depending on the on-site production process. The water quality indicators range from 6% to 15% sodium sulfate salinity, 200 to 1200 mg / L COD, and 40 to 200 mg / L NH3-N. The wastewater biochemical treatment process is: anoxic / aerobic + coagulation sedimentation tank + ozone oxidation + contact oxidation + secondary sedimentation tank. Under high-salt conditions, it is difficult to establish nitrification to remove ammonia nitrogen, making ammonia nitrogen treatment difficult. In addition, ordinary biochemical systems in high-salt environments cannot achieve COD standards. The experiment started with low-salinity operation and gradually switched to high-salinity wastewater. Fermentation broth containing LH-B.0008 halophilus was added to the anoxic / aerobic and contact oxidation tanks. The contact oxidation tank was operated after full aeration and biofilm formation. Two treatment sequences were set up to compare the addition of LH-B.0008 fermentation broth with and without LH-B.0008 fermentation broth, and other process parameters were the same.

[0063] The fermentation broth containing Haloxylon ammodendron LH-B.0008 was added to anoxic / aerobic and contact oxidation tanks, with concentrations of 1‰, 2‰, and 1‰ of the tank volume, respectively.

[0064] The range of influent and effluent water quality indicators for wastewater treatment systems with and without LH-B.0008 added after 50 days of operation is shown in Table 8.

[0065] Table 8. Treatment effect of the present invention, *Haloxylon ammodendron* LH-B.0008, on high-salt wastewater from the dismantling and recycling of new energy lithium batteries after stable operation.

[0066] The above description is merely a preferred embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any person skilled in the art can easily modify the technical solutions described in the foregoing embodiments or make equivalent substitutions for some of the technical features within the scope of the technology disclosed in the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A Chromobacterium acetoabidum strain LH-B.0008, characterized by: The strain is named as Halomonas arcis , and the accession number is: CGMCC NO:16316.

2. Use of the Chromobacterium aciculatum LH-B.0008 as claimed in claim 1, characterized in that, The use is that the Halomonas arcis LH-B.0008 is used for high-salinity wastewater treatment with salinity of 1% to 30% and / or high-temperature wastewater treatment at 38 to 45°C.

3. Use according to claim 2, characterized in that: The use is that the Halomonas arcis LH-B.0008 is used for high-salinity wastewater treatment with salinity of 1% to 30% and / or high-temperature wastewater treatment at 38 to 45°C.

4. Use according to claim 2, characterized in that: The synchronous denitrification and COD degradation are carried out under anoxic or aerobic conditions.

5. Use according to claim 2, characterized in that: The use of the Halomonas arcis LH-B.0008 is used for low-cost standard treatment of refractory wastewater COD by coupling biochemical unit and advanced oxidation process; the refractory wastewater is selected from papermaking wastewater, tanning wastewater or new energy lithium battery recycling wastewater.

6. Use of the Chromobacterium aciculatum LH-B.0008 as claimed in claim 1, characterized in that, The use is that the Halomonas arcis LH-B.0008 is used for low-salinity wastewater treatment with salinity less than 1%, and the wastewater includes nitrogen-containing and COD-containing wastewater, especially nitrogen-containing and COD-containing wastewater at 38 to 45°C high-temperature environment.

7. Use according to any one of claims 2 to 6, characterized in that: The wastewater is from carbon fiber production wastewater containing refractory acrylonitrile and high-concentration DMSO, papermaking wastewater, tanning wastewater and new energy lithium battery recycling wastewater.

8. Use according to any one of claims 2 to 6, characterized in that: The Halomonas arcis LH-B.0008 fermentation broth or the solid bacterial agent prepared by drying the fermentation broth of the Halomonas arcis LH-B.0008 is used for wastewater treatment.

9. Use according to claim 8, characterized in that: The preparation method of the Halomonas arcis LH-B.0008 fermentation broth is as follows: 0.5% sodium chloride is added in the medium 1 as fermentation medium, and the formula of the medium 1 is as follows: 10 g / L of tryptone, 5 g / L of yeast extract, 0.2 ml / L of methanol, 0.2 ml / L of ethylene glycol, 0.1 ml / L of glycerol, 0.1 ml / L of methylamine, 0.2 g / L of phenol, 0.1 ml / L of toluene, 1 ml / L of trace elements, and pH is 7.0 to 8.0; Primary fermentation: the slope strain of the Halomonas arcis LH-B.0008 is inoculated into the fermentation medium, and the culture is carried out at 37°C and 120 r / min for 48 h to obtain the primary fermentation broth; Secondary amplification fermentation: the primary fermentation broth is inoculated into 2 L of fermentation medium at 10%, and the culture is carried out at 37°C and 160 r / min for 48 h to obtain the secondary fermentation broth; Tertiary amplification fermentation: 2 L of the secondary fermentation broth is inoculated into 20 L of an aerobic fermentation tank for further amplification culture, and the fermentation medium is added for culture, the adding time is set to 36 h, the culture time is 48 h, the culture temperature is 37°C, the stirring speed is 160 r / min, and the dissolved oxygen is controlled to be 2.0 to 6.0 mg / L; the Halomonas arcis LH-B.0008 fermentation broth is obtained after 48 h.

10. Use according to claim 8, characterized in that: The preparation method of the solid bacterial agent of the Halomonas arcis LH-B.0008 is as follows: The concentrated bacteria solution of the Lecythophora hynnosa LH-B.0008 prepared by the method of claim 9 is concentrated by sedimentation, and is mixed with a carrier to prepare a solid bacteria agent; 75-85 parts of the concentrated bacteria solution and 15-25 parts of the carrier are stirred and mixed uniformly, and are dried in a fluidized bed at 45°C to obtain the solid bacteria agent; the carrier is any one or several of plant straw powder, rice husk powder, natural mineral carrier, and polysaccharide carrier.

Images