Rhodobacter sphaeroides for degrading 2, 4-dichlorophenol and producing hydrogen through fermentation and application of rhodobacter sphaeroides in wastewater degradation

Abstract

The invention discloses rhodobacter sphaeroides capable of degrading 2, 4-dichlorophenol and producing hydrogen through fermentation and application of the rhodobacter sphaeroides in wastewater degradation, and belongs to the technical field of wastewater treatment. The strain can be used for removing 2, 4-dichlorophenol (2, 4-DCP) and can be used for efficiently producing hydrogen. A microbial agent which is developed by utilizing the strain and is used for synchronously degrading 2, 4-dichlorophenol through adsorption-photocatalysis and microorganisms is combined with an advanced oxidation technology and a biological treatment technology, and hydrogen is obtained as a high-added-value product, so that biomass energy is fully utilized, renewable utilization of energy is realized, and environmental pollution is reduced. And the purposes of removing water pollutants and efficiently producing clean energy are achieved.

Description

Technical Field

[0001] The invention belongs to the technical field of wastewater treatment and relates to a Rhodobacterium sphaeroides strain capable of degrading 2,4-dichlorophenol and simultaneously producing hydrogen by fermentation, and application thereof in wastewater degradation. Background Art

[0002] Phenolic wastewater is a common pollutant in industrial wastewater. 2,4-Dichlorophenol (2,4-DCP), a typical chlorophenol, is highly toxic and difficult to degrade, posing a serious threat to the ecological environment and human health. Once it enters water bodies, it not only damages aquatic ecosystems but can also accumulate through the food chain, ultimately endangering human health. Therefore, developing efficient and environmentally friendly phenolic wastewater treatment technologies is of great practical significance.

[0003] Currently, methods for treating 2,4-DCP primarily include physical, chemical, and biological methods. While physical and chemical methods can remove 2,4-DCP to a certain extent, they often suffer from high energy consumption, high costs, and the potential for secondary pollution. In contrast, biological methods have attracted widespread attention due to their environmental friendliness and low costs. However, traditional biological treatment technologies often suffer from limitations such as low degradation efficiency and long treatment times when treating high-concentration or recalcitrant organic pollutants. Advanced oxidation processes (AOPs) have demonstrated significant advantages in the treatment of phenolic wastewater, offering high efficiency in degrading recalcitrant organic matter, rapid reaction times, and a wide range of applications. However, AOPs typically consume large amounts of energy and chemical reagents and may generate secondary pollution, limiting their large-scale application. Therefore, the search for green treatment technologies that can both efficiently degrade phenolic pollutants and achieve resource utilization has become a hot topic.

[0004] Rhodobacterium sphaeroides ( Rhodobacter sphaeroides) is a photosynthetic bacterium capable of decomposing organic matter to produce hydrogen under anaerobic light conditions. It is currently primarily used in bioenergy production, environmental remediation, synthesis of high-value-added products, and agricultural applications. Chinese Patent No. CN110343643B discloses a strain of Rhodobacter sphaeroides that can produce hydrogen through photofermentation using a wide range of carbon and nitrogen sources. Its cumulative hydrogen production reaches a maximum of 2954 mL / L, and it is used to degrade peanut shell hydrolyzate. Chinese Patent No. 2006101175136 discloses a strain of Rhodobacter sphaeroides that can be used with organic acids, sugars, or alcohols. Its hydrogen conversion rate exceeds 70% using butyrate as a substrate, but it does not involve pollutant degradation. CN105461082A reports the use of Rhodobacter sphaeroides in the treatment of soybean wastewater, primarily involving small molecule carbon and nitrogen sources. Soybean wastewater is non-toxic and harmless, and the goal is to obtain bacterial resources for use in agriculture, animal husbandry, or fisheries. Han Qingli et al. published a paper titled "Protective Effects of Rhodobacter sphaeroides on Zebrafish Exposed to Dichlorvos," reporting that Rhodobacter sphaeroides could remove over 90% of the organophosphorus pesticide dichlorvos at a concentration of 33.6 mg / L within 24 hours, providing scientific evidence for the water pollutant degradation capabilities of Rhodobacter sphaeroides. CN107513513B reported a strain of Rhodobacter sphaeroides that efficiently degraded furfural toxins, but lacked other properties. Jiao Haihua et al. published a paper titled "Effects of Rhodobacter sphaeroides on the Carcinogenic Risk of Polycyclic Aromatic Hydrocarbon-Contaminated Soil," which stated that the mechanism of action of Rhodobacter sphaeroides application is to activate the soil microbial ecosystem and promote the removal of PAHs in the soil.

[0005] Although Rhodobacter sphaeroides has achieved certain application results in the fields of energy and environment, the performance of the currently reported Rhodobacter sphaeroides strains in degrading organic pollutants and producing hydrogen needs to be further improved. Research on the degradation of specific pollutants (such as 2,4-dichlorophenol) and simultaneous hydrogen production has not been reported. The application effect and mechanism of Rhodobacter sphaeroides in complex wastewater treatment still need in-depth study. Summary of the Invention

[0006] Traditional biological treatment technologies are limited by low degradation efficiency and long treatment times when degrading high-concentration or difficult-to-degrade organic pollutants (such as 2,4-dichlorophenol). Existing Rhodobacter sphaeroides strains are insufficient in both organic pollutant degradation and hydrogen production, and there is a lack of research on the simultaneous degradation of 2,4-dichlorophenol and hydrogen production. The present invention aims to provide a strain of Rhodobacter sphaeroides that degrades 2,4-dichlorophenol and produces hydrogen through fermentation, and its application in wastewater degradation.

[0007] In order to achieve the above object, the present invention adopts the following technical solutions: The present invention provides a strain of Rhodobacterium sphaeroides that degrades 2,4-dichlorophenol and simultaneously produces hydrogen through fermentation. Rhodobacter sphaeroides)HTP1, characterized in that the strain is deposited in the General Microbiology Center of China Culture Collection Administration of Microorganisms, with a deposit number of CGMCC No. 33696, a deposit date of March 3, 2025, and a classification name of Rhodobacter sphaeroides.

[0008] The present invention provides a microbial agent for degrading 2,4-dichlorophenol and simultaneously producing hydrogen by fermentation, comprising a strain of Rhodobacterium sphaeroides ( Rhodobacter sphaeroides )HTP1.

[0009] The microbial agent contains Rhodobacterium sphaeroides ( Rhodobacter sphaeroides ) The viable cell count of HTP1 is 8×10 5 ~8×10 6 CFU / g.

[0010] The present invention provides a method for preparing the above-mentioned microbial agent for degrading 2,4-dichlorophenol and simultaneously fermenting and producing hydrogen, comprising: Step 1: Degrading 2,4-dichlorophenol and fermenting hydrogen to produce Rhodobacter sphaeroides ( Rhodobacter sphaeroides ) HTP1 is inoculated into liquid culture medium to obtain seed solution; Step 2: inoculating the seed liquid into the screening culture medium and culturing the culture medium until the strain reaches the logarithmic growth phase, centrifuging the culture medium, and resuspending the culture medium to obtain a microbial agent capable of degrading 2,4-dichlorophenol and simultaneously fermenting and producing hydrogen.

[0011] Further, in step 1, the liquid culture medium is composed of: 2.00 g / L succinic acid, 0.50 g / L ammonium sulfate, 0.10 g / L L-glutamic acid, 0.04 g / L L-aspartic acid, 1.00 g / L sodium chloride, 20 mL / L phosphate buffer, 20 mL / L nutrient solution, and 1 mL / L vitamin solution; the carbon source includes any one or combination of xylose, maltose, rhamnose, glucose, succinic acid, malic acid, acetic acid, butyric acid, and pretreated corn straw hydrolyzate; the components of the nutrient solution are: 10.00 g / L nitrilotriacetic acid, 29.50 g / L magnesium sulfate heptahydrate, 3.34 g / L calcium chloride dihydrate, 0.10 g / L ferrous sulfate heptahydrate, 0.01 g / L ammonium molybdate tetrahydrate, and 25 mL / L trace element solution; the components of the trace element solution are: 11.00 g / L zinc sulfate heptahydrate g / L, disodium ethylenediaminetetraacetic acid dihydrate 2.77 g / L, ferrous sulfate heptahydrate 5.00 g / L, boric acid 0.11 g / L, manganese sulfate 1.38 g / L, copper sulfate pentahydrate 0.39 g / L, cobalt nitrate hexahydrate 0.25 g / L; the components of the vitamin solution are: thiamine hydrochloride 0.50 g / L, niacin 1.00 g / L, and biotin 0.01 g / L.

[0012] In step 1, the culture temperature is 25-40° C., and the culture time is 3-6 days.

[0013] In step 2, the screening culture medium is composed of 100 mg / L ferric oxide.

[0014] In step 2, the seed solution inoculation ratio is 0.1% to 30% (mass volume percentage).

[0015] The above-mentioned strain of Rhodobacterium sphaeroides that degrades 2,4-dichlorophenol and produces hydrogen through fermentation ( Rhodobacter sphaeroides ) Application of HTP1 or one of the above-mentioned microbial agents for degrading 2,4-dichlorophenol and simultaneously producing hydrogen by fermentation in wastewater treatment.

[0016] The invention provides a method for degrading 2,4-dichlorophenol and simultaneously producing hydrogen by fermentation. The microbial agent for degrading 2,4-dichlorophenol and simultaneously producing hydrogen by fermentation is inoculated into a fermentation medium containing 2,4-dichlorophenol for anaerobic fermentation.

[0017] The fermentation medium comprises: 5.00-20.00 g / L of carbon source, 1.00 g / L of L-glutamic acid, 0.04 g / L of L-aspartic acid, 1.00 g / L of sodium chloride, 20 mL / L of phosphate buffer, 20 mL / L of nutrient solution, 1 mL / L of vitamin solution, and a pH of 5-10; the carbon source comprises any one or a combination of xylose, maltose, rhamnose, glucose, succinic acid, malic acid, acetic acid, butyric acid, and pretreated corn straw hydrolyzate; the nutrient solution comprises: 10.00 g / L of nitrilotriacetic acid, 29.50 g / L of magnesium sulfate heptahydrate, 3.34 g / L of calcium chloride dihydrate, 0.10 g / L of ferrous sulfate heptahydrate, 0.01 g / L of ammonium molybdate tetrahydrate, and 25 mL / L of trace element solution; the trace element solution comprises: 11.00 g / L of zinc sulfate heptahydrate g / L, disodium ethylenediaminetetraacetic acid dihydrate 2.77 g / L, ferrous sulfate heptahydrate 5.00 g / L, boric acid 0.11 g / L, manganese sulfate 1.38 g / L, copper sulfate pentahydrate 0.39 g / L, cobalt nitrate hexahydrate 0.25 g / L; the components of the vitamin solution are: thiamine hydrochloride 0.50 g / L, niacin 1.00 g / L, and biotin 0.01 g / L.

[0018] The inoculation ratio of the microbial agent is 5% to 30%; the anaerobic fermentation temperature is 25-40° C., the anaerobic fermentation light intensity is 800-5000 lux, and the anaerobic fermentation time is 0-96 h.

[0019] Furthermore, the inoculation ratio of the microbial agent is 20%, the anaerobic fermentation temperature is 30° C., and the anaerobic fermentation light intensity is 5000 lux.

[0020] Compared with the prior art, the present invention has the following beneficial effects: The present invention provides a strain of Rhodobacterium sphaeroides that degrades 2,4-dichlorophenol and simultaneously produces hydrogen through fermentation. Rhodobacter sphaeroides ) HTP1, which has the characteristics of rapid growth and wide carbon source utilization; the tolerance concentration of Rhodobacter sphaeroides HTP1 to 2,4-dichlorophenol is 500 mg / L, and the removal efficiency of phenol at 200 mg / L within 96 hours reaches 54.3%.

[0021] The microbial agent for degrading 2,4-dichlorophenol and simultaneously producing hydrogen by fermentation provided by the present invention is Rhodobacter sphaeroides ( Rhodobacter sphaeroides ) HTP1 is the main component, and it has multiple technical effects such as efficient degradation of pollutants, synchronous fermentation to produce hydrogen, rapid growth, wide utilization of carbon sources and environmental friendliness. This bacterial agent has broad application prospects and important practical value in the fields of industrial wastewater treatment, renewable energy production and environmental remediation.

[0022] The preparation method provided by the present invention realizes the efficient preparation of a microbial agent for degrading 2,4-dichlorophenol and simultaneously fermenting and producing hydrogen through steps such as high-efficiency seed liquid preparation, screening and enrichment of logarithmic phase strains, and centrifugation and resuspension processes. The entire preparation process includes steps such as inoculation, cultivation, centrifugation and resuspension, is relatively simple to operate, is easy to produce on a large scale, does not involve the addition of harmful chemicals, and conforms to an environmentally friendly production concept.

[0023] The application provided by the present invention, the sphaeroides red bacterium HTP1 and the microbial agent prepared therefrom have a high efficiency in degrading 2,4-dichlorophenol, can significantly reduce the concentration of pollutants in wastewater in a relatively short period of time, and the high efficiency in degradation helps to quickly purify wastewater and reduce environmental pollution. While degrading 2,4-dichlorophenol, the HTP1 strain can ferment and produce hydrogen, realizing waste resource utilization. The hydrogen production process not only reduces processing costs, but also provides a new way for the production of renewable energy. The sphaeroides red bacterium HTP1 and the microbial agent provided by the present invention, which can degrade 2,4-dichlorophenol and produce hydrogen by fermentation, show significant advantages in wastewater treatment, such as high efficiency in pollutant degradation, simultaneous fermentation and hydrogen production, environmental friendliness and wide applicability.

[0024] The method for degrading 2,4-dichlorophenol and simultaneously fermenting hydrogen provided by the present invention can further maximize the resource utilization of wastewater through anaerobic fermentation, improve the economy and feasibility of the treatment effect, and provide a new, efficient and environmentally friendly treatment solution for the field of wastewater treatment. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 For the present invention, the spherical red bacteria ( Rhodobacter sphaeroides ) Scanning electron micrograph of HTP1; Figure 2 For the present invention, the spherical red bacteria ( Rhodobacter sphaeroides ) Phylogenetic tree of HTP1 based on 16S rDNA sequences; Figure 3 For the present invention, the spherical red bacteria ( Rhodobacter sphaeroides ) Cumulative biogas and hydrogen production by HTP1 using different carbon sources; Figure 4 For the present invention, the spherical red bacteria ( Rhodobacter sphaeroides ) Growth curve of HTP1; Figure 5 For the present invention, the spherical red bacteria ( Rhodobacter sphaeroides ) Cumulative hydrogen production and removal efficiency of HTP1 in fermentation medium containing different concentrations of 2,4-dichlorophenol; Figure 6 For the present invention, the spherical red bacteria ( Rhodobacter sphaeroides) Growth curves of HTP1 and bacterial agents in fermentation medium containing 200 mg / L 2,4-dichlorophenol; Figure 7 For the present invention, the spherical red bacteria ( Rhodobacter sphaeroides ) Cumulative hydrogen production and removal rate of HTP1 in fermentation medium containing different concentrations of 2,4-dichlorophenol; Figure 8 For the present invention, the spherical red bacteria ( Rhodobacter sphaeroides ) Cumulative hydrogen production of ATCC49419 in fermentation medium with / without 2,4-dichlorophenol. DETAILED DESCRIPTION

[0026] The following will clearly and completely describe the concept of the present invention and the technical effects produced in conjunction with the embodiments, so as to fully understand the purpose, characteristics and effects of the present invention. Obviously, the embodiments described are only part of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, other embodiments obtained by those skilled in the art without creative work are within the scope of protection of the present invention. The test methods used in the embodiments are all conventional methods unless otherwise specified; the materials, reagents, etc. used, unless otherwise specified, can all be reagents and materials obtained from commercial channels.

[0027] The present invention uses Rhodobacter sphaeroides ( Rhodobacter sphaeroides HTP1 was deposited at the China General Microbiology Center under the China Culture Collection Administration, with accession number CGMCC No. 33696. Rhodobacter sphaeroides ATCC 49419 was purchased from the Shanghai Collection of Microorganisms Center (SHBCC) and can be purchased through other public channels, including the China General Microbiology Center (CGMCC).

[0028] Example 1 This embodiment provides a strain of Rhodobacterium sphaeroides that degrades 2,4-dichlorophenol and produces hydrogen through fermentation. Rhodobacter sphaeroides ) Isolation, purification and identification of HTP1. The specific process is as follows: (1) Separation and purification Fresh chicken manure was sampled from the Zhuque Flower and Bird Market in Xi'an, Shaanxi Province (108.94°E, 34.24°N), stored in sterile 15 mL centrifuge tubes, and stored at 4°C. The chicken manure was transferred to the screening medium and incubated anaerobically at 30°C under light conditions for 12-72 hours until the color of the screening medium changed from transparent to deep red. The bacterial solution was then diluted to 10% with sterile water. -4 , 10 -5, 10 -6 , ball-spread on a solid plate of screening medium, and culture the strain at 25-40℃ for 12-48 h; pick a single colony with an inoculating loop, streak on a solid plate of screening medium, and culture at 25-40℃ for 12-48 h; repeat the above streaking three times on the screening medium to obtain a single colony; inoculate a single colony into the screening medium, and culture at 25-40℃ until the strain reaches the logarithmic phase of growth.

[0029] (2) Identification 1. Morphological identification The morphology of the strain was observed by scanning electron microscopy through plate culture. The colony morphology of the strain on the plate, the colony morphology under a stereo microscope and the bacterial morphology under a scanning electron microscope are shown in the attached figure. Figure 1 shown.

[0030] Microscopically, the cells are oval, have flagella, and no spores. The strain is Gram-negative and can grow under a variety of conditions, such as aerobic, anaerobic, and dark conditions. When cultured at 25-40°C for 12-48 hours, the strain forms red colonies with smooth surfaces, raised protrusions, and neat edges, with a colony diameter of 1-1.5 mm.

[0031] 2. Molecular Biology Identification The bacterial genome to be tested was extracted using a bacterial genome extraction kit (SK8225) from Sangon Biotech (Shanghai) Co., Ltd., and then a pair of universal bacterial 16S rDNA sequencing primers (27F: AGAGTTTGATCMTGGCTCAG (as shown in SEIQ NO. 2) and 1492R: GGTTACCTTGTTACGACTT (as shown in SEIQ NO. 3) were used to PCR amplify the 16S rDNA of HTP1. The amplification system is shown in Table 1. The reaction program was: 01 95°C for 4 min, 02 95°C for 20 s, 03 55°C for 20 s, 04 72°C for 1 min, 05 72°C for 10 min, 06 4°C, with a total of 35 cycles from 02 to 04. The amplified product was sequenced by Sangon Biotech Co., Ltd. The length of the amplified sequence of this Rhodobacterium sphaeroides HTP1 based on the 16S rDNA sequence was 1357 kp (as shown in SEIQ NO. 1). The full-length sequence and developmental tree are shown in Table 1. Figure 2 As shown, according to the obtained 16S rDNA sequence, the BLAST comparison analysis with NCBI was carried out, and the phylogenetic tree based on the 16S rDNA sequence was used to obtain the position and phylogenetic relationship of the strain in the biological evolution process. Rhodobacteraceae ) family has a close genetic relationship with Rhodobacter sp. JA460.

[0032] The sequencing results have been uploaded to the National Center for Biotechnology Information (NCBI, USA) with the accession number SUB14316799.

[0033] Table 1: PCR amplification system (20 μL system)

[0034] 3. Physiological and biochemical characteristics (1) Carbon source utilization The strain in the logarithmic growth phase was inoculated into the fermentation medium at a 10% inoculation ratio. The initial pH of the fermentation medium was 7.0. The culture was kept at 30°C and 2000 lux. The fermentation performance of the strain under different carbon source types (10 g / L) was compared. The results are shown in Figure 3 .

[0035] The fermentation medium comprises: 5.00-20.00 g / L of carbon source, 1.00 g / L of L-glutamic acid, 0.04 g / L of L-aspartic acid, 1.00 g / L of sodium chloride, 20 mL / L of phosphate buffer, 20 mL / L of nutrient solution, 1 mL / L of vitamin solution, and a pH of 5-10; the carbon source comprises any one or a combination of xylose, maltose, rhamnose, glucose, succinic acid, malic acid, acetic acid, butyric acid, and pretreated corn straw hydrolyzate; the nutrient solution comprises: 10.00 g / L of nitrilotriacetic acid, 29.50 g / L of magnesium sulfate heptahydrate, 3.34 g / L of calcium chloride dihydrate, 0.10 g / L of ferrous sulfate heptahydrate, 0.01 g / L of ammonium molybdate tetrahydrate, and 25 mL / L of trace element solution; the trace element solution comprises: 11.00 g / L of zinc sulfate heptahydrate g / L, disodium ethylenediaminetetraacetic acid dihydrate 2.77 g / L, ferrous sulfate heptahydrate 5.00 g / L, boric acid 0.11 g / L, manganese sulfate 1.38 g / L, copper sulfate pentahydrate 0.39 g / L, cobalt nitrate hexahydrate 0.25 g / L; the components of the vitamin solution are: thiamine hydrochloride 0.50 g / L, niacin 1.00 g / L, and biotin 0.01 g / L.

[0036] By the attached Figure 3 The data show that R. sphaeroides HTP1 can metabolize a variety of carbon sources to produce hydrogen. For sugars as substrates, R. sphaeroides HTP1 efficiently metabolizes rhamnose to produce hydrogen. Bacteroides ruminicola”, the metabolic pathway of rhamnose in microorganisms is relatively direct and efficient, and it can be quickly decomposed to produce a large amount of ATP, which is beneficial to maintaining the metabolism and hydrogen production of microorganisms. In addition, rhamnose can be extracted from waste at low cost. When acids are used as substrates, Rhodobacter sphaeroides HTP1 metabolizes succinate to produce hydrogen more efficiently. Therefore, wastes such as succinate production wastewater and straw hydrolyzate can be used for photofermentation hydrogen production by Rhodobacter sphaeroides HTP1, thereby obtaining low-energy consumption and highly compatible biohydrogen energy.

[0037] (2) Growth curve Rhodobacterium sphaeroides ( Rhodobacter sphaeroides ) HTP1 was inoculated into liquid culture medium and cultured at a constant temperature of 30°C and 2000 lux. Samples were taken at regular intervals and their absorbance at 660 nm was measured to draw a growth curve of the strain, as shown in Figure 2. Figure 4 shown.

[0038] The components of the liquid culture medium are: 2.00 g / L succinic acid, 0.50 g / L ammonium sulfate, 0.10 g / L L-glutamic acid, 0.04 g / L L-aspartic acid, 1.00 g / L sodium chloride, 20 mL / L phosphate buffer, 20 mL / L nutrient solution, and 1 mL / L vitamin solution; the components of the nutrient solution are: 10.00 g / L nitrilotriacetic acid, 29.50 g / L magnesium sulfate heptahydrate, 3.34 g / L calcium chloride dihydrate, 0.10 g / L ferrous sulfate heptahydrate, 0.01 g / L ammonium molybdate tetrahydrate, and 25 mL / L trace element solution; the components of the trace element solution are: 11.00 g / L zinc sulfate heptahydrate, 2.77 g / L disodium ethylenediaminetetraacetic acid dihydrate, 5.00 g / L ferrous sulfate heptahydrate, 0.11 g / L boric acid, and 1.38 g / L manganese sulfate. g / L, copper sulfate pentahydrate 0.39 g / L, cobalt nitrate hexahydrate 0.25 g / L; the components of the vitamin solution are: thiamine hydrochloride 0.50 g / L, niacin 1.00 g / L, and biotin 0.01 g / L.

[0039] By the attached Figure 4 The data show that Rhodobacter sphaeroides HTP1 has the characteristic of rapid growth. A single colony is inoculated into liquid culture medium and cultured for 24-36 hours to obtain seed liquid with suitable photobiomass density for hydrogen production application.

[0040] (3) Removal of 2,4-dichlorophenol and hydrogen production performance 2,4-Dichlorophenol reacts with 4-aminoantipyrine (4-AAP) and potassium ferricyanide (K3[Fe(CN)6]) under alkaline conditions to generate red quinone compounds with a maximum absorption wavelength of around 510 nm. The concentration of 2,4-dichlorophenol is quantified by colorimetry.

[0041] The amount of hydrogen gas was calculated by multiplying the hydrogen concentration by the gas production volume, which was determined by volumetric measurement under standard atmospheric pressure. Hydrogen concentration was measured using a TCD (thermal conductivity detector) in a gas chromatograph equipped with a 4 m × 3 mm stainless steel Hayesep tube. Argon was used as the carrier gas at a flow rate of 20 mL / min, the detector temperature was 200°C, the filament temperature was set at 230°C, the injection port was set at 100°C, and the column oven temperature was 50°C. The sample to be tested and the standard sample were injected into the injector at the same injection volume. The peak area ratio between the sample to be tested and the standard sample was the hydrogen concentration ratio.

[0042] Take the spherical red bacteria in the logarithmic growth phase ( Rhodobacter sphaeroides ) HTP1 bacterial suspension was inoculated at a 10% inoculum ratio into a fermentation medium with an initial pH of 7.0 and 10 g / L glucose as the carbon source. Cultures were maintained at 30°C and 5000 lux of light. 2,4-Dichlorophenol removal and fermentation hydrogen production by the strains were compared at 2,4-Dichlorophenol concentrations of 0, 25, 50, 100, and 200 mg / L. Detailed results are shown in the Appendix. Figure 5 shown.

[0043] The tolerance concentration of 2,4-dichlorophenol of Rhodobacter sphaeroides HTP1 was 500 mg / L, and the removal efficiency of 200 mg / L 2,4-dichlorophenol reached 54.3% within 96 h. The hydrogen production in the fermentation broth containing 0, 25, 50, 100, and 200 mg / L 2,4-dichlorophenol was 2565, 2173, 1513, 1250, and 953 mL / L, respectively.

[0044] The fermentation medium is a liquid culture medium containing 100 mg / L of ferric oxide, and the composition of the liquid culture medium is: 2.00 g / L of succinic acid, 0.50 g / L of ammonium sulfate, 0.10 g / L of L-glutamic acid, 0.04 g / L of L-aspartic acid, 1.00 g / L of sodium chloride, 20 mL / L of phosphate buffer, 20 mL / L of nutrient solution, and 1 mL / L of vitamin solution.

[0045] By the attached Figure 5 The data shows that with increasing amounts of 2,4-dichlorophenol added, the cumulative hydrogen production of Rhodobacter sphaeroides initially increases and then decreases. While degrading 2,4-dichlorophenol, Rhodobacter sphaeroides produces hydrogen, but the hydrogen production and removal rate are affected by the 2,4-dichlorophenol concentration. Within a certain concentration range (e.g., 25 mg / L), Rhodobacter sphaeroides exhibits high hydrogen production and removal rates. High concentrations of 2,4-dichlorophenol inhibit Rhodobacter sphaeroides, resulting in decreased hydrogen production and removal rates.

[0046] Based on the above biological and physiological and biochemical characteristics, the above strain Rhodobacter sphaeroides ( Rhodobacter sphaeroides ) HTP1 was deposited on March 3, 2025, at the Budapest Treaty International Microbiological Depository: China General Microbiology Center (CGMCC), address: Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, with the deposit number: CGMCC No. 33696. The proposed classification name is: Rhodobacter sphaeroides ( Rhodobacter sphaeroides ).

[0047] Example 2 Based on Example 1, this example provides a microbial agent for degrading 2,4-dichlorophenol and simultaneously fermenting hydrogen. The specific preparation process is as follows: Step 1: isolate the strain Rhodobacter sphaeroides obtained in Example 1. Rhodobacter sphaeroides ) HTP1 is activated and inoculated into liquid culture medium to obtain seed solution; The liquid culture medium is composed of: 2.00 g / L succinic acid, 0.50 g / L ammonium sulfate, 0.10 g / L L-glutamic acid, 0.04 g / L L-aspartic acid, 1.00 g / L sodium chloride, 20 mL / L phosphate buffer, 20 mL / L nutrient solution, and 1 mL / L vitamin solution.

[0048] Step 2: inoculate 10% of the seed solution into a fermentation medium and culture the culture medium until the strain reaches the logarithmic phase of growth, centrifuge, and resuspend to obtain a microbial agent that degrades 2,4-dichlorophenol and simultaneously ferments to produce hydrogen.

[0049] The fermentation medium is a liquid medium containing 100 mg / L of ferric oxide.

[0050] Example 3 The bacterial solution of Example 1 or the bacterial agent of Example 2 grown to the logarithmic phase was collected by centrifugation at 7500 rpm for 3 minutes, washed three times with fermentation medium, and then inoculated into a fermentation medium with an initial pH of 6-9 at an inoculum ratio of 5% to 30%. 2,4-Dichlorophenol was added to the fermentation medium, and fermentation was carried out at 25-40°C under anaerobic conditions and a light intensity range of 800-5000 lux for 0-96 hours.

[0051] The fermentation medium comprises the following components: 5.00-20.00 g / L of carbon source, 1.00 g / L of L-glutamic acid, 0.04 g / L of L-aspartic acid, 1.00 g / L of sodium chloride, 20 mL / L of phosphate buffer, 20 mL / L of nutrient solution, and 1 mL / L of vitamin solution. The components of the nutrient solution are: 10.00 g / L nitrotriacetic acid, 29.50 g / L magnesium sulfate heptahydrate, 3.34 g / L calcium chloride dihydrate, 0.10 g / L ferrous sulfate heptahydrate, 0.01 g / L ammonium molybdate tetrahydrate, and 25 mL / L trace element solution; the components of the trace element solution are: 11.00 g / L zinc sulfate heptahydrate, 2.77 g / L disodium ethylenediaminetetraacetic acid dihydrate, 5.00 g / L ferrous sulfate heptahydrate, 0.11 g / L boric acid, 1.38 g / L manganese sulfate, 0.39 g / L copper sulfate pentahydrate, and 0.25 g / L cobalt nitrate hexahydrate; the components of the vitamin solution are: 0.50 g / L thiamine hydrochloride, 1.00 g / L nicotinic acid, and 0.01 g / L biotin.

[0052] A tolerance test was conducted on high-performance hydrogen-producing strains or bacterial agents, that is, 0, 50, 100, 200, 500, and 1000 mg / L of 2,4-dichlorophenol was added to the fermentation medium, the OD value of the strain was monitored, and the supernatant was taken by centrifugation to measure the 2,4-dichlorophenol concentration in the culture medium.

[0053] The tolerance concentration of 2,4-dichlorophenol of the microbial agent combined with ferric oxide to degrade 2,4-dichlorophenol and simultaneously produce hydrogen by fermentation is 1.5 times that of Rhodobacter sphaeroides HTP1, which is 750 mg / L. Figure 6 In the fermentation broth containing 200 mg / L of 2,4-dichlorophenol, the detectable biooptical density value of the microbial agent that degrades 2,4-dichlorophenol and produces hydrogen simultaneously is always higher than that of Rhodobacter sphaeroides HTP1, confirming that the adsorption-advanced oxidation performance of ferric oxide for 2,4-dichlorophenol is beneficial to reducing the pollutant concentration in the fermentation system and providing more metabolizable substances for biomass accumulation.

[0054] The photocatalytic hydrogen production performance of ferric oxide itself was not detected under the corresponding light source and culture medium conditions ( Figure 7At the same dosage as the inoculant, it removed 0.4% of 200 mg / L 2,4-dichlorophenol within 96 hours. With the addition of Rhodobacter sphaeroides, the synergistic effect of the two increased the removal efficiency to 86.8%. Furthermore, because hematite enhances the light response of the fermentation system and photogenerated electron transfer, namely, the maintenance of Rhodobacter sphaeroides enzyme activity, the fermentation hydrogen production properties were also improved. In an environment without 2,4-dichlorophenol, the cumulative hydrogen production reached 2822 mL / L, 1.1 times that of Rhodobacter sphaeroides HTP1 alone. In an environment with 200 mg / L 2,4-dichlorophenol, the cumulative hydrogen production reached 1546 mL / L, only 1.6 times that of Rhodobacter sphaeroides HTP1.

[0055] Comparative Example: Use an inoculating loop to pick Rhodobacter phaeroides ATCC49419 purchased from the Shanghai Collection of Microorganisms (SHBCC) and streak onto a solid plate containing screening medium. Incubate at 25-40°C for 12-48 hours. Repeat this streak three times on the screening medium to obtain a single colony. Inoculate the single colony onto the screening medium and incubate at 25-40°C until the strain reaches the logarithmic phase of growth. A strain in the logarithmic phase of growth is inoculated at a 10% inoculum ratio into a fermentation medium with an initial pH of 7.0 and 10 g / L glucose as the carbon source. Incubate at 30°C under 5000 lux illumination.

[0056] See attached Figure 8 The control strain was able to produce hydrogen via glucose photofermentation, with a cumulative hydrogen production of 708 mL / L over 96 hours, only 28.3% of that of HTP1. However, when exposed to 200 mg / L 2,4-dichlorophenol, the control strain died in the 2,4-dichlorophenol solution, demonstrating no phenol degradation or hydrogen production. Therefore, R. sphaeroides HTP1 exhibited significantly higher hydrogen production and 2,4-dichlorophenol tolerance than the control strain.

[0057] The above content is only for explaining the technical idea of ​​the present invention and cannot be used to limit the protection scope of the present invention. Any changes made on the basis of the technical solution in accordance with the technical idea proposed by the present invention shall fall within the protection scope of the claims of the present invention.

Claims

1. Rhodobacter sphaeroides HTP1 that degrades 2,4-dichlorophenol and simultaneously ferments to produce hydrogen, characterized in that, Rhodobacter sphaeroides The Rhodobacter sphaeroides strain is deposited in the General Microbiology Center of the China Microbial Culture Collection Center, with the deposit number CGMCC No. 33696, and the deposit date is March 3, 2025.

2. A microbial agent for degrading 2,4-dichlorophenol and simultaneously fermenting hydrogen, characterized in that, A strain of Rhodobacter sphaeroides Rhodobacter sphaeroides that degrades 2,4-dichlorophenol and simultaneously ferments to produce hydrogen, including the one described in claim 1 Rhodobacter sphaeroides ) HTP1.

3. The microbial inoculum for degrading 2,4-dichlorophenol and simultaneously fermenting hydrogen production according to claim 2, characterized in that, The viable count of Rhodobacter sphaeroides ( Rhodobacter sphaeroides ) HTP1 in the microbial inoculum is 8×10 5 ~8×10 6 CFU / g.

4. The preparation method of a microbial agent for degrading 2,4-dichlorophenol and simultaneously fermenting hydrogen, as described in claim 2 or 3, is characterized in that including: Step 1, inoculate Rhodobacter sphaeroides HTP1 ( Rhodobacter sphaeroides ) which can degrade 2,4-dichlorophenol and ferment hydrogen simultaneously as described in claim 1 into a liquid medium for cultivation to obtain a seed solution; Rhodobacter sphaeroides ​ Step 2: Inoculate the seed liquid into the screening medium and culture it until the logarithmic phase of strain growth, then centrifuge and resuspend to obtain a microbial agent for degrading 2,4-dichlorophenol and simultaneously fermenting hydrogen.

5. The preparation method of a microbial agent for degrading 2,4-dichlorophenol and simultaneously fermenting hydrogen production according to claim 4, characterized in that, In Step 1, the culture temperature is 25 - 40 °C, and the culture time is 3 - 6 days.

6. The preparation method of a microbial agent for degrading 2,4-dichlorophenol and simultaneously fermenting hydrogen production according to claim 4, characterized in that In Step 2, the inoculation amount of the seed liquid is 0.1% - 30% of the volume of the screening medium.

7. The preparation method of a microbial agent for degrading 2,4-dichlorophenol and simultaneously fermenting hydrogen production according to claim 4, characterized in that, In Step 2, the screening medium contains 100 mg / L of iron(III) oxide.

8. Use of the Rhodobacter sphaeroides strain HTP1 for degrading 2,4-dichlorophenol and producing hydrogen by fermentation as described in claim 1, or a microbial agent for degrading 2,4-dichlorophenol and producing hydrogen by fermentation simultaneously as described in any one of claims 2 to 3 in wastewater treatment. Rhodobacter sphaeroides ​ 9. A method for degrading 2,4-dichlorophenol while fermenting to produce hydrogen, characterized in that, Inoculate the microbial agent for degrading 2,4-dichlorophenol and simultaneously fermenting hydrogen described in Claim 2 into a fermentation medium containing 2,4-dichlorophenol for anaerobic fermentation.

10. The method for degrading 2,4-dichlorophenol and simultaneously fermenting hydrogen production according to claim 9, characterized in that, The inoculation ratio of the microbial agent is 5% - 30%; the anaerobic fermentation temperature is 25 - 40 °C, the anaerobic fermentation light intensity is 800 - 5000 lux, and the anaerobic fermentation time is 0 - 96 h.

Images