Method for degrading azo dye and simultaneously producing hydrogen by utilizing Enterobacter sp.

Abstract

The invention discloses a method for degrading an azo dye and simultaneously producing hydrogen by utilizing Enterobacter sp., and belongs to the field of dye wastewater treatment. By screening an enterobacter strain with a special metabolic pathway, the strain realizes hydrogen production while degrading azo dyes (such as methyl red), breaks through the limitation of mutual independence of pollutant degradation and hydrogen production processes in the traditional technology, and remarkably improves the resource utilization efficiency, the strain decolorizes methyl red, the decolorization rate can reach 98% or above within 12 hours, hydrogen is produced at the same time, and the production cost is reduced. The strain has the advantages of obvious effect, simple culture method, high growth speed and difficult variation, is not limited to the degradation and hydrogen production of azo dyes, can be possibly expanded to the field of treatment of other organic pollutants, has wide prospects in industrial application of dye wastewater treatment and biological hydrogen production, and has wide application prospects. And a new technical thought is provided for pollutant degradation-clean energy.

Description

Technical Field

[0001] The present invention belongs to the field of dye wastewater treatment, and specifically relates to a method for treating dye wastewater by using Enterobacter Enterobacter sp.) A method for efficiently degrading azo dyes and simultaneously producing hydrogen. Background Art

[0002] Azo dyes are among the most widely used synthetic dyes in the textile, printing and dyeing, and papermaking industries. Methyl red, a typical monoazo dye, is difficult to effectively degrade in the natural environment using conventional physical and chemical methods due to its stable benzene ring and azo bond (-N=N-) in its molecular structure. Traditional treatment methods such as adsorption, chemical oxidation, and photocatalysis suffer from low efficiency, high energy consumption, and the generation of secondary pollutants (such as aromatic amine intermediates). They may even produce more toxic derivatives, posing a potential threat to ecosystems and human health.

[0003] Microbial degradation of dye wastewater has attracted widespread attention due to its environmental friendliness, economic viability, and potential for resource utilization. During microbial degradation of dyes, the dye decolorization rate is an important indicator of the strain's degradation capacity. Converting the chemical energy of complex organic carbon sources into bioenergy (hydrogen production, methane production, etc.) is another key indicator of microbial treatment of dye wastewater and renewable energy technologies. Currently, existing technologies primarily focus on single processes: pollutant degradation or hydrogen production. For example, efficient treatment of dye wastewater has been achieved by screening and domesticating bacterial strains capable of efficiently degrading azo dyes; and hydrogen production efficiency has been improved by optimizing the microbial metabolic pathways. However, relatively few studies have examined strains that possess both azo reduction and hydrogen production metabolic pathways.

[0004] In practice, industrial wastewater, such as textile wastewater, contains not only high concentrations of azo dyes but also potentially complex components such as heavy metals, salts, and additives. These components not only inhibit microbial growth and metabolic activity but also disrupt the biodegradation of synthetic dyes. Therefore, developing a bacterial strain capable of simultaneously achieving efficient azo dye degradation and sustained hydrogen production under complex environmental conditions is crucial for promoting the transformation of industrial wastewater treatment towards resource and energy utilization. Summary of the Invention

[0005] In view of the current state of the art that the main focus is on the single process of pollutant degradation or hydrogen production, and the lack of strains that have both azo reduction and hydrogen production metabolic pathways, the present invention aims to provide a method for utilizing Enterobacter ( Enterobacter sp.) to degrade azo dyes and simultaneously produce hydrogen. By screening and domesticating bacterial strains with special metabolic pathways, they are able to degrade the azo dye methyl red while converting the chemical energy of organic carbon sources into bioenergy such as hydrogen, thereby achieving resource utilization of dye wastewater and production of renewable energy.

[0006] In order to achieve the above object, the present invention adopts the following technical solutions: The present invention provides a strain of Enterobacter that degrades azo dyes and produces hydrogen simultaneously ( Enterobacter sp.) HD01, which was deposited in the China General Microbiological Culture Collection Center with the deposit number CGMCC No.34230 and the deposit date was April 17, 2025.

[0007] The gene sequence of the strain is shown in SEQ ID NO.3.

[0008] The present invention provides the above-mentioned Enterobacter that degrades azo dyes and produces hydrogen simultaneously ( Enterobacter Application of HD01 in the degradation of azo dyes and simultaneous hydrogen production.

[0009] The present invention provides a method for utilizing Enterobacter ( Enterobacter A method for degrading azo dyes and simultaneously producing hydrogen, comprising: (1) The Enterobacter ( Enterobacter sp.) were streaked onto screening medium plates and cultured to obtain single colonies; (2) Pick a single colony grown on the screening medium plate, inoculate it into LB liquid medium, and enrich and culture it to obtain Enterobacter ( Enterobacter sp.) culture medium; (3) Enterobacteriaceae ( Enterobacter sp.) culture medium was inoculated into the fermentation medium and cultured until the final bacterial concentration was expressed as OD 600 When the pH reaches 0.4 to 0.6, fermentation and hydrogen production are continued until the hydrogen production is completed.

[0010] In step (1), the screening culture medium is composed of: 10 g / L glucose, 3 g / L tryptone, 100 mg / L methyl red, 20 mL / L nutrient solution, 20 mL / L phosphate buffer and 1 mL / L vitamin solution; wherein the vitamin solution includes 0.50 g / L thiamine hydrochloride, 1.00 g / L niacin and 0.01 g / L biotin.

[0011] In step (1), the culture temperature is 15-40°C and the culture time is 36-54 hours.

[0012] In step (2), the culture temperature is 15-40°C, and the culture is carried out on a shaking table at 200-300 rpm for 20-26 hours.

[0013] In step (3), the Enterobacter ( Enterobacter sp.) culture broth accounts for 2% to 20% of the volume of the fermentation broth.

[0014] Furthermore, in step (3), the fermentation culture medium is composed of: 10 g / L glucose, 3 g / L tryptone, 20 mL / L nutrient solution, 20 mL / L phosphate buffer, 25-500 mg / L azo dye and 1 mL / L vitamin solution.

[0015] Furthermore, the nutrient solution includes 10.00 g / L of nitrotriacetic 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.

[0016] Furthermore, the trace element solution includes 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; Furthermore, the vitamin solution includes 0.50 g / L of thiamine hydrochloride, 1.00 g / L of niacin, and 0.01 g / L of biotin.

[0017] The azo dye is any one of methyl red, Congo red and methyl orange.

[0018] The fermentation temperature is 34-36° C., and the fermentation time is 20-26 hours.

[0019] Compared with the prior art, the present invention achieves the following technical effects: The present invention provides a strain of Enterobacter that degrades azo dyes and produces hydrogen at the same time ( Enterobacter sp.) HD01, which was identified by 16S rDNA and belongs to Enterobacter sp., has extremely high activity and a broad-spectrum decolorization ability for dyes. At the same time, it has efficient hydrogen production ability for organic carbon sources such as glucose. For decolorization of methyl red, the decolorization rate can reach more than 98% in 12 hours, and hydrogen is produced at the same time, with an amount of 559 mL / L, which is a significant effect; this strain breaks through the limitation of the traditional technology that pollutant degradation and hydrogen production processes are independent of each other, and achieves hydrogen production while degrading azo dyes (such as methyl red), which significantly improves resource utilization efficiency; the strain can achieve a decolorization rate of methyl red of more than 98% in 12 hours, and produce hydrogen at the same time, with a significant effect, indicating that it has efficient degradation and hydrogen production capabilities, and therefore it is necessary to preserve it in accordance with legal requirements.

[0020] The application provided by the present invention breaks through the limitations of traditional technologies, improves resource utilization efficiency, and has significant environmental and economic benefits. The strain has significant effects in degrading azo dyes and producing hydrogen, such as a methyl red decolorization rate of over 98% within 12 hours and a hydrogen production of 559 mL / L.

[0021] The present invention provides the use of Enterobacter ( Enterobacter sp.) to degrade azo dyes and produce hydrogen simultaneously. A strain of Enterobacter with a special metabolic pathway was screened out. This strain can produce hydrogen while degrading azo dyes (such as methyl red), breaking through the limitation of traditional technologies that pollutant degradation and hydrogen production are independent of each other, and improving resource utilization efficiency. The process of Enterobacter degrading azo dyes and producing hydrogen is both biological and environmentally friendly. Compared with traditional chemical treatment methods, microbial treatment of dye wastewater does not cause secondary pollution and has relatively low treatment costs. The culture method of Enterobacter is simple, the growth rate is fast, and it is not easy to mutate. It can be used as a model strain for studying the dual effects of bacteria on dye decolorization and biological hydrogen production mechanisms. It is not only suitable for the degradation and hydrogen production of azo dyes, but may also be expanded to the treatment of other organic pollutants. It has great prospects for industrial application in dye wastewater treatment and biological hydrogen production. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 This is a phylogenetic tree diagram of the dye-degrading and hydrogen-producing bacteria screened in the present invention; Figure 2 This is an optical microscopic image of the dye-degrading and hydrogen-producing bacteria screened by the present invention; Figure 3 This is a scanning electron micrograph of the dye-degrading and hydrogen-producing bacteria screened by the present invention; Figure 4 The degradation effect of the bacteria of the present invention on methyl red at different concentrations; Figure 5 This is a diagram showing the hydrogen production kinetics of the bacteria of the present invention under different concentrations of methyl red; Figure 6 This is a comparison diagram of the effects of the bacteria of the present invention before and after degrading methyl red. DETAILED DESCRIPTION

[0023] In order to enable those skilled in the art to better understand the solutions of the present invention, the technical solutions in the embodiments of the present invention will be clearly and completely described below in conjunction with the drawings in the embodiments of the present invention. Obviously, the embodiments described are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts should fall within the scope of protection of the present invention.

[0024] If no specific experimental steps or conditions are specified in the examples, the conventional experimental steps or conditions described in the literature in this field can be used. If the manufacturer of the reagents or instruments is not specified, they are all commercially available conventional reagents.

[0025] The LB liquid culture medium used in the present invention is: 10 g / L tryptone, 5 g / L yeast extract, 10 g / L NaCl, pH=7.0, and dilute to 1 L with deionized water, and sterilize by high pressure at 121°C for 20 to 30 minutes.

[0026] Example 1 (1) Screening, separation and purification Soil samples were obtained from multiple landfills, and the soil samples were added to a syringe filled with sterile screening culture medium at an inoculum volume of 1:50. The samples were placed in an incubator at 15-40°C and cultured until the culture medium turned colorless and gas was produced. The bacterial solution was added with different volumes of sterile water for gradient dilution, and the dilutions were spread on a solid plate of screening culture medium and cultured anaerobically. After colonies grew on the plate, colonies of different colors and sizes were picked and streaked onto a new solid plate of screening culture medium. The streaking was repeated several times until a single colony was found on the same plate.

[0027] Use an inoculating loop to pick a single strain from the plate, inoculate it into LB liquid medium, grow it overnight, and collect the cells by centrifugation. The collected cells are then added to a syringe filled with sterile screening medium and incubated in a 15-40°C incubator until the culture medium turns colorless and gas is produced. Decolorization efficiency is measured using a UV-visible spectrophotometer, and hydrogen concentration is measured using a gas chromatograph. The optimal strain is selected based on decolorization and hydrogen production. Samples are collected at regular intervals, centrifuged, and the supernatant is collected. The absorbance at the dye's maximum absorption peak is measured using a UV-visible spectrophotometer (HACH, DR 6000, USA). Decolorization efficiency (%) is calculated as (C0 - C1) / C0 × 100, where C0 is the OD value at 0 h of exposure and C1 is the OD value at 24 h of exposure.

[0028] The screening culture medium consists of: 10 g / L glucose, 3 g / L tryptone, 100 mg / L methyl red, 20 mL / L nutrient solution, 20 mL / L phosphate buffer, and 1 mL / L vitamin solution.

[0029] The amount of hydrogen was obtained by multiplying the hydrogen concentration by the gas production. The gas production was determined by volumetric determination under standard atmospheric pressure. The hydrogen concentration was measured using a TCD (thermal conductivity detector) equipped in a gas chromatograph (GC, Agilent, 8860). The GC was 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 hot filament temperature was set to 230°C, the injection port temperature was 100°C, and the column oven temperature was 50°C. The sample to be tested and the standard sample were injected into the injector with the same injection volume. The peak area ratio between the sample to be tested and the standard sample was the hydrogen concentration ratio.

[0030] Use an inoculation loop to pick the bacterial strain on the activated plate culture medium and inoculate it into a centrifuge tube containing LB culture medium. Shake it in a shaker at 35°C and 250 rpm overnight. Collect the bacterial liquid that has grown to the logarithmic phase by centrifugation at 7500 rpm for 3 minutes. Wash it three times with hydrogen-producing culture medium to obtain milky white, uncontaminated strain cells.

[0031] (2) Identification of strains 1. Molecular Biology Identification Total DNA was extracted from the strain using the SK8225 bacterial genomic extraction kit provided by Sangon Biotech (Shanghai) Co., Ltd., and PCR amplification was performed. The primers used for amplification of the 16S rDNA sequence of HD01 were: forward primer 27F: 5'-AGAGTTTGATCCTGGCTCAG-3' (SEQ ID NO. 1); reverse primer 1492R: 5'-GGTTACCTTGTTACGACTT-3' (SEQ ID NO. 2). The PCR reaction conditions were as follows: pre-denaturation at 95°C for 5 min; 30 cycles of denaturation at 94°C for 30 s, annealing at 57°C for 30 s, and extension at 72°C for 1.5 min; extension at 72°C for 10 min, and storage at 4°C. The amplified products were sequenced.

[0032] Table 1: HD01 based on 16S rDNA sequence

[0033] Cluster analysis was performed using MEGA 6.0 software and it was found that the strain Enterobacter sp. DSM30060 belong to the same branch. Therefore, the strain Enterobacter sp. DSM30060 were highly similar, and the phylogenetic tree of strain HD01 based on 16S rDNA sequence by the neighbor-joining method, e.g. Figure 1 As shown, the position and genetic relationship of the strain in the biological evolution process were obtained, HD01 and Enterobacter sp. DSM 30060 is a close relative.

[0034] The strain was analyzed for physiological and biochemical characteristics and 16S rDNA sequence, and compared with NCBI BLAST. According to the Bergey Manual of Bacterial Identification (8th edition), the strain was identified for physiological and morphological characteristics. It was determined that the strain belonged to the genus Enterobacter. The species name was Enterobacter. Enterobacter Genus ( Enterobacter ), species called Enterobacter ( Enterobacter sp.) strain HD01.

[0035] 2. Bacterial morphological observation (1) Optical microscopic observation of bacteria The bacteria were collected according to the above method, fixed by conventional flame fixation, and then placed under an optical microscope for staining experiment observation. Figure 2 shown.

[0036] (2) Scanning electron microscopy (SEM) observation of bacteria Take 3.6 mL of bacterial solution and add 0.4 mL of 25% glutaraldehyde. Fix at 4°C for 24 h. Then take 0.3 mL of the mixture and add 1.5 mL of sterile water to rinse twice and ethanol once. Centrifuge at 10,000 rpm for 2 min. Dilute 6 times before adding the sample. Observe the results in the attached table. Figure 3 shown.

[0037] Based on the above biological characteristics, the strain HD01 was identified as Enterobacter 。 The strain has been deposited in the Budapest Treaty International Microbiological Depository: China General Microbiology Center, China Culture Collection Administration, Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, 100101, China. The deposit date is April 17, 2025, with the deposit number CGMCC No. 34230. The proposed taxonomic name is Enterobacter ( Enterobacter sp.).

[0038] Example 2 Decolorization and biohydrogen production effects of the strain on azo dyes A single colony grown on the screening medium plate of Example 1 was picked and inoculated into LB liquid medium. The culture was shaken overnight at 35°C and 250 rpm in a shaker. The bacterial liquid that grew to the logarithmic phase was used to obtain Enterobacter ( Enterobacter sp.) culture medium; take Enterobacter ( Enterobacter sp.) culture medium, and inoculated it into the fermentation culture medium at an inoculum amount of 10% of the fermentation culture medium volume, and the culture medium without strain was used as the control group.

[0039] The fermentation culture medium is composed of: 10 g / L glucose, 3 g / L tryptone, 20 mL / L nutrient solution, 20 mL / L phosphate buffer, 25-500 mg / L methyl red and 1 mL / L vitamin solution; The nutrient solution includes 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 includes 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 vitamin solution includes 0.50 g / L of thiamine hydrochloride, 1.00 g / L of niacin, and 0.01 g / L of biotin.

[0040] The device was placed in a magnetic stirring pot in a 35°C incubator and decolorized and hydrogen was produced at a speed of 200 rpm. The pH was 6.0 and the experiment was carried out for 24 h. The results are shown in the attached Figures 4 - 6 As shown in the figure, the decolorization rate increased with reaction time at all concentrations, ultimately reaching a dynamic equilibrium. Simultaneously, strain HD01 produced hydrogen. When the methyl red concentration was ≤100 mg / L, the degradation rate exceeded 98% within 12 hours, with hydrogen production ≥559 mL / L. The fermentation broth was visually observed to change color from red to colorless.

[0041] 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. A strain of Enterobacter that degrades azo dyes and produces hydrogen ( Enterobacter sp.) HD01, characterized in that The strain is deposited in the China General Microbiological Culture Collection Center with the deposit number CGMCC No.34230 and the deposit date is April 17, 2025.

2. A strain of Enterobacter that degrades azo dyes and produces hydrogen according to claim 1 ( Enterobacter sp.) HD01, characterized in that The gene sequence of the strain is shown in SEQ ID NO.

3.

3. A strain of Enterobacter that degrades azo dyes and produces hydrogen according to any one of claims 1 or 2 ( Enterobacter Application of HD01 in the degradation of azo dyes and simultaneous hydrogen production.

4. A method of using Enterobacter Enterobacter sp.) a method for degrading azo dyes while producing hydrogen, characterized in that: include: (1) The Enterobacterium according to claim 1 ( Enterobacter sp.) were streaked onto screening medium plates and cultured to obtain single colonies; (2) Pick a single colony grown on the screening medium plate, inoculate it into LB liquid medium, and culture it to obtain Enterobacter ( Enterobacter sp.) culture medium; (3) Enterobacteriaceae ( Enterobacter sp.) culture medium was inoculated into the fermentation medium and cultured until the final bacterial concentration was expressed as OD 600 When the pH reaches 0.4 to 0.6, fermentation and hydrogen production are continued until the hydrogen production is completed.

5. according to claim 4, utilize Enterobacter ( Enterobacter sp.) a method for degrading azo dyes while producing hydrogen, characterized in that: In step (1), the culture temperature is 15-40°C and the culture time is 36-54 hours.

6. according to claim 4, utilizing Enterobacter ( Enterobacter sp.) a method for degrading azo dyes while producing hydrogen, characterized in that: In step (2), the culture temperature is 15-40°C, and the culture is carried out on a shaking table at 200-300 rpm for 20-26 hours.

7. according to claim 4, utilizing Enterobacter ( Enterobacter sp.) a method for degrading azo dyes while producing hydrogen, characterized in that: In step (3), the Enterobacter ( Enterobacter sp.) culture broth accounts for 2% to 20% of the volume of the fermentation broth.

8. according to claim 4, utilizing Enterobacter ( Enterobacter sp.) a method for degrading azo dyes while producing hydrogen, characterized in that: The azo dye is any one of methyl red, Congo red and methyl orange.

9. according to claim 4, utilizing Enterobacter ( Enterobacter sp.) a method for degrading azo dyes while producing hydrogen, characterized in that: The fermentation temperature is 34-36° C., and the fermentation time is 20-26 hours.

10. according to claim 4, utilizing Enterobacter ( Enterobacter sp.) degrading azo dyes and simultaneously producing hydrogen, wherein the fermentation broth comprises: 10 g / L glucose, 3 g / L tryptone, 20 mL / L nutrient solution, 20 mL / L phosphate buffer, 25-500 mg / L azo dye, and 1 mL / L vitamin solution.

Images