Method of bacterial transformation of microalgal extracellular dissolved organic matter to enhance antibiotic photodegradation

Abstract

The application discloses a method for enhancing antibiotic photodegradation by bacterial transformation of microalgae extracellular dissolved organic matter, comprising the following steps: (1) culturing microalgae in a culture medium, centrifuging to collect the supernatant of the culture solution and filtering to remove bacteria, and obtaining microalgae extracellular dissolved organic matter (DOM); (2) inoculating bacteria into the microalgae extracellular dissolved organic matter obtained in step (1) for culture, so that the bacteria metabolically transform the microalgae extracellular dissolved organic matter (DOM), centrifuging the culture solution and filtering to remove bacteria after the culture is completed, and obtaining bacterial transformation of microalgae extracellular dissolved organic matter; and (3) adding the bacterial transformation of microalgae extracellular dissolved organic matter obtained in step (2) to antibiotic-containing wastewater, and enhancing the photodegradation of the antibiotic under light conditions. The application can improve the photosensitivity of algal DOM by bacterial transformation, promote the indirect photodegradation of the antibiotic under light conditions, and is conducive to reducing the risk of drug resistance.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, and in particular relates to a method for enhancing the photodegradation of antibiotics by transforming extracellular soluble organic matter in microalgae using bacteria. Background Technology

[0002] Antibiotics have relatively stable molecular structures and significantly different physicochemical properties. Numerous studies have shown that long-term antibiotic residues in the environment not only have toxic effects on aquatic organisms and soil microorganisms, but may also disrupt the structure of environmental microbial communities, inducing the generation and spread of antibiotic-resistant bacteria and antibiotic resistance genes (ARGs). Therefore, achieving efficient removal of antibiotics from complex wastewater systems while simultaneously reducing the risk of resistance during treatment is of great significance.

[0003] Biological methods for antibiotic removal primarily rely on the adsorption or metabolic transformation of microorganisms, offering the advantage of mild conditions. However, traditional methods may lead to the accumulation of drug resistance over long-term operation, and their treatment effectiveness is easily affected by fluctuations in water quality, requiring further improvement in engineering adaptability. Microalgae can utilize nutrients such as nitrogen and phosphorus in wastewater for growth and exhibit a certain removal capacity for various organic pollutants. Existing research mainly focuses on the direct adsorption, bioaccumulation, or metabolic transformation of pollutants by microalgae, while paying less attention to the role of the large amount of dissolved organic matter (DOM) released during microalgae growth. Under light conditions, DOM can absorb light energy and undergo photochemical reactions to generate triple excited-state DOM (…). 3 DOM*), singlet oxygen ( 1 Active intermediates such as O2 drive or regulate the indirect photodegradation of organic pollutants. In antibiotic-containing systems, DOM has a significant impact on the environmental behavior of antibiotics: on the one hand, DOM can alter the mobility and bioavailability of antibiotics through complexation or adsorption; on the other hand, its photochemical activity can mediate the indirect photodegradation of antibiotics. Existing studies have shown that DOM from different sources and with different compositions exhibits significant differences in photochemical reactivity, with DOMs that are more aromatic and humified generally having stronger photosensitizing effects.

[0004] Microalgae are a significant source of DOM (degradable organic matter) in aquatic environments, continuously releasing algal DOM during their growth. However, this type of DOM is primarily composed of relatively simple components such as proteins and polysaccharides, with low humification levels and limited photochemical activity under natural conditions. Therefore, relying solely on algal DOM is often insufficient to support efficient photochemical removal of antibiotics. To enhance the treatment capacity of microalgal systems in complex wastewater, symbiotic bacterial-algal technology has been gradually introduced into the wastewater treatment field. Existing research primarily focuses on the synergistic effect of nutrient cycling and biomass growth, or on using bacteria as the primary agents for direct pollutant degradation. However, during the metabolic process of bacteria using DOM as a carbon source, the composition and structure of DOM may be significantly altered, further regulating its aromaticity, humification level, and photochemical properties. The impact of this process on the indirect photodegradation of antibiotics, as well as the evolution of DOM structure to enhanced function, lacks systematic research and technical pathways in current technologies. Therefore, there is an urgent need to propose a method that utilizes bacterial transformation to regulate the release of DOM from microalgae, enhancing its photochemical activity, thereby promoting the indirect photodegradation and removal of antibiotics to meet the treatment requirements of complex wastewater systems for high efficiency, stability, and low resistance risk. Summary of the Invention

[0005] To address the problems in existing technologies, such as the risk of inducing drug-resistant bacteria and gene accumulation in complex wastewater systems by biological antibiotic removal methods, and the limited photosensitivity of microalgae systems making it difficult to promote efficient indirect photodegradation of antibiotics, this invention provides a method for enhancing antibiotic photodegradation by bacterial transformation of microalgal extracellular soluble organic matter. Compared to untransformed microalgal extracellular DOM, this invention improves the ability of microalgal extracellular DOM to mediate indirect photodegradation of antibiotics and helps reduce dependence on the direct degradation pathway of antibiotics by microorganisms.

[0006] This invention introduces specific bacteria that utilize the extracellular DOM released during microalgal growth as the primary carbon source. Through bacterial metabolic transformation, the less photosensitive components of the algal DOM are converted into more aromatic and conjugated humic substances, significantly enhancing its light absorption capacity and bioactive species generation potential. Under light conditions, the bacterially transformed DOM continuously generates triple-excited organic matter and singlet oxygen, efficiently attacking the key structures of antibiotics through indirect photochemical reactions, achieving stable degradation. This invention can promote the indirect photodegradation of antibiotics in complex aquatic systems without the addition of photosensitizers or strong oxidants, while also helping to reduce the potential risks of antibiotic resistance gene and bacterial accumulation resulting from long-term reliance on direct microbial degradation.

[0007] The technical solution of the present invention is as follows: A method for enhancing antibiotic photodegradation through bacterial transformation of extracellular soluble organic matter in microalgae includes the following steps: (1) Cultivate microalgae in culture medium, centrifuge to collect culture supernatant and filter to obtain microalgal extracellular soluble organic matter (DOM). (2) Inoculate the bacteria into the microalgal extracellular soluble organic matter obtained in step (1), and culture the microalgal extracellular soluble organic matter as a growth substrate so that the bacteria can metabolize and transform the microalgal extracellular soluble organic matter. After the culture is completed, centrifuge and filter the culture medium to obtain the microalgal extracellular soluble organic matter transformed by bacteria. (3) The bacterial-transformed microalgal extracellular soluble organic matter obtained in step (2) is added to the antibiotic-containing wastewater to enhance the photodegradation of antibiotics under light conditions.

[0008] Preferably, in step (1), the microalgae are selected from any one or more of Chlorella, Chlamydomonas, Scenedesmus obliquus, Anabaena aeruginosa, and Microcystis aeruginosa.

[0009] Preferably, in step (1), the culture medium is BG11 medium; the conditions for microalgae culture include: culture temperature 25±1℃, light intensity 2000~3000 Lux, no additional aeration, culture time 8~16 days; and filtration using a 0.1~0.5 μm filter membrane. More preferably, the microalgae culture time is 16 days.

[0010] Preferably, in step (2), the bacteria are selected from any one or more of Bacillus cereus NCU LJ-1, Pseudomonas humidus, and Sphingomonas zeylans. More preferably, the bacteria are Bacillus cereus NCU LJ-1.

[0011] Preferably, in step (2), after the bacteria are cultured to the logarithmic growth phase, they are inoculated into the extracellular dissolved organic matter of microalgae at a volume percentage of 0.5-2%. The culture conditions include: culture temperature of 35-38°C, culture time of 1-4 days, culture in the dark or away from light; and filtration using a 0.1-0.5 μm filter membrane.

[0012] Preferably, in step (3), the antibiotic includes any one or more of sulfathiazole, sulfadiazine, sulfamethoxazole, and lincomycin. More preferably, the antibiotic is lincomycin or sulfathiazole.

[0013] Preferably, in step (3), the amount of bacteria that transform the extracellular dissolved organic matter of microalgae in the wastewater is 10 to 500 mg / L based on total organic carbon (TOC), and the photodegradation conditions include: pH 6.5 to 7.0, temperature 25±1℃, light intensity 40000 to 50000 Lux, and time 1 to 24 h.

[0014] The present invention also provides a bacterial-transformed microalgal extracellular soluble organic compound obtained by the above method.

[0015] The Bacillus cereus NCU LJ-1 strain provided by this invention has been deposited at the China Center for Type Culture Collection (CCTCC) with accession number CCTCC NO: M 20251470. The suggested classification name is... Bacillus cereus NCU LJ-1, deposited at No. 299 Bayi Road, Wuchang District, Wuhan City, Hubei Province, on June 24, 2025, was isolated and purified by the inventors from the anaerobic fermentation broth of Zhenghe Ecology, Xinyu City, Jiangxi Province in May 2023.

[0016] Preferably, the 16S rRNA gene sequence of Bacillus cereus NCU LJ-1 is shown in SEQ ID NO: 1.

[0017] Preferably, single colonies of Bacillus cereus NCU LJ-1 are obtained by dilution plating method, inoculated into LB medium, and preserved at 4°C.

[0018] Preferably, the biological characteristics of the Bacillus cereus NCU LJ-1 include: the colonies are milky white, with a smooth surface, round shape, and neat edges.

[0019] The present invention also provides a microbial preparation comprising the bacteria that transform the extracellular soluble organic matter of microalgae, or the Bacillus cereus.

[0020] The present invention also provides the above-mentioned method for enhancing the photodegradation of antibiotics by transforming microalgal extracellular soluble organic matter with bacteria, and the application of the above-mentioned bacteria transforming microalgal extracellular soluble organic matter, or the above-mentioned Bacillus cereus, in enhancing the indirect photodegradation of antibiotics.

[0021] Preferably, enhancing the indirect photodegradation process of antibiotics includes the following steps: 1) Microalgae continuously release and accumulate extracellular DOM during their growth process. This DOM is equivalent to a natural photosensitizer. It absorbs light energy to form an excited state and triggers the generation of reactive oxygen species (ROS) through energy transfer, thereby mediating the indirect photodegradation of antibiotics. The photosensitizing effect gradually increases with the extension of microalgae culture time.

[0022] 2) Bacteria remodel the extracellular DOM structure of microalgae through metabolic transformation, shifting it from biologically derived components to components with higher aromaticity, conjugation, and humification.

[0023] 3) The extracellular DOM of microalgae transformed by bacteria is mainly excited by light to form bioactive species. 3 DOM* and 1O2, through selective oxidation, allows active species to preferentially attack positions in molecules with high electron density where energy or electron transfer can occur, thereby initiating transformations such as hydroxylation, rearrangement, breakage, and ring opening. This promotes the photochemical transformation of antibiotic molecular structures and reduces their persistent residual risk in the aquatic environment.

[0024] 4) In 3 DOM* and 1 Driven by O2, antibiotics undergo selective oxidative transformation, significantly improving the overall indirect photodegradation efficiency. This indicates that bacterial transformation in this invention can effectively enhance the photodegradation ability of algal DOM for some antibiotics.

[0025] The beneficial effects of this invention are:

[0026] 1. This invention enhances the photochemical reactivity of microalgal extracellular soluble organic matter (DOM) under light conditions by metabolic transformation of microalgae by bacteria, thereby enhancing the indirect photodegradation effect of antibiotics, with a more significant promoting effect on lincomycin and sulfathiazole.

[0027] 2. This invention enables the extracellular DOM of microalgae to evolve from biologically derived components to humic-like components with high aromaticity and high conjugation through bacterial transformation, which is conducive to the occurrence of photosensitivity reactions.

[0028] 3. The bacterial transformation of microalgal extracellular DOM in this invention mainly occurs through its generation under light. 1 O2 and 3 DOM* mediates indirect photodegradation of STZ, while bacteria further enhance this process through metabolic transformation, demonstrating stronger photosensitivity and the potential for generating bioactive species. This reduces the reliance on direct bacterial degradation of antibiotics from a technical perspective, which is beneficial in reducing the possibility of drug resistance accumulation during long-term operation.

[0029] 4. This invention enhances the humification degree and photosensitivity of the extracellular DOM of microalgae through bacterial metabolic transformation, enabling it to continuously generate DOM under light. 3 DOM* and 1 O2 and other active intermediates enhance the indirect photodegradation of antibiotics. Because... 1 O2 and 3 DOM* has a universal oxidation and energy transfer effect on organic pollutants containing aromatic rings, heterocycles, and substituents (such as -NH2, -SO2). Therefore, this technical route is not only applicable to sulfathiazole (STZ), but also to antibiotics with similar structures or belonging to the same family that can be photodegraded. Attached Figure Description

[0030] Figure 1This is a comparison of the physiological response and removal efficiency of Chlorella monoculture to different concentrations of sulfathiazole (STZ) in Example 1 ((a) Optical density (OD) of Chlorella monoculture system). 680 (b) Photosynthetic activity of Chlorella (Fv / Fm); (c) STZ removal rate in Chlorella monoculture system; (d) TOC generation in Chlorella monoculture system under STZ loading conditions. Figure 2 This is a schematic diagram of the photoreactor device in Example 2; Figure 3 The effect of Chlorella vulgaris on STZ removal in Example 2 under heterotrophic + co-trophic (a) and co-trophic (b) modes; Figure 4 This is a comparison of the photochemical degradation effects of Chlorella cDOM, DOM16, and DOM8 on STZ in Example 3; Figure 5 The identification of Bacillus cereus NCU LJ-1 strain in Example 4 (phylogenetic tree of the strain (a), colony morphology of the strain on LB medium (b), fluorescent staining observation image of the strain (c); colony morphology of the strain on MYP medium (d), colony morphology of the strain on TSSB medium (e)). Figure 6 The changes in TOC concentration in the solution of Bacillus cereus NCU LJ-1 cultured in DOM16 at different times (0-4 days) in Example 6, and the effects of cultures with different culture times (0 days, 1 day, 3 days) on the photochemical degradation of STZ; Figure 7 The formation of ∙OH in the DOM16(a) and DOM16_B3(b) solutions of Example 7; Figure 8 In the DOM16(a) and DOM16_B3(b) solutions of Example 7 1 O2 generation status; Figure 9 In the DOM16 (a) and DOM16_B3 solutions (b) of Example 7 3 DOM generation status; Figure 10 The 3D-EEM spectra of different DOM solutions with 10 mg / L TOC in Example 7 are shown (from left to right: DOM8, DOM16, DOM16_B1 and DOM16_B3 solutions). Figure 11The results of PARAFAC analysis of the humic acid-like component and the Chlorella extracellular metabolite component in Example 7 are shown in the following figures: EEMs plot (a), loading plot (b, c), and percentage of each component in DOM16, DOM16_B1, and DOM16_B3 (d, component 1 is the humic acid-like component, and component 2 is the Chlorella extracellular metabolite component). Figure 12 Van Krevelen plots of the four solutions (ADE, DOM8, DOM16, DOM16_B3) in Example 7, colored with the GFE index; Figure 13 The molecular composition of the four solutions (ADE, DOM8, DOM16, DOM16_B3) in Example 7. Detailed Implementation

[0031] The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0032] Unless otherwise specified, all reagents involved in the embodiments of this invention are commercially available products and can be purchased through commercial channels.

[0033] The present invention is a Chlorella (classification and nomenclature) Chlorella sp., accession number FACHB-31), Chlamydomonas (classification and nomenclature) Chlamydomonas sp . Accession number FACHB-3612), Scenedesmus obliquus (classification and nomenclature) Scenedesmus obliquus (Accession number FACHB-12), nitrogen-fixing anthocyanin (classification and nomenclature) Anabaena azotica (Accession number FACHB-119), Microcystis aeruginosa (classification and nomenclature) Microcystis aeruginosa All samples (accession number FACHB-315) were purchased from the Freshwater Algae Gene Bank (FACHB) of the National Aquatic Germplasm Resource Bank of the Institute of Hydrobiology, Chinese Academy of Sciences, located at No. 7, Shandong Hunan Road, Luojia Mountain, Wuhan.

[0034] The *Sphingosine monocytogenes* strain of this invention is classified and named as follows: Sphingomonas zeaeNCU J1, deposited at the China Center for Type Culture Collection (CCTCC) on June 23, 2025, with accession number CCTCC NO: M 20251462, was isolated by the inventors in June 2024 from a water sample containing only aspartame carbon source and contaminated with bacteria in the laboratory of the Engineering Research Center for Biomass Conversion of the Ministry of Education at Nanchang University, Nanchang City, Jiangxi Province. This strain has been published in patent application CN120829860A.

[0035] The present invention is a moist fibrinolytic bacterium (classification and nomenclature). Cellulomonas uda The strain (strain number: BNCC336468) was purchased from Beina Chuanglian Biotechnology Co., Ltd. and stored in LB medium.

[0036] The BG11 culture medium formula of this invention is as follows: sodium nitrate (NaNO3) 1.500 g / L, dipotassium hydrogen phosphate (K2HPO4) 0.040 g / L, magnesium sulfate heptahydrate (MgSO4·7H2O) 0.075 g / L, calcium chloride dihydrate (CaCl2·2H2O) 0.036 g / L, citric acid (C6H8O7) 0.006 g / L, ferric ammonium citrate (C6H8FeNO7) 0.006 g / L, disodium ethylenediaminetetraacetate (EDTA·2Na) 0.001 g / L, sodium carbonate (Na2CO3) 0.02 g / L, and trace elements (A5) 1 mL / L. Its formula is: boric acid (H3BO3) 2.86 g / L, manganese chloride tetrahydrate (MnCl2·4H2O) 1.81 g / L, and zinc sulfate (ZnSO4) 0.222 g / L. The BG11 medium contains 0.39 g / L sodium molybdate (Na2MoO4), 0.079 g / L copper sulfate pentahydrate (CuSO4·5H2O), and 0.0494 g / L cobalt nitrate hexahydrate (Co(NO3)2·6H2O). The BG11 expansion medium is based on the BG11 medium and additionally supplemented with 1.0 g / L ammonium chloride (NH4Cl), 1.0 g / L sodium acetate (CH3COONa), 0.252 g / L dipotassium hydrogen phosphate (K2HPO4), and 0.25 g / L potassium dihydrogen phosphate (KH2PO4).

[0037] The LB medium formulation of this invention is as follows: 10.0 g / L peptone, 5.0 g / L yeast extract, 10.0 g / L sodium chloride, and pH adjusted to 7.0 using 1 mol / L NaOH.

[0038] The MYP culture medium formula of the present invention is as follows: peptone 10 g / L, mannitol 10 g / L, beef extract powder 1 g / L, sodium chloride 10 g / L, phenol red 0.025 g / L, agar 15 g / L, pH value 7.2±0.1.

[0039] The TSSB culture medium formula of the present invention is as follows: tryptone 15 g / L, plant peptone 5 g / L, sodium chloride 5 g / L, anhydrous dipotassium hydrogen phosphate 2.5 g / L, glucose 2.5 g / L, agar 12 g / L, pH 7.2±0.2.

[0040] Example 1: Physiological response and removal effect of Chlorella monoculture on different concentrations of sulfathiazole (STZ)

[0041] Pick Chlorella ( Chlorella Single colonies of *Chlorella* sp. were inoculated into sterile BG11 medium and cultured to the logarithmic growth phase. After 2-3 activation cycles, the colonies were inoculated into BG11 medium again to obtain enhanced *Chlorella*. 100 mL of BG11 medium was added to a 250 mL Erlenmeyer flask, and *Chlorella* was inoculated to achieve an initial concentration of 2.50 × 10⁻⁶. 6 Cells / mL (biomass 0.04 g / L, dry weight) were added to the culture medium to achieve concentrations of 0 mg / L, 0.5 mg / L, 5 mg / L, and 20 mg / L, respectively. The pH of the culture was then adjusted to 7. The culture temperature was 25±1℃, the light intensity was 2500 Lux, and the medium was manually shaken twice daily without additional aeration. The culture was carried out for 18 days, with three replicates. The optical density (OD) of the Chlorella monoculture system was measured every two days. 680 pH, STZ concentration, and Chlorella photosynthetic activity (Fv / Fm) were measured. The STZ removal rate (%) was calculated by detecting STZ concentration: STZ removal rate (%) = 100% × (initial STZ concentration - real-time STZ concentration) / initial STZ concentration. Total organic carbon (TOC) was measured every 4 days. Results are shown in Tables 1-5 and... Figure 1 .

[0042] The results showed that sulfathiazole inhibited the growth of Chlorella in a concentration-dependent manner, with OD... 680 The OD value represents the biomass of Chlorella. As the concentration of STZ in the treatment group increases, the OD value also increases. 680 The values ​​gradually decrease (see Table 1 and ). Figure 1 ).

[0043] High concentrations (20 mg / L) of STZ inhibited light energy conversion efficiency and interfered with the metabolic homeostasis of Chlorella during the first two days of culture, resulting in a decrease in Fv / Fm. However, it showed significant physiological adaptation and recovery ability during the fourth to eighth days of culture (see Table 3).

[0044] pH may alter adsorption-desorption behavior. Under neutral conditions, STZ is in a protonated state, which is conducive to electrostatic adsorption. As the number of culture days increases, the pH of the system gradually increases and then tends to stabilize (see Table 2). The gradual increase in pH can induce STZ deprotonation, leading to weakened adsorption or even desorption, which is manifested in the rebound of STZ concentration from day 4 to day 10 of culture (see Table 4).

[0045] On day 18, the STZ removal rate decreased with increasing initial concentration, indicating that the system has the potential to handle higher concentrations of STZ. TOC accumulated significantly in the culture medium with increasing culture time (see Table 5), suggesting that algal DOM may play an important role in the indirect photodegradation of STZ.

[0046] Table 1. OD of Chlorella at different concentrations of STZ 680

[0047] Table 2 pH of Chlorella at different concentrations of STZ

[0048] Table 3. Photosynthetic activity (Fv / Fm) of Chlorella under different concentrations of STZ.

[0049] Table 4. STZ removal rate (%) of Chlorella at different STZ concentrations

[0050] Table 5. TOC content (mg / L) of Chlorella at different STZ concentrations.

[0051] Example 2: Photodegradation effect of sulfathiazole mediated by Chlorella DOM

[0052] Microalgae can release algal DOM into the environment through efflux metabolism, promoting STZ degradation. Algal DOM can also mediate antibiotic-catalyzed degradation under light conditions. Therefore, this study investigates the effect of algal DOM on STZ photodegradation. The steps are as follows: (1) Photoreactor setup: Five 600×600 mm LED flat panel lights (color temperature 5700K, rated power 32W, with 36 built-in 0.9W LED modules) are fixedly assembled into a [model name missing]. Figure 2 The light intensity at a distance of 0-5 cm above the bottom panel of the photoreactor shown is approximately 45,000 Lux.

[0053] (2) Microalgae culture: Select a single colony of Chlorella and inoculate it into sterile BG11 medium and culture it to the logarithmic phase. After 2-3 activations, inoculate it into BG11 medium to obtain enhanced Chlorella. The culture temperature is 25±1℃, the light intensity is 2500 Lux, and the medium is manually shaken twice a day without additional aeration.

[0054] Two treatment groups were set up in the experiment: one group was a heterotrophic-multitrophic group that was initially cultured in the dark and then switched to light culture; the other group was a multitrophic group that was cultured under full light. The culture medium used was BG11 supplemented with 2 g / L sodium acetate to provide an organic carbon source. After adding 100 mL of BG11 medium supplemented with sodium acetate to a 250 mL Erlenmeyer flask, Chlorella vulgaris was inoculated and its OD was increased. 680 The value was 0.10, and then STZ stock solution was added to bring the STZ concentration in the culture medium to 5 mg / L. For the heterotrophic + multitrophic group, conical flasks were wrapped in aluminum foil for dark culture from day 0 to 10. From day 10 onwards, the aluminum foil was removed, and culture was switched to light. The multitrophic group was cultured under light throughout days 0 to 18. The experiment was repeated three times, with culture conditions consistent with those described in Example 1. STZ concentration changes were monitored every two days. The results are shown in Tables 6 and 7. Figure 3 .

[0055] The results showed that no significant STZ removal was observed during the dark culture period from day 0 to day 10. However, when the culture was switched to light conditions starting from day 10, the STZ removal rate increased rapidly within 2 days, demonstrating a significant light-induced removal effect. The Chlorella ditrophic group cultured under full light conditions also showed sustained STZ removal capacity. These results confirm that algal DOM released by Chlorella can mediate the photochemical degradation of STZ under light conditions.

[0056] Table 6 STZ removal rate (%) of Chlorella under heterotrophic + concomitant mode

[0057] Table 7 STZ removal rate of Chlorella in the ditrophic model (%)

[0058] Example 3: Comparison of the photodegradation capabilities of intracellular and extracellular DOM in Chlorella vulgaris for sulfathiazole.

[0059] The algal DOM in the environment comes partly from organic matter directly emitted during the growth of Chlorella (extracellular DOM), and partly from intracellular organic matter released during the apoptosis or lysis of Chlorella (intracellular DOM). Therefore, the degradation effects of different Chlorella DOMs on STZ were further compared. The specific steps are as follows: (1) Chlorella was cultured in BG11 with 2500 Lux light and 25°C without additional aeration.

[0060] (2) Preparation of different DOM solutions: Chlorella biomass was harvested by centrifugation on days 8 and 16, respectively. The supernatant was filtered through a 0.22 μm microporous membrane to remove the original microorganisms and obtain Chlorella extracellular DOM (DOM8 and DOM16). Chlorella cells harvested on day 16 were washed three times with ultrapure water and then redissolved in ultrapure water. After three freeze-thaw cycles at -70℃, the Chlorella cells were disrupted using an ultrasonic disruptor to release intracellular organic matter. After centrifugation and filtration through a 0.22 μm microporous membrane to remove the original microorganisms, Chlorella intracellular DOM (cDOM16) was obtained. All obtained DOMs were stored at -20℃ for later use to prevent microbial activity and material degradation.

[0061] (3) Take 30 mL of cDOM16, DOM16 and DOM8 solutions respectively, add STZ stock solution to make the initial concentration 1 mg / L, and then adjust the pH of the sample to 7. A 90 mm diameter petri dish was used as the reaction vessel, and the sides were sealed with sealing film to prevent water evaporation during the reaction. A sample inlet was prepared by piercing the petri dish lid with a syringe needle, and the sample solution was injected into the petri dish using a 5 mL syringe. All samples were placed in the photoreactor described in Example 2 for photoreaction (light intensity 45000 Lux, 25±1 ℃) for 6 h. The experiment was set up in 3 replicates, and 1.00 mL of sample was drawn from the petri dish every 1 h using a syringe to monitor the change in STZ concentration. The results are shown in Table 8 and Figure 4 .

[0062] The results showed that the photochemical degradation capacity of *Chlorella* DOM obtained from different sources and at different cultivation stages varied significantly. The TOC concentration of DOM16 was 3.75 times that of DOM8, and after 6 h of reaction, the STZ removal rate of DOM16 was approximately 2.58 times that of DOM8, indicating that the accumulation of extracellular DOM enhances the STZ photodegradation capacity of *Chlorella* with prolonged cultivation time. The TOC concentration of cDOM16 was 32.03 times and 8.53 times that of DOM8 and DOM16, respectively. Figure 4 However, its STZ removal rate was lower than that of DOM16, indicating that the ability of DOM to mediate STZ photodegradation depends not only on TOC concentration but also on the source of DOM. Overall, extracellular DOM obtained after 16 days of culture showed a stronger promoting effect on STZ photodegradation.

[0063] Table 8. Removal rates (%) of STZ by DOM8, DOM16 and cDOM16 of Chlorella

[0064] Example 4: Acquisition and identification of NCU LJ-1, a symbiotic bacterium that can synergistically enhance STZ removal.

[0065] 1. Enrichment of indigenous microbial communities The inventors enriched indigenous microbial communities from untreated anaerobic fermentation broth (ADE) of Zhenghe Ecology in Xinyu, Jiangxi Province. The enrichment culture medium is shown in Table 9.

[0066] Table 9 Enrichment Culture Medium Formulation

[0067] 2. Synergistic enhancement of STZ removal by indigenous microbiota Pick Chlorella Chlorella sp . Single colonies were inoculated into sterile BG11 medium and cultured to the logarithmic growth phase. After 2-3 activation cycles, they were inoculated into BG11 medium again to obtain enhanced Chlorella. 100 mL of BG11 medium was added to a 250 mL Erlenmeyer flask, and the activated Chlorella in the logarithmic growth phase was inoculated, resulting in an OD of... 680 The initial concentration of STZ was 0.10, and STZ stock solution was added to the culture medium to bring the initial STZ concentration to 5 mg / L. The pH of the culture was then adjusted to 7. Another group was inoculated with 1% (v / v) of activated indigenous bacterial seed culture (the seed culture contained LB medium components, which could maintain the basic activity of bacteria in the initial culture stage). A control group, BG11, was also set up without inoculation of Chlorella and indigenous bacteria. The culture temperature was 25±1℃, the light intensity was 2500 Lux, and the medium was manually shaken twice daily without additional aeration. The culture lasted for 18 days, and STZ concentration was measured every 2 days. The experiment was repeated in triplicate, and the STZ removal rate after 12 days was calculated. The results are shown in Table 10.

[0068] The results showed that photolysis and hydrolysis alone failed to effectively degrade STZ in BG11 medium. Compared with the Chlorella monoculture system, the STZ removal rate was significantly improved within 12 days after Chlorella coexisted with indigenous bacteria enriched from untreated anaerobic fermentation broth (ADE). This indicates that the indigenous bacteria may promote STZ removal by coupling with the metabolic processes of Chlorella and altering the composition, structure, or photochemical reaction characteristics of DOM in the system.

[0069] Table 10. STZ removal rate in different culture systems

[0070] 3. Acquisition and identification of symbiotic bacterium NCU LJ-1 In May 2023, the inventors enriched indigenous bacterial communities from untreated anaerobic fermentation broth at Zhenghe Ecology in Xinyu, Jiangxi Province. Further purification was achieved using LB medium supplemented with 2% agar via plate spreading. The results are shown in [Figure number missing]. Figure 5 Finally, a dominant strain was obtained. The colonies of this strain were milky white, with a smooth surface, round shape, and neat edges, which conformed to the typical colony morphology characteristics of Bacillus spp. ( Figure 5 (b) The results of fluorescence staining observation (Figure 1) Figure 5 c) shows that the cells of this strain are rod-shaped, consistent with the typical morphological characteristics of Bacillus. Differential cultures were performed using mannitol-polymyxin agar (MYP) and tryptone-soybean-blood agar (TSSB), respectively. On MYP agar plates, the colonies of strain B1 were slightly pink, indicating that it does not ferment mannitol (…). Figure 5 (d) A white to pink precipitate ring appears around the colony, indicating that it has phospholipase activity. On TSSB medium, the colonies of this strain are light gray and exhibit complete hemolysis. Figure 5 The above physiological and biochemical characteristics are consistent with those of Bacillus cereus (e). Bacillus cereus Typical traits of ).

[0071] The strain was identified by 16S rRNA sequencing, and the 16S rDNA sequence of the strain is shown in SEQ ID NO: 1. Then, similar sequences were obtained from the NCBI database for BLAST alignment analysis, and a phylogenetic tree was constructed. Figure 5 (a, B1), the results showed that strain B1 was related to the Bacillus cereus group (e.g., a, B1). Bacillus cereus ATCC 14579 and Bacillus cereus strain CCM 2010 is located upstream on the same branch, indicating that strain B1 has a high phylogenetic relationship with the Bacillus cereus group. Furthermore, strain B1 is related to strain CCM 2010 in the phylogenetic tree. Bacillus albus, Bacillus paramobilis and Bacillus mycoides Bacillus strains clustered closely together, indicating that they belong to the genus Bacillus. Bacillus ), and with Bacillus cereus They are closely related by blood.

[0072] Based on the combined 16S rRNA sequencing analysis and physiological and biochemical characteristics, this strain was confirmed to belong to *Bacillus cereus*. The relevant sequencing data has been submitted to the NCBI database and a accession number (PV355407) has been obtained. This strain has been deposited at the China Center for Type Culture Collection (CCTCC), with accession number CCTCC NO: M 20251470. The suggested taxonomic name is... Bacillus cereus NCULJ-1, deposited at No. 299 Bayi Road, Wuchang District, Wuhan City, Hubei Province, on June 24, 2025.

[0073] Example 5: Photodegradation effect of Bacillus cereus NCU LJ-1 monoculture on sulfathiazole The experiment consisted of one experimental group (LB_B) and two control groups (LB, BG11). 100.00 mL of LB medium was added to each of the 250 mL Erlenmeyer flasks in the LB_B and LB groups, while 100.00 mL of BG11 medium was added to the BG11 group. STZ stock solution was added to all treatment groups to ensure an initial STZ concentration of 5.00 mg / L. 1.00 mL of Bacillus cereus in the logarithmic growth phase was used… Bacillus cereus The NCU LJ-1 seed culture was centrifuged, washed with sterile ultrapure water, and resuspended in 0.50 mL of sterile water, then inoculated into Erlenmeyer flasks for the LB_B group. Equal volumes of sterile water were added to the LB and BG11 control groups. The pH of all cultures was adjusted to approximately 7.00 using 1 M HCl and 1 M NaOH solutions. The experimental culture period was set at 96 h, with three replicates. The 0–48 h period was incubated in the dark at 37°C, and the 48–96 h period was incubated under light conditions consistent with those described in Example 2 (light intensity 45000 Lux, 25±1°C). 1.00 mL of culture medium was collected every 24 h for OD monitoring. 600 The changes in pH and STZ concentration are shown in Tables 11-13.

[0074] The results showed that Bacillus cereus NCU LJ-1 grew rapidly in LB medium, with an OD of 0–48 h (dark phase). 600 The pH level increased significantly, but no STZ removal was observed during the same period, indicating that this strain did not exhibit significant direct biodegradation ability. Simultaneously, the pH of the system showed an upward trend during cultivation, reflecting the release of alkaline products and changes in the culture environment accompanying its growth and metabolism. During the light exposure phase (48–96 h), no STZ removal was observed in the BG11 group, indicating that the contribution of direct photolysis or hydrolysis of STZ was negligible under the experimental conditions. Furthermore, after 96 h of light exposure, the STZ removal rate in the LB control group was significantly higher than that in the LB_B group of Bacillus cereus NCU LJ-1.

[0075] The above results indicate that the LB medium itself may possess certain photosensitizing activity due to the presence of organic components such as peptone and yeast extract, thereby interfering with STZ removal. Therefore, Example 5 primarily illustrates that Bacillus cereus NCULJ-1 does not primarily remove STZ through direct biodegradation. Subsequent examples will further demonstrate that the main role of NCU LJ-1 is to metabolize and transform algal DOM, enhancing its photochemical reactivity and thus strengthening the system's indirect photodegradation ability for STZ.

[0076] Table 11 OD of Bacillus cereus NCU LJ-1 in different culture media 600

[0077] Table 12 pH values ​​of Bacillus cereus NCU LJ-1 in different culture media

[0078] Table 13 STZ removal rate (%) of Bacillus cereus NCU LJ-1 in different culture media

[0079] Example 6: Comparison of the photodegradation effect of Bacillus cereus NCU LJ-1 transformation products on sulfathiazole using Chlorella extracellular DOM as a matrix.

[0080] Based on the fact that DOM16 of *Chlorella vulgaris* significantly enhanced the photodegradation ability of STZ compared to DOM8 in Example 3, the extracted DOM16 solution (TOC 30.82 mg / L, pH 9.56) was used as the growth substrate for *Bacillus cereus* to investigate the transformation of *Chlorella vulgaris* DOM by *Bacillus cereus* NCU LJ-1 and its enhancing effect on the photodegradation of sulfathiazole. The steps are as follows: DOM16 was obtained according to the method in Example 3. The DOM16 solution was pre-filtered through a 0.22 μm filter membrane to remove as many original microorganisms as possible. Four experimental groups were set up with fermentation times (1 day, 2 days, 3 days, and 4 days). 100 mL of DOM16 solution was added to each 250 mL Erlenmeyer flask. 1.00 mL of Bacillus cereus NCU LJ-1 seed culture in the logarithmic growth phase was taken, centrifuged, washed with sterile ultrapure water, and resuspended in 0.50 mL of sterile water before being inoculated into the DOM16 fermentation system. The cultures were fermented in a constant temperature and humidity incubator at 37℃ for 1, 2, 3, and 4 days in the dark. After fermentation, the cultures were sterilized by passing the solutions through a 0.22 μm filter membrane to obtain Bacillus cereus-transformed DOM16 solutions with different fermentation times, labeled as DOM16_B1, DOM16_B2, DOM16_B3, and DOM16_B4, respectively. Their TOC content was measured, and the results are shown in Table 14. Figure 6 .

[0081] Subsequently, 30 mL of DOM16, DOM16_B1, and DOM16_B3 solutions were taken respectively, and STZ stock solution was added to adjust the STZ concentration to 1 mg / L. The pH of the samples was then adjusted to 7. Following the method in Example 2, the samples were injected into petri dishes for photocatalytic reaction (light intensity 45000 Lux, 25±1 ℃). The experiment was repeated three times, with a reaction time of 6 h. 1 mL samples were taken before and after the reaction to detect changes in STZ concentration. The STZ removal rates are shown in Table 15. Figure 6 .

[0082] The results showed that the TOC concentration of DOM16 continuously decreased from day 0 to day 4, indicating that Bacillus cereus NCU LJ-1 continuously grew and consumed organic matter during this process. Further comparison of the photodegradation effect of DOM solutions obtained at different fermentation times on STZ revealed that, compared with unconverted DOM16 (fermentation day 0), DOM16_B1 and DOM16_B3 obtained after 1 and 3 days of fermentation both showed improved removal rates of 1 mg / L STZ within 6 hours. This indicates that although bacterial transformation reduced the total organic carbon content in the DOM system, it enhanced its ability to mediate STZ photodegradation. This suggests that bacterial metabolism transformed DOM from a component with weaker photosensitivity to a component with stronger photosensitivity, thereby enhancing the indirect photodegradation effect on STZ.

[0083] Table 14 TOC content (mg / L) of DOM16 solution transformed by Bacillus cereus at different fermentation times.

[0084] Table 15 STZ removal rate (%) of Bacillus cereus transformed into DOM16 solution at different fermentation times

[0085] Example 7: Mechanism analysis of the synergistic enhancement of sulfathiazole photodegradation by algal-derived DOM and Bacillus cereus transformation of DOM.

[0086] This embodiment provides a comprehensive analysis of the active species contribution, steady-state concentration of active species, and photochemical characteristics and molecular composition evolution of different DOMs in the photodegradation of sulfathiazole mediated by algal DOM16 and DOM16_B3 transformed from Bacillus cereus.

[0087] First, DOM16 was prepared according to the method of Example 3, and DOM16_B3 was prepared according to the method of Example 6, for later use.

[0088] 1. Free radical inhibition experiment Take 30 mL of DOM16 and DOM16_B3 respectively, and set up the experimental setup as follows: a) Raw group: control group without inhibitor; b) SA group: Add triple excited-state DOM ( 3 DOM*) quencher sorbic acid (SA) up to 1.0 mmol / L; c) FFA group: Add singlet oxygen ( 1 O2) quencher furfuryl alcohol (FFA) up to 5.0 mmol / L; d) IPA group: Isopropanol (IPA), a hydroxyl radical (∙OH) scavenger, was added to a concentration of 100 mmol / L.

[0089] STZ stock solution was added to each group to make the initial STZ concentration 1 mg / L. The pH of the solution was adjusted to 7. Then, each sample was injected into a culture dish and placed in the photoreactor set in Example 2 (light intensity 45000 Lux, 25±1 ℃) for 6 h. The experiment was repeated 3 times. After the reaction, samples were taken, filtered through a 0.22 μm filter membrane, and the residual STZ concentration was measured. The STZ removal rate was calculated.

[0090] The results are shown in Table 16. The DOM16 control group (Raw) without any inhibitors showed some STZ degradation. When any inhibitors were added... 1 After using the O2 scavenger FFA, the degradation of STZ was almost completely inhibited. Furthermore, the addition of... 3 The degradation rate of STZ was significantly reduced after treatment with DOM* scavenger SA. This indicates that... 1 O2 and 3 DOM* is the main active species in the photolysis of STZ. However, after adding the ∙OH scavenger IPA, the degradation rate of STZ did not decrease significantly compared with the control group, indicating that ∙OH contributes little to the photolysis of STZ. The same phenomenon was observed in DOM16_B3. Based on these results, it is speculated that the indirect photodegradation pathway of STZ mediated by DOM16 and DOM16_B3 may involve the activation of organic matter in DOMs under light to generate… 3 DOM*, then 3 Further generation of DOM* 1 O2 is used to achieve the oxidative degradation of STZ.

[0091] Table 16 STZ removal rate (%) under different active species inhibition conditions of DOM16 and DOM16_B3

[0092] 2. Determination of active species during photolysis The DOM solutions (DOM16, DOM16_B3) were diluted with ultrapure water to a TOC concentration of 10 mg / L. Different chemical probes were added to 30 mL of the DOM solution: 2,4,6-trimethylphenol (TMP, 10 μmol / L, used for characterization). 3 DOM*), furfuryl alcohol (FFA, 10 μmol / L, used for characterization) 1 O2) and p-chlorobenzoic acid (pCBA, 4 μmol / L, used to characterize ∙OH), and adjust the pH to 7.0; according to the method in Example 2, each sample was injected into a petri dish for photocatalytic reaction (light intensity 45000 Lux, 25±1 ℃), and the experiment was set up for 3 replicates. The initial concentration of the probe (C0) was detected, and then the probe concentration (C0) was detected every 20 min.t ) changes, In(C) at different times t The results of / C0) are shown in Tables 17-19 and Figures 7-9 Different DOM solutions 1 The steady-state concentration of O2 is shown in Table 20.

[0093] Calculate the active species based on probe decay kinetics. 1 The steady-state concentration of O2.

[0094] The steady-state concentration of the active species is calculated as follows: The steady-state concentration [Radical]ss of the active species follows the formula: .

[0095] in, The concentration of the probe at time t (i.e., C) t ), The initial concentration of the probe (i.e., C0). Let be the second-order reaction rate constant for the reaction between the probe and the active species. When the probe is FFA and the active species is... 1 In the presence of O2, the second-order reaction rate constant is 1.0 × 10⁻⁶. 8 M -1 s -1 , Let In(C) be the first-order reaction rate constant between a certain active species and a probe. t / C0) slope as a function of t (unit: s) (in this embodiment, the time unit also needs to be converted).

[0096] The steady-state concentration [Radical]ss of the active species is calculated using the formula: .

[0097] The results show that active species were generated in both DOM solutions. 3 DOM*、 1 O2, and DOM16_B3 transformed by Bacillus cereus has higher 3 DOM*、 1 O2 generation capability. Among them, DOM16_B3's... 1 The steady-state concentration of O2 was significantly higher than that of unconverted DOM (DOM16).

[0098] Table 17 OH- formation in DOM16 and DOM16_B3 solutions

[0099] Table 18 DOM16 and DOM16_B3 solutions 1 O2 generation

[0100] Table 19 DOM16 and DOM16_B3 solutions 3 DOM* generation status

[0101] Table 20 DOMs solutions with 10 mg / L TOC 1 O2 steady-state concentration (mol / L)

[0102] 3. Photochemical characteristics of DOM To investigate the compositional characteristics and evolution of DOMs from different sources, 3D-EEM was used to characterize the DOM solutions (DOM8, DOM16, DOM16_B1, and DOM16_B3). Before 3D-EEM testing, each DOM solution was diluted with ultrapure water to a TOC concentration of 10.00 mg / L. Specifically, a fluorescence spectrophotometer was used for testing, with excitation wavelengths (Ex) ranging from 200 to 450 nm and emission wavelengths (Em) ranging from 250 to 600 nm. 3D-EEM can indicate the main fluorescent component types in the DOM, such as proteins and humic substances, by the position and intensity of fluorescence peaks. The EEM spectra are typically divided into five regions as shown in Table 21 to identify different components.

[0103] Table 21 Classical region division of fluorescence spectra

[0104] See results Figure 10 The main fluorescence peak of DOM16 is located in region IV, and its components are mainly soluble metabolites released by Chlorella. After bacterial fermentation, the fluorescence peak of DOM16_B3 migrates from region IV to region V. This phenomenon indicates that bacteria can consume easily degradable organic matter released by Chlorella and gradually accumulate to form complex organic matter with a high degree of humification.

[0105] The PARAFAC analysis results are shown below. Figure 11 From a, b, and c, two typical components were obtained: a humic acid-like component and a Chlorella extracellular metabolite component. The proportions of these two components in DOM16, DOM16_B1, and DOM16_B3 are shown in [reference needed]. Figure 11 See d in Table 22. Bacterial fermentation promotes DOM humification and increases the contribution of humic components. This phenomenon indicates that the action of Bacillus cereus NCU LJ-1 significantly transforms the composition of DOM from easily degradable algal metabolites to recalcitrant humic acid-like substances, consistent with the aforementioned conclusion regarding changes in EEM fluorescence peaks.

[0106] Typical fluorescence index parameters of DOM16, DOM16_B1, and DOM16_B3, including biogenic index (BIX), fluorescence index (FI), and humification index (HIX), were measured. The results are shown in Table 23. The BIX values ​​of DOM16 were all above 0.7, and the FI values ​​were close to 1.8, indicating that algal DOM is mainly composed of active metabolites and has strong biogenic characteristics. After bacterial fermentation treatment, the HIX values ​​of DOM16_B1 and DOM16_B3 increased significantly while the FI values ​​decreased, further demonstrating that bacterial transformation of DOM weakens its biogenic characteristics, enhances its humic characteristics, and increases its aromaticity and conjugation degree.

[0107] Table 22. Proportions of the two components in DOM16, DOM16_B1, and DOM16_B3

[0108] Table 23 Fluorescence indices of DOM16, DOM16_B1, and DOM16_B3

[0109] 4. FTICR-MS Molecular-Level Response Mechanism FTICR-MS peaks of four solutions (ADE, DOM8, DOM16, and DOM16_B3) were collected and Van Krevelen plots were generated to compare the relative proportions of different molecular classes. Results are shown below. Figures 12-13 According to Table 24, the samples were rich in lignins, unsaturated hydrocarbons, proteins, lipids, amino sugars, and fused aromatic hydrocarbons, while the abundance of carbohydrates and tannins was relatively low. Figure 12 ).Depend on Figure 13As shown in Table 24, lipids and proteins account for nearly 30% of the composition of ADE, lignin accounts for 30.69%, and the remaining categories account for a relatively small proportion. This indicates that anaerobic digestion fluid and DOM contain not only a large amount of microbial metabolites (proteins) but also a considerable proportion of humic substances (lignins). In contrast, algal DOM is mainly composed of proteins, with proteins accounting for 78.59% in DOM8, while lipids and lignins account for only 5.69% and 10.64%, respectively. With the culture period extended to 16 days, the proportion of proteins in DOM16 decreased to 66.40%, while lignin increased to 20.18%, indicating that the algae's own metabolism can gradually improve the aromaticity of DOM in the culture medium to some extent. After 3 days of fermentation with Bacillus cereus NCU LJ-1, the proportion of proteins in DOM16_B3 decreased to 40.13%, lipids decreased from 8.27% to 5.33%, while lignin increased from 20.18% to 38.76%, and fused hydrocarbons increased from 1.28% to 2.17%. The proportions of amino sugars and carbohydrates also increased, indicating that bacteria preferentially consume easily degradable proteins and lipids and generate or enrich lignin and fused aromatic hydrocarbons with higher aromaticity. Bacterial transformation causes algal DOM to evolve from low-aromaticity, low-conjugation components to high-aromaticity, high-conjugation humic-like components, improving the light absorption capacity and triplet excitation efficiency of DOM, thereby enhancing its aromaticity. 1 The generation of active species such as O2 ultimately achieves synergistic enhanced photodegradation of STZ.

[0110] Table 24 Molecular composition (%) of the four solutions (ADE, DOM8, DOM16, DOM16_B3)

[0111] Example 8: Comparison of the photodegradation effect of Chlorella DOM transformed by different bacteria (Bacillus cereus NCU LJ-1, Moistened Cellulosum, and Sphingosporium zeylans) on sulfathiazole.

[0112] (1) DOM16 acquisition and preprocessing: Extracellular DOM16 of Chlorella was obtained according to the method in Example 3 and was used for later use.

[0113] (2) Preparation of seed culture of different bacteria: Bacillus cereus NCU LJ-1, Fibromosyne humidus, and Sphingomonas zeylinum were taken respectively and inoculated into LB liquid medium. They were cultured at 37°C in the dark or in the dark until the logarithmic growth phase to prepare the seed culture of the corresponding bacteria.

[0114] (3) Transformation of DOM16 by different bacteria: Following the method for preparing DOM16_B3 in Example 6, 100 mL of DOM16 solution was added to each 250 mL Erlenmeyer flask; 1 mL of the logarithmic growth phase seed culture of each bacterium was taken, centrifuged, washed with sterile ultrapure water, and resuspended in 0.50 mL of sterile water, and then inoculated into the corresponding DOM16 fermentation system. All cultures were incubated in a 37℃ incubator in the dark for 3 days. An uninoculated DOM16 solution was selected as a control, with all other conditions the same.

[0115] (4) After fermentation, the samples were sterilized through a 0.22 μm filter membrane to obtain different DOM solutions after 3 days of fermentation. STZ mother liquor was added to each solution to make the STZ concentration 1 mg / L, and the pH of the samples was adjusted to 7. According to the method in Example 2, the samples were injected into petri dishes for photocatalytic reaction (light intensity 45000 Lux, 25±1 ℃). The experiment was set up in 3 replicates, with a reaction time of 6 h. 1 mL samples were taken before and after the reaction to detect the change in STZ concentration.

[0116] The results are shown in Table 25. Compared with DOM without bacterial inoculation, the removal capacity of STZ was improved after different bacteria transformed Chlorella DOM, with removal rates ranging from 31.64±0.51% to 35.48±0.79%, indicating that the introduction of bacteria can improve the degradation efficiency of STZ.

[0117] Table 25 STZ removal rate (%) after different bacteria transformed Chlorella DOM16

[0118] Example 9 Comparison of STZ photodegradation effects before and after treatment with mixed bacteria on different microalgal extracellular DOM and mixed algal extracellular DOM

[0119] (1) Obtaining extracellular DOM of single algae and mixed algae: Four microalgae, *Scenedesmus obliquus*, *Chlamydomonas*, *Anabaena aeruginosa*, and *Microcystis aeruginosa*, were cultured in BG11 medium to the logarithmic growth phase. 2 ml of each culture solution was centrifuged, resuspended in 1 mL of BG11 medium, and inoculated into 200 mL of BG11 medium. *Anabaena aeruginosa*, being a suspended aggregate, was harvested by centrifugation and inoculated into 200 mL of BG11 medium at a wet weight. After 16 days of culture, the algal cells were removed by centrifugation, and the supernatant was filtered through a 0.22 μm microporous membrane for sterilization to obtain the extracellular DOM of a single algal species. Except for *Anabaena aeruginosa*, the other three microalgae were cultured separately to the logarithmic growth phase, and 2 ml of their culture solution was centrifuged, resuspended in 1 mL of BG11 medium, and then mixed and inoculated into 200 mL of BG11 medium. Since nitrogen-fixing algae grow in suspension aggregates, after centrifugation and harvesting, they were inoculated at 2 g / L (wet weight) into the BG11 medium inoculated with the above three microalgae. After inoculation, a mixed algal solution was obtained and cultured for 16 days. Then, the algal cells were removed by centrifugation, and the supernatant was filtered through a 0.22 μm microporous membrane for sterilization to obtain the mixed algal extracellular DOM.

[0120] (2) Preparation of mixed bacterial culture: Single bacteria of Bacillus cereus NCU LJ-1, Cnidosa humidus, and Sphingomonas zeylans were picked and cultured in LB medium to the logarithmic growth phase. The culture conditions were 37℃, protected from light or in the dark. 1 mL of the seed culture of the logarithmic growth phase mixed bacterial culture was taken, centrifuged, washed with sterile ultrapure water, and resuspended in 0.5 mL of sterile water to obtain the bacterial suspension for inoculation.

[0121] (3) Preparation of DOM16_B3 for single algae transformation by mixed bacteria and DOM16_B3 for mixed algae transformation by mixed bacteria: Take the DOM16_B3 for single algae and DOM16_B3 for mixed algae obtained in step (1) and dispense them into conical flasks. Inoculate the bacterial suspension of mixed bacteria obtained in step (2) and culture for 3 days at 37°C in the dark or in the dark. The mixed bacteria use the DOM16_B3 for single algae and DOM16_B3 for mixed algae transformation as substrates. After the culture is completed, centrifuge to remove the bacterial cells and filter through a 0.22 μm microporous membrane to remove bacteria, and obtain the DOM16_B3 solution for single algae and DOM16_B3 solution for mixed algae transformation by mixed bacteria.

[0122] (4) Photodegradation of STZ: 30 mL of extracellular DOM from single algae, extracellular DOM from mixed algae, extracellular DOM from mixed bacteria transformed into extracellular DOM16_B3 from single algae, and extracellular DOM16_B3 from mixed bacteria transformed into extracellular DOM16_B3 from mixed algae were placed in culture dishes, and STZ stock solution was added to make the initial concentration 1 mg / L, and the pH of the system was adjusted to 7.0. The samples were placed in the photoreactor of Example 2 and irradiated at 25±1℃ and 45000 Lux for 6 h, with 3 replicates for each group.

[0123] (5) Sample determination: Each sample was taken before and after the reaction, filtered through a 0.22 μm microporous membrane, and the STZ residual concentration was determined by HPLC. The STZ removal rate was calculated, and the differences in photodegradation enhancement of different microalgae by mixed bacteria were compared. The results are shown in Table 26. The TOC content of each sample was also detected, and the results are shown in Table 27.

[0124] The results showed that mixed bacteria could reduce the TOC of extracellular DOM of different microalgae. The photodegradation ability of STZ by extracellular DOM of Scenedesmus obliquus, Microcystis aeruginosa and Chlamydomonas was increased after treatment with mixed bacteria, while the photodegradation ability of STZ by DOM of other microalgae after treatment with mixed bacteria was not significantly improved.

[0125] Table 26 STZ removal rate (%) before and after treatment with mixed bacteria for different microalgae DOM

[0126] Table 27 TOC content (mg / L) of different microalgae before and after DOM treatment with mixed bacteria

[0127] Example 10: Comparison of the photodegradation capacity of extracellular DOM of Chlorella, mixed algae transformed with Bacillus cereus NCU LJ-1, and the extracellular DOM of original Chlorella and mixed algae for different types of antibiotics.

[0128] (1) Preparation of Chlorella extracellular DOM16 and Bacillus cereus transformed Chlorella extracellular DOM16_B3: First, Chlorella extracellular DOM16 was prepared according to the method of Example 3, and Bacillus cereus transformed Chlorella extracellular DOM16_B3 was prepared according to the method of Example 6 for later use.

[0129] (2) Preparation of mixed algal extracellular DOM16 and Bacillus cereus transformation of mixed algal extracellular DOM16_B3: First, prepare mixed algal extracellular DOM16 according to the method of Example 9. Then, refer to the method of preparing mixed algal extracellular DOM16_B3 solution after mixed bacteria transformation in Example 9 to prepare Bacillus cereus transformation of mixed algal extracellular DOM16_B3 for later use.

[0130] (3) The four antibiotics and their preparation and addition methods are as follows: the three sulfonamides (sulfathiazole, sulfadiazine, and sulfamethoxazole) are dissolved in dilute sodium hydroxide; lincomycin is directly prepared into a solution using its hydrochloride salt. After preparing the above four antibiotics into 1 g / L solutions according to the corresponding methods, water is added to prepare antibiotic stock solutions with a concentration of 10 mg / L.

[0131] (4) Take 49.5 mL of Chlorella extracellular DOM16, Bacillus cereus-transformed Chlorella extracellular DOM16_B3, mixed algae extracellular DOM16, and Bacillus cereus-transformed mixed algae extracellular DOM16_B3 into a culture dish, adjust the pH to 7.0, and add 0.5 mL of the four antibiotic stock solutions prepared in step (3) to obtain different reaction solutions with an antibiotic concentration of 0.1 mg / L. Replace the DOM solution in the above antibiotic treatment groups with ultrapure water, and set up a blank control group with the other conditions the same.

[0132] (5) The sample obtained in step (4) was placed in the photoreactor of Example 2 and irradiated at 25±1℃ and 45000 Lux for 6 h. Three replicates were set up for each group. Samples were taken before and after the reaction. After the samples were filtered through a 0.22 μm microporous membrane, the residual concentration of each antibiotic was determined by high performance liquid chromatography (HPLC). The real-time antibiotic concentration / initial antibiotic concentration (C) of each antibiotic was calculated based on the concentrations before and after the reaction. t / C0). And compare the photodegradation enhancement effects of DOM16_B3 and DOM16 on different classes of antibiotics.

[0133] The results are shown in Table 28. After 6 hours of light exposure, the corresponding values ​​of the four antibiotics in the pure water treatment group showed relatively small changes, indicating that the direct photolytic effect of the antibiotics themselves was limited under the conditions of this embodiment. After adding Chlorella extracellular DOM16, the C6 values ​​of sulfadiazine, sulfathiazole, and lincomycin... t The C0 values ​​were 0.58±0.04, 0.90±0.28, and 0.92±0.12, respectively, all showing a decreasing trend compared to the pure water treatment group. This indicates that algal DOM can participate in the indirect photodegradation process of antibiotics and exhibits a certain photodegradation-promoting effect on some antibiotics. Furthermore, the extracellular DOM16_B3 of *Chlorella vulgaris* obtained after transformation with *Bacillus cereus* NCU LJ-1 reduced the C0 of sulfathiazole and lincomycin. tThe C0 values ​​further decreased to 0.85±0.07 and 0.63±0.04, indicating that the photochemical promoting effect of algal DOM on some antibiotics was enhanced after bacterial transformation. For the mixed algal extracellular DOM16, it also showed a certain photodegradation promoting effect on sulfadiazine and lincomycin. However, the mixed algal extracellular DOM16_B3 transformed by Bacillus cereus did not show a further enhancement in its photodegradation promoting effect on lincomycin. This suggests that the system composition is more complex after mixing DOM from different sources, and there may be phenomena such as competition for active components, light absorption interference, or quenching of active species, thus affecting its photodegradation effect on antibiotics.

[0134] Table 28 C-values ​​of Chlorella and mixed algae treated with different antibiotics using extracellular DOM16 and DOM16_B3. t / C0

[0135] Finally, it should be noted that the above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art can still modify the technical solutions described in the foregoing embodiments or make equivalent substitutions for some of the technical features. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A method for enhancing the photodegradation of antibiotics by bacterial transformation of extracellular soluble organic matter in microalgae, characterized in that: Includes the following steps: (1) Cultivate microalgae in a culture medium, collect the supernatant of the culture medium by centrifugation and filter to obtain the extracellular soluble organic matter of microalgae; (2) Inoculate the bacteria into the microalgal extracellular soluble organic matter obtained in step (1) and culture it. After the culture is completed, centrifuge and filter the culture medium to obtain the bacterial transformation of microalgal extracellular soluble organic matter. (3) The bacterial-transformed microalgal extracellular soluble organic matter obtained in step (2) is added to the antibiotic-containing wastewater to enhance the photodegradation of antibiotics under light conditions; In step (1), the microalgae are selected from any one or more of Chlorella, Chlamydomonas, Scenedesmus obliquus, Anabaena aeruginosa, and Microcystis aeruginosa. In step (2), the bacteria are selected from any one or more of Bacillus cereus, Pseudomonas humifusa, and Sphingomonas zeylans, and the Bacillus cereus is classified as follows: Bacillus cereus NCU LJ-1, with accession number CCTCC NO:M 20251470, the strain number of the *Potentilla hygroscopica* is BNCC336468, and the accession number of the *Sphingosporium zeylans* is CCTCC NO:M 20251462. In step (3), the antibiotics include any one or more of sulfathiazole, sulfadiazine, and lincomycin.

2. The method according to claim 1, characterized in that: In step (1), the culture medium is BG11 medium; the conditions for microalgae culture include: culture temperature 25±1℃, light intensity 2000~3000 Lux, no additional aeration, culture time 8~16 days; and filtration using a 0.1~0.5 μm filter membrane.

3. The method according to claim 1, characterized in that: In step (2), after the bacteria are cultured to the logarithmic growth phase, they are inoculated into the extracellular dissolved organic matter of microalgae at a volume percentage of 0.5-2%. The culture conditions include: culture temperature of 35-38℃, culture time of 1-4 days, culture in the dark or away from light; and filtration using a 0.1-0.5 μm filter membrane.

4. The method according to claim 1, characterized in that: In step (3), the amount of bacteria that transform the extracellular dissolved organic matter of microalgae in the wastewater is 10 to 500 mg / L based on the total organic carbon. The photodegradation conditions include: pH 6.5 to 7.0, temperature 25±1℃, light intensity 40000 to 50000 Lux, and time 1 to 24 h.

5. A bacterial-transformed microalgal extracellular soluble organic compound obtained by the method of claim 1.

6. A microbial preparation, characterized in that: Includes the bacterial transformation of extracellular dissolved organic matter of microalgae as described in claim 5.

7. The application of the method of claim 1, the bacterial transformation of extracellular soluble organic matter of microalgae as described in claim 5, or Bacillus cereus in enhancing the indirect photodegradation of antibiotics, characterized in that: The classification of Bacillus cereus is named as follows: Bacillus cereus NCU LJ-1, with accession number CCTCC NO:M 20251470, contains antibiotics including any one or more of sulfathiazole, sulfadiazine, and lincomycin. The enhanced antibiotic photodegradation is achieved by bacteria transforming extracellular soluble organic matter from microalgae to enhance the photodegradation of the antibiotic.

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