Method for treating aquaculture wastewater with antibiotics by microalgae and bacteria in cooperation

Abstract

The application discloses a method for treating antibiotics in aquaculture wastewater by microalgae and bacteria, and belongs to the technical field of biological treatment of aquaculture wastewater. Chlorella microalgae and a tetracycline-degrading dominant bacterial population obtained by tetracycline concentration gradient acclimation and screening from activated sludge in an aquaculture farm are inoculated into a sequencing batch photobioreactor at a ratio of 3-8:1, and the algal-bacterial symbiotic granular sludge is formed by self-assembly under alternating light and dark operation and selection pressure of gradually shortened settling time. Under the condition of no additional carbon source, the O2 / CO2 / DOC closed-loop circulation of intra-granular algal-bacterial mutualism is utilized to realize the simultaneous and efficient removal of tetracycline antibiotics, ammonia nitrogen and total phosphorus in aquaculture wastewater. Under the optimal conditions, the removal rate of tetracycline is 92%, the removal rate of ammonia nitrogen is 97%, the removal rate of total phosphorus is 88%, and the protein content of the harvested biomass is not less than 44%, which can be used as a feed protein raw material, so that the dual benefits of wastewater treatment and resource utilization are realized.

Description

Technical Field

[0001] This invention belongs to the field of biological treatment technology for aquaculture wastewater, specifically involving a symbiotic granular sludge system constructed by Chlorella microalgae and tetracycline-degrading dominant bacteria, which achieves simultaneous and efficient removal of tetracycline antibiotics, ammonia nitrogen and total phosphorus from aquaculture wastewater through a synergistic treatment method of microalgae and bacteria. Background Technology

[0002] Aquaculture is a vital industry for ensuring the supply of high-quality animal protein. However, the extensive use of antibiotics during the aquaculture process has led to aquaculture wastewater becoming a complex source of pollution containing antibiotics and high concentrations of nitrogen and phosphorus nutrients. Tetracycline antibiotics are the most widely used in aquaculture due to their broad-spectrum antibacterial properties and low cost. However, once discharged into the aquatic environment, they not only threaten the safety of aquatic ecosystems but also induce the generation and spread of antibiotic resistance genes, posing a serious public health risk. Currently, conventional biological treatment processes such as activated sludge processes, constructed wetlands, and membrane bioreactors can remove nitrogen and phosphorus nutrients from wastewater to some extent, but their ability to remove dissolved antibiotics is limited. Tetracycline removal rates are typically less than 30%, and the nitrogen and phosphorus levels in the treated effluent still struggle to consistently meet emission standards. Therefore, developing efficient biological treatment processes that can simultaneously achieve antibiotic degradation and deep nitrogen and phosphorus removal has become a critical technical challenge that urgently needs to be addressed in the field of aquaculture wastewater treatment.

[0003] In recent years, microalgae-bacterial granular sludge (MBGS) has attracted widespread attention as a novel biological treatment technology. This technology utilizes the symbiotic relationship between microalgae and bacteria within granular sludge to couple photosynthetic oxygen production with organic matter degradation, demonstrating significant advantages in wastewater nutrient removal and low-carbon operation. In their paper "Microalgae simultaneously promote antibiotic removal and antibioticresistance genes / bacteria attenuation in algal-bacterial granular sludge system," published in the *Journal of Hazardous Materials* (Vol. 438, 2022, p. 129286), Liu et al. inoculated microalgae onto existing bacterial granular sludge to form an algae-bacterial granular sludge system. In synthetic wastewater containing a mixture of tetracycline and sulfadiazine (2–4 mg / L), they achieved a tetracycline removal rate of 79.0% and a sulfadiazine removal rate of 94.0%. However, this scheme uses naturally enriched rather than targeted screening microbial communities as degrading functional bacteria. When the tetracycline concentration increases to the actual level of aquaculture wastewater (5~15 mg / L), the degradation efficiency decreases significantly, and the synergistic effect of antibiotic degradation and simultaneous deep removal of nitrogen and phosphorus is not achieved.

[0004] US Patent Application Publication No. US10189732B2 discloses an algae-bacterial granule for wastewater treatment and biomass feedstock production. This method cultivates algae in a wastewater system under static light conditions to form algae-bacterial granules, utilizing the symbiotic relationship between microalgae and bacteria to remove organic matter and nutrients from the wastewater while simultaneously producing recyclable biomass. However, this approach does not address the treatment of antibiotic-containing wastewater, does not introduce targeted screening bacteria with antibiotic degradation capabilities, and lacks targeted optimization of the algae-bacterial inoculation ratio and functional stratification design during granulation. Therefore, it is unsuitable for the synergistic treatment of antibiotic and nitrogen-phosphorus compound pollution in aquaculture wastewater.

[0005] A comprehensive analysis of existing technologies reveals the following core bottlenecks in the treatment of antibiotics in aquaculture wastewater using microalgae-bacteria granular sludge systems: Current MBGS systems generally use pre-cultured bacterial granular sludge or activated sludge as a substrate, with microalgae passively inoculated onto the granular surface. The dominant role of the granular structure belongs to the bacteria. This bacteria-dominated, algae-assisted construction method leads to three key problems: First, the proportion of antibiotic-degrading bacteria in the naturally enriched bacterial community is low and uncontrollable, resulting in a sharp drop in degradation efficiency under high tetracycline concentrations. Second, the carbon source metabolism of heterotrophic degrading bacteria relies on organic carbon in the wastewater or external carbon sources (such as glucose and sodium acetate), increasing operating costs and carbon emissions. Third, antibiotic-degrading bacteria, nitrifying bacteria, denitrifying bacteria, and polyphosphate-accumulating bacteria are randomly distributed within the granules rather than functionally stratified, lacking spatial coupling optimization among the three biological treatment functions (antibiotic degradation, denitrification, and phosphorus removal). The root cause of the above three problems lies in the fact that existing technologies have not recognized that microalgae can replace bacteria as the core of granular structures, thereby utilizing the oxygen concentration gradient formed by photosynthesis to drive the spontaneous stratified colonization of functional bacterial communities, achieving optimized and improved structural self-organization and functional spatial configuration. Summary of the Invention

[0006] The purpose of this invention is to address the core technical bottlenecks of existing microalgae-bacterial granular sludge systems in the treatment of antibiotics in aquaculture wastewater, namely, the uncontrollable function of degrading bacteria, dependence on external carbon sources, and the lack of spatial coupling optimization of the three treatment functions. This invention provides a method for the synergistic treatment of antibiotics in aquaculture wastewater by microalgae and bacteria. This method uses Chlorella microalgae as the core of the granular structure and pre-screened tetracycline-degrading dominant bacteria as directional functional components. Through a reverse-designed algae-bacterial inoculation ratio and a self-assembly granulation process driven by alternating light and dark conditions and sedimentation selection pressure, the simultaneous and efficient removal of tetracycline antibiotics, ammonia nitrogen, and total phosphorus from aquaculture wastewater is achieved without the addition of external carbon sources.

[0007] To achieve the above-mentioned objectives, the present invention adopts the following technical solution:

[0008] A method for the synergistic treatment of antibiotics in aquaculture wastewater by microalgae and bacteria includes the following steps:

[0009] Step 1. Microalgae of the genus *Chlorella* and tetracycline-degrading dominant bacterial communities obtained from activated sludge of an aquaculture farm through tetracycline concentration gradient acclimation and screening were co-inoculated in a sequencing batch photobioreactor at an algae-to-bacteria cell ratio of 3–8:1. The screening method for the tetracycline-degrading dominant bacterial communities was as follows: using activated sludge from the aeration tank of an aquaculture farm as the inoculum and tetracycline as the sole carbon source, the communities were acclimated and cultured in three stages at concentration gradients of 2, 5, and 10 mg / L, with each stage of acclimation and culture lasting no less than 7 days. The final stable bacterial community was selected as the tetracycline-degrading dominant bacterial community. Identification using 16S rRNA high-throughput sequencing revealed that the dominant genera of the degrading dominant bacterial community included at least two of the genera *Stenotrophomonas maltophilia*, *Hydrogenophaga*, and *Sphingobacterium*.

[0010] Step 2. Cultivate the sludge using an alternating light and dark cycle, with a light period of 10–14 h and a dark period of 10–14 h. Gradually shorten the settling time during cultivation to apply selection pressure. The settling time is gradually reduced from an initial 25–35 min to 3–8 min, decreasing by 3–5 min every 3–5 days, driving the self-assembly of the *Chlorella* microalgae and the tetracycline-degrading dominant bacterial community to form algal-bacterial symbiotic granular sludge. The granular sludge has a particle size of 1.5–3 mm, with a polysaccharide content of not less than 80 mg / g VSS in the tightly bound extracellular polymeric material (TB-EPS) and a protein-to-polysaccharide mass ratio of 0.3–0.5 in the loosely bound extracellular polymeric material (LB-EPS).

[0011] Step 3. The aquaculture wastewater containing tetracycline antibiotics, ammonia nitrogen, and total phosphorus is introduced into the sequencing batch photobioreactor (SBR) and treated under conditions of 4000–8000 lux light intensity, pH 7.0–8.5, and hydraulic retention time of 18–36 h. The concentration of tetracycline antibiotics in the wastewater is 1–20 mg / L, ammonia nitrogen is 20–80 mg / L, and total phosphorus is 3–15 mg / L. Without an external carbon source, oxygen is produced by the photosynthesis of microalgae within the granular sludge, which then powers the outer aerobic bacteria to degrade the tetracycline antibiotics. The carbon dioxide produced by bacterial metabolism is then used to assimilate with the photosynthetic nutrients of the microalgae, forming a symbiotic relationship between algae and bacteria. This process treats the aquaculture wastewater, achieving the simultaneous removal of tetracycline antibiotics, ammonia nitrogen, and total phosphorus.

[0012] Furthermore, the preferred value for the algae-bacteria cell ratio is 5:1.

[0013] Furthermore, the harvested algae and fungi biomass, after being dried, has a protein content of no less than 44% by mass. The algae and fungi biomass can be used as a feed protein raw material, achieving the dual benefits of wastewater treatment and biomass resource utilization.

[0014] Furthermore, after the aforementioned treatment, the concentrations of tetracycline antibiotics, ammonia nitrogen, and total phosphorus in the effluent meet the secondary standard requirements for aquaculture wastewater discharge.

[0015] Furthermore, the aquaculture wastewater is freshwater aquaculture wastewater.

[0016] Compared with the prior art, the present invention has the following beneficial effects:

[0017] (1) This invention proposes for the first time a strategy for constructing algae-bacterial symbiotic granular sludge with Chlorella microalgae as the core of the granular structure and pre-screened tetracycline-degrading dominant bacterial community as the directional functional component. This breaks through the existing microalgae-bacterial granular sludge technology, which is dominated by bacteria in granulation and passively attached by microalgae. It realizes the optimization of the self-organization of the granular structure and the functional spatial configuration. The reverse inoculation design with an algae-bacterial cell ratio of 3~8:1 enables a large number of Chlorella cells to secrete tightly bound extracellular polymeric polysaccharides to form a three-dimensional gel skeleton. The pre-screened degradation bacteria spontaneously colonize and stratify along the dissolved oxygen gradient driven by photosynthesis within the granule. An aerobic degradation zone is formed in the outer layer (DO>2 mg / L), a nitrification zone is formed in the middle layer (DO=0.5~2 mg / L), and a denitrification and biological phosphorus removal zone is formed in the core layer (DO<0.5 mg / L). This enables the three biological treatment functions of tetracycline degradation, denitrification and phosphorus removal to be spatially coupled within a single granule.

[0018] (2) The functional bacterial groups obtained by the present invention through three-stage domestication and screening of tetracycline concentration gradient have stable degradation activity against high concentrations of tetracycline (1~20 mg / L). The closed-loop O2 / CO2 / DOC symbiotic cycle of algae and bacteria enables the degrading bacteria to maintain metabolism with dissolved organic carbon released by microalgae under conditions without external carbon source, eliminating dependence on external carbon source, significantly reducing operating costs and carbon emissions. Under the optimized process conditions of light intensity 6000 lux, pH 7.5~8.2, and hydraulic retention time 24 h, the tetracycline removal rate reached 92%, the ammonia nitrogen removal rate reached 97%, and the total phosphorus removal rate reached 88% for simulated aquaculture wastewater containing 10 mg / L tetracycline, 50 mg / L ammonia nitrogen, and 8 mg / L total phosphorus. The effluent quality meets the secondary standard requirements for aquaculture tailwater discharge.

[0019] (3) The algae and bacteria biomass harvested by this invention has a protein content of not less than 44% by mass, and can be used as a low-value feed protein raw material. It realizes the dual benefits of aquaculture wastewater treatment and biomass resource utilization, and provides an integrated process solution for green and low-carbon biological treatment of aquaculture tailwater with nitrogen and phosphorus removal and antibiotic removal functions. Attached Figure Description

[0020] Figure 1 The curves show the changes in particle size and sedimentation velocity over culture time during the granulation process.

[0021] Figure 2 The bar chart shows the comparison of tetracycline removal rate, ammonia nitrogen removal rate, and total phosphorus removal rate between Example 1, Example 2, and Comparative Examples 1-3. Detailed Implementation

[0022] The technical solution of the present invention will be further described in detail below with reference to the accompanying drawings and embodiments. The following embodiments are only used to illustrate the present invention and are not intended to limit the scope of the present invention.

[0023] A method for the synergistic treatment of antibiotics in aquaculture wastewater by microalgae and bacteria includes the following steps: Chlorella is inoculated into BG-11 medium and cultured under conditions of 25±1°C, 4000 lux light intensity, and a 12 h / 12 ​​h light / dark cycle until the algal cell density reaches 1×10⁻⁶ cells / years. 7 The culture medium, with a cell / mL concentration of at least [number] cells, is prepared for use. The preferred Chlorella microalgae used in this invention are *Chlorella vulgaris*, obtained from the algae collection of the Institute of Hydrobiology, Chinese Academy of Sciences. The main components of the BG-11 culture medium include: NaNO3 1.5 g / L, K2HPO4·3H2O 0.04 g / L, MgSO4·7H2O 0.075 g / L, CaCl2·2H2O 0.036 g / L, citric acid 0.006 g / L, ferric ammonium citrate 0.006 g / L, Na2EDTA 0.001 g / L, Na2CO3 0.02 g / L, and trace element solution. Sterilization conditions are 121°C for 20 min. *Chlorella pyrenoidosa* or *Chlorella sorokiniana* can also be used, with appropriate adjustments to the culture conditions as described above.

[0024] Activated sludge from the aeration tanks of aquaculture farms was collected as inoculum. The activated sludge was taken from the wastewater treatment system of a freshwater fishpond that had been operating for more than one year and had previously used tetracycline antibiotics. The collected activated sludge was inoculated at a rate of 10% (volume fraction) into an inorganic salt culture medium containing no organic carbon source. Using tetracycline as the sole carbon and energy source, a three-stage acclimatization culture was conducted at concentration gradients of 2 mg / L → 5 mg / L → 10 mg / L. Each acclimatization culture was carried out in a constant-temperature shaking incubator at 30°C and 150 r / min for at least 7 days. After each acclimatization stage, the culture solution was transferred at a 10% inoculum to a fresh inorganic salt culture medium containing the next concentration gradient of tetracycline. The culture was continuously passaged at a tetracycline concentration of 10 mg / L for at least three generations. The strains that were stable after passage and exhibited a tetracycline degradation rate of at least 80% were selected as the dominant tetracycline-degrading strains.

[0025] The dominant tetracycline-degrading bacterial communities identified through screening were subjected to 16S rRNA high-throughput sequencing. Total genomic DNA was extracted from the bacterial communities and amplified by PCR using universal primers 338F (5'-ACTCCTACGGGAGGCAGCAG-3') and 806R (5'-GGACTACHVGGGTWTCTAAT-3'). The amplified products were purified and subjected to paired-end sequencing using the Illumina MiSeq platform. After quality control, splicing, and chimera removal, the sequencing data were clustered into operational taxonomic units (OTUs) at a 97% similarity level, and species annotation was performed based on the Silva database. The results showed that the dominant genera of the degrading bacterial community included *Stenotrophomonas maltophilia* (relative abundance 15%–25%), *Hydrogenophaga* (relative abundance 10%–20%), and *Sphingobacterium* (relative abundance 8%–15%), with a combined relative abundance of no less than 40%. *Stenotrophomonas maltophilia* belongs to the phylum Proteobacteria and is a functional genus reported in the literature to be closely related to tetracycline biotransformation; *Hydrogenophaga* has a strong ability to degrade aromatic compounds under aerobic conditions; and *Sphingobacterium* was identified as a core functional bacterium in various antibiotic degradation systems.

[0026] The *Chlorella* microalgae suspension cultured in the above steps and the tetracycline-degrading dominant bacterial suspension were co-inoculated into a photo-sequencing batch reactor (PSBR) at an algae-to-bacterial cell ratio of 3–8:1. A preferred algae-to-bacterial cell ratio was 5:1. The PSBR had an effective volume of 5 L, a height-to-diameter ratio of 5:1, and was surrounded by LED light sources. Each operating cycle included five stages: influent, light reaction, dark reaction, settling, and drainage. The light reaction time was 10–14 h, and the dark reaction time was 10–14 h, with a preferred light / dark cycle of 12 h / 12 ​​h. The settling time started at 30 min and decreased by 4 min every 4 days, gradually decreasing to 5 min after approximately 21 days. The drainage ratio was 50%.

[0027] Under the above operating conditions, the algae-bacterial co-culture system undergoes a three-stage evolution process from flocculent to aggregated to dense granulation. Days 1-7 are the induction period, during which *Chlorella* cells secrete large amounts of extracellular polymeric substances (EPS). Among these, the polysaccharide components of tightly bound EPS (TB-EPS) form a three-dimensional gel network with glucuronic acid and mannose as the main chains. Pre-screened degrading bacteria, through their surface hydrophobic outer membrane proteins (OmpA family), undergo hydrophobic-electrostatic adsorption with the algal polysaccharide backbone, initially forming algae-bacterial micro-aggregates. Days 8-14 are the aggregation growth period, during which the gradually shortening settling time eliminates loosely structured flocculent sludge, retaining algae-bacterial aggregates with good settling performance. The aggregate particle size increases to 0.5-1.5 mm. Days 15-21 are the mature granulation period, with particle size stabilizing at 1.5-3 mm, a dense structure, and a settling velocity reaching 15-25 m / h. Figure 1 As shown, the particle size and settling velocity exhibit an S-shaped growth curve during the granulation process, which is initially slow and then accelerates.

[0028] EPS from mature granular sludge was characterized using a fractional thermal extraction method. After centrifugation and washing, the granular sludge was resuspended in phosphate buffer (pH 7.0), stirred at 4°C for 1 h, and then centrifuged (4000 r / min, 10 min). The supernatant collected was the loosely bound EPS (LB-EPS). The precipitate was resuspended, heated in a 60°C water bath for 30 min, and centrifuged again to collect the supernatant, which was the tightly bound EPS (TB-EPS). The polysaccharide content was determined using the phenol-sulfuric acid method (with glucose as a standard), and the protein content was determined using the Bradford method (with bovine serum albumin as a standard). The results showed that the polysaccharide content of TB-EPS in mature granules was 87.5 ± 4.2 mg / g VSS, and the protein / polysaccharide mass ratio of LB-EPS was 0.38 ± 0.05.

[0029] Light intensity has a significant impact on the granulation process and treatment efficiency. When light intensity is below 3000 lux, the photosynthetic rate of Chlorella is insufficient, EPS secretion is low, the granulation cycle is prolonged to over 35 days, and the granule structure is loose. Within the light intensity range of 4000–8000 lux, the granulation cycle is 18–25 days, and the granule structure is dense. When light intensity exceeds 10000 lux, high light intensity leads to photoinhibition of Chlorella, reducing photosynthetic efficiency. Simultaneously, excessive proliferation of algal cells on the granule surface causes thickening of the outer layer and insufficient internal oxygen supply. Therefore, this invention limits the light intensity to 4000–8000 lux, preferably 6000 lux. The pH value is naturally maintained within the range of 7.0–8.5 by CO2 consumption during photosynthetic carbon fixation, usually without the need for external acid / alkali adjustment. A hydraulic retention time of 18–36 h ensures sufficient contact time for tetracycline in the granular sludge to complete the enzymatic degradation reaction.

[0030] Mature granular sludge exhibits functional stratification from the core to the outer layer. The core region of the granules is mainly composed of Chlorella cells and their secreted TB-EPS polysaccharide gel. Oxygen from photosynthesis diffuses outward from the core in this region, with a DO concentration below 0.5 mg / L, creating an anaerobic / hypoxic microenvironment where denitrifying bacteria and polyphosphate-accumulating bacteria colonize. The middle layer of the granules has a DO concentration of 0.5–2 mg / L, where nitrifying bacteria such as ammonia-oxidizing bacteria (AOB) and nitrite-oxidizing bacteria (NOB) colonize, oxidizing ammonia nitrogen to nitrate nitrogen. The outer layer of the granules directly contacts the wastewater, with a DO concentration above 2 mg / L. Pre-screened tetracycline-degrading dominant bacteria (centered on Stenotrophomonas maltophilia and Hydrogen-eating bacteria) colonize here, utilizing the continuously supplied DO from microalgal photosynthesis to enzymatically degrade tetracycline under aerobic conditions. This photosynthetic-driven, self-layered microecological structure enables three functions—tetracycline degradation, nitrification-denitrification denitrification, and biological phosphorus removal—to be achieved simultaneously within a single particle.

[0031] One of the core features of this invention lies in its operational capability without an external carbon source. This technological advantage relies on a closed-loop O2 / CO2 / DOC cycle formed by the symbiotic relationship between algae and bacteria within the particles. During the light-reacting phase, Chlorella fixes CO2 and releases O2 through photosynthesis. The O2 diffuses from the particle core to the outer layer, providing a continuous oxygen supply for the outer aerobic degrading bacteria and the middle nitrifying bacteria, replacing the mechanical aeration in traditional wastewater treatment processes. Simultaneously during the dark reaction and light-reacting phases, the outer aerobic bacteria utilize O2 to metabolize organic matter and degrade tetracycline. The generated CO2 diffuses back to the particle core region and is re-fixed and utilized by Chlorella.

[0032] A reverse inoculation design with an algae-to-bacterial cell ratio of 5:1 (more algae than bacteria) ensured that the dissolved organic carbon (DOC, including small molecule metabolites such as sugar alcohols, organic acids, and amino acids) produced by Chlorella photosynthesis was sufficient to sustain the basal metabolic needs of a small number of tetracycline-degrading bacteria. The quorum sensing signaling molecules N-acyl homoserine lactones (AHLs, such as C6-HSL and 3-oxo-C8-HSL) produced by the degrading bacteria induced upregulation of the rbcL gene encoding the large subunit of ribulose-1,5-bisphosphate carboxylase / oxygenase in Chlorella, promoting increased photosynthetic carbon fixation efficiency; and downregulation of genes related to glycolysis and the tricarboxylic acid cycle, inhibiting heterotrophic metabolism in algal cells, allowing more photosynthetically fixed carbon to flow extracellularly, providing a continuous carbon source for the outer degrading bacteria. Ammonia nitrogen (NH4) in the wastewater... + -N) and phosphate (PO4) 3- The nitrogen and phosphorus from these sources are directly assimilated and absorbed by Chlorella, serving as nitrogen and phosphorus sources for protein and nucleic acid synthesis, thus achieving biological fixation and removal. This multi-element closed loop of O2 / CO2 / DOC / N / P allows the entire system to operate stably without the need for an external organic carbon source.

[0033] Tetracycline (C) 22 H 24 The degradation of N2O8 (molecular weight 444.44) in the algae-bacterial granular sludge system of this invention is mainly achieved through the following three enzymatic pathways.

[0034] Pathway I – TetX-mediated regioselective hydroxylation at the C11a position. TetX is a FAD-dependent flavin monooxygenase (EC 1.14.13-) that uses NADPH and molecular oxygen as cosubstrate to catalyze the regioselective hydroxylation of tetracycline at the C11a position of the A / B ring junction. The reaction mechanism is as follows: NADPH reduces the flavin cofactor FAD to FADH2, which reacts with O2 to generate the active intermediate FAD-4a-peroxide. The latter electrophilically hydroxylates the enol at the C11a-C12 position to generate 11a-hydroxytetracycline (C11a-C12- ... 22 H 24 N2O9 (molecular weight 460.44). The C11a position is occupied by sp. 2 Hybridization transforms into sp 3 After hybridization, the Mg of tetracycline 2+ The chelating and ribosome-binding abilities are significantly weakened, resulting in the loss of antibacterial activity. This hydroxylated product can spontaneously form a stable C6-C12 hemiacetal under acidic conditions.

[0035] Pathway II – AlkB-type oxidative demethylation pathway. AlkB (EC 1.14.11.33) is a DNA oxidative demethylase that catalyzes the stepwise demethylation of the N-dimethylamino group at the C4 position in the tetracycline degradation system, successively generating 4-demethyltetracycline (C4). 21 H22 N2O8 (molecular weight 430.41) and 4-bis(demethyltetracycline) (C 20 H 20 N2O8 (molecular weight 416.38) releases one molecule of formaldehyde with each demethylation.

[0036] Pathway III – Dehydration-Deacylation-Ring-Opening Complex Pathway. prpD (2-methylcitrate dehydratase, EC4.2.1.79) catalyzes the β-elimination dehydration reaction of the secondary hydroxyl group at C6, generating dehydrated tetracycline (C6). 22 H 22 N2O7 (molecular weight 426.42). Subsequently, the C2 amide group is hydrolyzed to generate deamidated tetracycline, and the A ring undergoes ring-opening via dioxygenase-catalyzed oxidative cleavage, degrading stepwise into low-molecular-weight organic acids, alcohols, and amines. LC-MS / MS analysis detected the intermediate and final products of the above three pathways in the effluent of the system of this invention. Toxicity assessment of the degradation products (using the luminescent bacterium Vibrio Fischer as an indicator organism) showed that the biotoxicity of all degradation products was lower than that of the tetracycline parent compound.

[0037] Example 1

[0038] Following the above method, *Chlorella vulgaris* and a tetracycline-degrading dominant bacterial community obtained through three-stage domestication and screening from activated sludge in the aeration tank of a freshwater carp farm were used as materials. The algae-to-bacterial cell ratio was 5:1, and co-cultured and granulated in a 5 L sequencing batch photobioreactor. The lighting conditions were LED white light at an intensity of 6000 lux, with a light / dark cycle of 12 h / 12 ​​h. The settling time started at 30 min, decreasing by 4 min every 4 days, and reaching 5 min after 21 days. After granulation, simulated aquaculture wastewater was introduced for treatment. The wastewater quality parameters were: tetracycline 10 mg / L, NH4+ 10 mg / L. + -N 50 mg / L, TP 8 mg / L, COD 150 mg / L, pH 7.5~8.2. Hydraulic retention time 24 h, no external carbon source, continuous operation for 30 days.

[0039] The treatment results of Example 1 are shown in Table 1. During the stable operation phase (days 8-30), the average removal rate of tetracycline was 92.3 ± 2.1%, and the removal rate of NH4 was... + The average removal rates of nitrogen (N) and phosphorus (TP) were 97.1 ± 1.5%, 88.4 ± 2.8%, and 85.6 ± 3.2%, respectively. The tetracycline concentration in the effluent was 0.77 ± 0.21 mg / L, and the NH4+ concentration was... +The concentration of -N was 1.45±0.75 mg / L, and the concentration of TP was 0.93±0.22 mg / L, meeting the Class II standard requirements of the "Aquaculture Wastewater Discharge Standard" (draft for comments) (tetracycline <2 mg / L, NH4 <2 mg / L). + -N<5 mg / L, TP<1.5 mg / L). The harvested algal and fungal biomass was dried at 105°C to constant weight, and the protein content was determined by the Kjeldahl method to be 47.3%, which meets the basic quality requirements for feed protein raw materials.

[0040] Example 2

[0041] Following the method of Example 1, but adjusting the algae-to-bacterial cell ratio to 3:1 and 8:1, granulation culture and wastewater treatment were performed, respectively. The results showed that at an algae-to-bacterial ratio of 3:1, the granulation time was extended to 28 days, the tetracycline removal rate was 86.5 ± 3.4%, and NH4+ removal was reduced. + -N removal rate was 93.8±2.1%, and TP removal rate was 82.6±3.5%. At an algae-to-bacteria ratio of 8:1, with a granulation time of 25 days, tetracycline removal rate was 89.1±2.7%, and NH4+ removal rate was... + The removal rate of -N was 95.2±1.8%, and the removal rate of TP was 85.3±3.0%. Compared with the optimal algae-to-bacterial ratio of 5:1, the treatment efficiency of both parameters decreased, but remained within an acceptable range, verifying the feasibility of the algae-to-bacterial ratio range of 3~8:1. The granulation speed was slower at an algae-to-bacterial ratio of 3:1, presumably due to insufficient EPS production from the algae source to quickly form a gel framework; the tetracycline removal rate decreased slightly at an algae-to-bacterial ratio of 8:1, presumably due to a lower initial inoculum size of the degrading bacteria, leading to a delayed establishment of the outer functional membrane layer.

[0042] Comparative Example 1 – Bacterial-dominated granulation control

[0043] Activated sludge from aquaculture farms was directly inoculated into a sequencing batch reactor (SBR) without the addition of Chlorella or pre-screened degrading bacteria. Aerobic granular sludge (AGS) was cultured under the same SBR operating parameters and settling selective pressure conditions as in Example 1. After granulation, the same simulated aquaculture wastewater was introduced for treatment. Results showed that granulation took approximately 45 days to complete, with a particle size of 1–2 mm. Tetracycline removal rate was only 35.2 ± 5.8% (mainly dependent on adsorption), and NH4+ removal rate was [not specified]. + The removal rate of nitrogen (N) was 72.3±4.5%, and the removal rate of total phosphorus (TP) was 51.6±5.2%. Due to the lack of pre-screened degrading bacteria and microalgae photosynthetic oxygen supply, the aerobic granular sludge had extremely limited biodegradation capacity for tetracycline, and the lack of carbon source resulted in poor denitrification.

[0044] Comparative Example 2 – Control without pre-screening microbial communities

[0045] Common Chlorella and activated sludge from aquaculture farms (without tetracycline acclimation screening) were co-inoculated in a sequencing batch photobioreactor at a cell ratio of 5:1. Granulation culture and wastewater treatment were performed according to the same operating parameters as in Example 1. The results showed that granulation could be completed within 23 days (similar to Example 1), but the tetracycline removal rate was only 58.6 ± 6.3%, significantly lower than the 92.3% in Example 1. This result indicates that the proportion of tetracycline-degrading bacteria in the naturally enriched activated sludge microbiota is insufficient, failing to provide effective biodegradation capacity under high tetracycline concentrations (10 mg / L), thus verifying the necessity of pre-screening targeted bacterial communities for efficient tetracycline removal. NH4 + The nitrogen removal rate was 91.5±3.2%, and the phosphorus removal rate was 80.1±4.0%, which was relatively small compared with Example 1. This indicates that the nitrogen and phosphorus removal functions are mainly undertaken by the algae-bacteria symbiotic system itself, and the dependence on bacterial pre-screening is low.

[0046] Comparative Example 3 – External Carbon Source Control

[0047] Granular sludge for algae-bacteria co-culture was constructed according to the method in Example 1, but sodium acetate was added as an external carbon source during the wastewater treatment stage, and the COD / N ratio was adjusted to 8:1. The results showed that the tetracycline removal rate was 93.8±1.9%, and NH4+ removal was... + The removal rate of nitrogen (N) was 98.2 ± 0.8%, and the removal rate of phosphorus (TP) was 91.5 ± 2.0%. Compared with the carbon-source-free operation in Example 1, the removal rates of each component increased by only 1-3%, with no statistically significant difference (p>0.05). This result confirms the conclusion of this invention that the algal-bacterial symbiotic O2 / CO2 / DOC closed-loop cycle can effectively replace external carbon sources. External carbon sources have a very low marginal contribution to the treatment effect and instead increase operating costs and carbon emissions.

[0048] The test data are summarized and analyzed as follows:

[0049] Table 1. Comparison of treatment effects between Examples 1 and 2 and Comparative Examples 1-3

[0050] From Table 1 and Figure 2As can be seen, Example 1 achieved simultaneous removal of 92.3% tetracycline, 97.1% ammonia nitrogen, and 88.4% total phosphorus without an external carbon source. Example 2 achieved simultaneous removal of 86.5% tetracycline, 93.8% ammonia nitrogen, and 82.6% total phosphorus without an external carbon source. All indicators were significantly better than those of Comparative Example 1 (pure aerobic granular sludge) and Comparative Example 2 (without pre-screened bacteria), and there was no statistically significant difference compared with Comparative Example 3 (with an external carbon source). The tetracycline removal rate of Comparative Example 1 was only 35.2%, and it mainly relied on adsorption rather than biodegradation, confirming the irreplaceable role of the combination of microalgae photosynthetic oxygen supply and pre-screened degrading bacteria in the efficient biodegradation of tetracycline. The tetracycline removal rate of Comparative Example 2 was 58.6%, which was 33.7% lower than that of Example 1. This significant difference confirms the key role of functional bacteria pre-screening in the controllability of tetracycline degradation function. The results of Comparative Example 3, on the other hand, verified the feasibility of the algal-bacterial symbiotic O2 / CO2 / DOC closed-loop cycle replacing external carbon sources.

[0051] During 30 days of continuous operation, the granular sludge structure of Example 1 remained stable, with no granular disintegration or filamentous bulking observed. The mixed liquor suspended solids (MLSS) concentration remained at 4.5–5.5 g / L, and the sludge volume index (SVI) was [not specified]. 30 The TB-EPS polysaccharide content remained stable at 35–50 mL / g, indicating that the granular sludge has good settling performance and long-term operational stability. The TB-EPS polysaccharide content within the granules remained at 85–95 mg / g VSS during operation, and the LB-EPS protein / polysaccharide ratio remained at 0.35–0.45, with no significant shift in EPS composition.

[0052] The scope of protection of this invention is not limited to the specific parameter values ​​disclosed in the above-described embodiments. Any technical solution that is based on the core inventive concept of this invention and obtained through equivalent substitution or adjustment of engineering parameters should fall within the scope of protection of this invention.

Claims

1. A method for the synergistic treatment of antibiotics in aquaculture wastewater by microalgae and bacteria, characterized in that, Includes the following steps: Step 1: Microalgae of the genus Chlorella and tetracycline-degrading dominant bacterial groups obtained from activated sludge of aquaculture farms through tetracycline concentration gradient acclimation and screening are co-inoculated in a sequencing batch photobioreactor at an algae-to-bacteria cell ratio of 3-8:

1. The screening method for the tetracycline-degrading dominant bacterial groups is as follows: using activated sludge from the aeration tank of aquaculture farms as the bacterial source and tetracycline as the sole carbon source, the bacteria are obtained through three-stage acclimation culture at concentration gradients of 2, 5, and 10 mg / L, with each acclimation culture cycle lasting no less than 7 days. The final stable bacterial group after passage is taken as the tetracycline-degrading dominant bacterial group. Step 2: Cultivate in a light-dark alternating operation mode, and gradually shorten the settling time during the cultivation process to apply selective pressure, driving the Chlorella microalgae and the tetracycline-degrading dominant bacterial community to self-assemble into algae-bacteria symbiotic granular sludge. Step 3: Introduce the aquaculture wastewater containing tetracycline antibiotics, ammonia nitrogen and total phosphorus into the sequencing batch photobioreactor. Under the condition of no external carbon source, the microalgae in the granular sludge use photosynthetic oxygen production to supply the outer aerobic bacteria to degrade tetracycline antibiotics, and the carbon dioxide and nutrients produced by bacterial metabolism are supplied to the microalgae in turn, forming an algal-bacterial symbiotic relationship to treat the aquaculture wastewater. In the algae-bacteria symbiotic granular sludge, the dissolved oxygen gradient formed by oxygen production through microalgae photosynthesis drives the stratified colonization of bacterial communities. In the outer layer of the granules where the dissolved oxygen is higher than 2 mg / L, the dominant tetracycline-degrading bacteria colonize to form an aerobic degradation zone. In the middle layer of the granules where the dissolved oxygen is 0.5~2 mg / L, nitrifying bacteria colonize to form a nitrification zone. In the core layer of the granules where the dissolved oxygen is lower than 0.5 mg / L, denitrifying bacteria and polyphosphate-accumulating bacteria colonize to form a denitrification and biological phosphorus removal zone. This allows the degradation of tetracycline antibiotics, denitrification, and phosphorus removal to be spatially coupled within a single granule, achieving the simultaneous removal of tetracycline antibiotics, ammonia nitrogen, and total phosphorus. The dominant bacterial community for tetracycline degradation was identified by 16S rRNA high-throughput sequencing, and the dominant bacterial genera included at least two of Stenotrophomonas maltophilia, Hydrogen-eating bacteria and Sphingobacterium spp. In step 2, the light exposure time and dark period time of the light-dark alternating operation mode are 10-14 h and 10-14 h respectively; the settling time is gradually shortened from the initial 25-35 min to 3-8 min, and shortened by 3-5 min every 3-5 days.

2. The method according to claim 1, characterized in that, The particle size of the algae-bacteria symbiotic granular sludge formed in step 2 is 1.5~3 mm; in the granular sludge, the polysaccharide content of the tightly bound extracellular polymer is not less than 80 mg / g VSS, and the protein to polysaccharide mass ratio in the loosely bound extracellular polymer is 0.3~0.

5.

3. The method according to claim 2, characterized in that, In step 3, the light intensity of the photobioreactor is 4000~8000 lux, the pH is 7.0-8.5, and the hydraulic retention time is 1836 h; the concentration of tetracycline antibiotics in the aquaculture wastewater is 1~20 mg / L, the concentration of ammonia nitrogen is 20~80 mg / L, and the concentration of total phosphorus is 3~15 mg / L.

4. The method according to claim 1, characterized in that, The ratio of algae to bacteria cells is 5:

1.

5. The method according to claim 1, characterized in that, The harvested algae and fungi biomass, after processing and drying, has a protein content of not less than 44% by mass, and is used as a feed protein raw material.

6. The method according to claim 3, characterized in that, After the treatment, the concentrations of tetracycline antibiotics, ammonia nitrogen, and total phosphorus in the effluent meet the secondary standards for aquaculture wastewater discharge.

7. The method according to claim 1, characterized in that, The aquaculture wastewater is freshwater aquaculture wastewater.

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