IntroductionIncreasing pollution from swine wastewater poses a serious threat to environmental safety and human health. Nutrients (e.g. nitrogen and phosphorus) and heavy metals (especially copper and zinc) are core risk factors for swine wastewater, limiting its resource utilisation. Accordingly, eco-friendly and effective wastewater treatment methods are urgently required1,2. The activated sludge method is currently the primary method used in livestock wastewater treatments3. However, the mechanical aeration required and the necessity of adding a carbon source result in high energy consumption and costs4. Recently, swine wastewater treatment based on microalgae cultivation has received widespread attention5,6. Microalgae offer the advantages of a short growth cycle and high photosynthetic efficiency and can effectively remove nutrients such as nitrogen and phosphorus as well as heavy metal ions from water7,8. In addition, microalgae can use photosynthesis to obtain both carbon sources and the required input energy, thereby considerably reducing the use of chemical reagents and resultant by-products, such as sludge4,9. Microalgae exhibit great potential for carbon fixation and emission reduction. In addition, microalgal biomass has a wide range of resource utilisation pathways. It can be used to generate biofuels through biohydrogen production and biopower generation10,11 and can also be converted to biofertilizers, e.g., through aerobic composting, thereby improving soil nutrients, water-holding capacity12, and reducing the cost of wastewater management. Therefore, microalgae water purification technology is an eco-friendly and economically feasible solution with broad application prospects.Microorganisms play an important role in wastewater purification using microalgae13. Microorganisms such as microalgae and bacteria are the most basic ecological functional units in microalgae-bacterial consortia, and there are extensive interactive connections, such as nutrient exchange, signal transduction, and gene transfer14,15,16. Thereby, how to enhance its purification performance has always been a research hotspot, particularly studying community succession and functional changes in microalgae-bacteria consortia which is of great importance for process optimisation and evaluation.In addition to lighting pattern, temperature, and nutrient component, trace elements, such as iron, are also important parameters affecting the activity of microalgae-bacteria consortia. Iron is an essential trace element required in various biological processes, including cell growth, chlorophyll synthesis, and electron transfer. Previous studies have indicated an appropriate amount of nanoscale zero-valent iron can significantly promote the growth and nutrient absorption in microalgae17. Owing to its unique superparamagnetic properties, nanoscale magnetite aids in the sedimentation and separation of microalgae18, and these studies have mainly focused on the impact of adding iron-based materials on material type, particle size, and additive dosage19. The effects of adding nano-sized iron materials on the community structure and function of microalgae-bacteria consortia remain largely unexplored.In this study, the performance of microalgae-bacteria consortia in purifying swine wastewater were compared, and the biological status of microalgae upon the addition of different nano-sized iron materials was also explored. With the help of high-throughput sequencing technology and metabolic pathway studies, the response of microalgae-bacteria consortia to nano-sized iron material particles was systematically explored, facilitating future improvement of swine waste water purification by improving the effects of nano-sized iron material additions, and subsequently supplementing the microalgae-bacteria consortia.Materials and methodsInoculum and materialsThe microalgae used in this study was Desmodesmus sp. CHX1 (phylum Chlorophyta, CGMCC No.6649)20, which was cultured in BG11 medium, until it reached the logarithmic phase, for inoculation in subsequent experiments. After long-term acclimatisation and cultivation, it exhibited excellent adaptability and purification of swine wastewater21,22. The nano-sized iron materials used in the experiments included nanoscale zero-valent iron (Nano-ZVI) and nanoscale magnetite (Nano-Fe3O4), both with a particle size of 50 nm; they were purchased directly from the reagent supplier (Macklin Bio-Chem Technology Co., Ltd., Shanghai, China).Swine wastewaterSwine wastewater was obtained from the effluent of the anaerobic tank of a wastewater treatment project of a large-scale swine farm in Hangzhou, Zhejiang Province, China23 and stored at 4 °C for future use. The basic physical and chemical properties of the swine wastewater used in this study are listed in Table 1.Table 1 Basic properties of swine wastewater.Full size tableExperimental design and procedureThe experiment was conducted in 500 mL conical flasks containing 400 mL swine wastewater. The samples were divided into three treatment groups—R1, R2, and R3. R1 was the control treatment, inoculated with microalgae only; R2 was inoculated with microalgae, and 50 mg·L−1 Nano-ZVI was added; R3 was inoculated with microalgae, and 50 mg·L−1 Nano-Fe3O4 was added. The conical flasks were placed in a temperature-controlled light-shaking incubator (QHZ-98B, Huamei Biochemical Instruments Ltd., Taicang, China) for cultivation. The rotation speed was 150 rpm, the temperature was 25 ± 2 °C, the light intensity was 8,000 Lux (provided by H-tube fluorescent tubes), and the light cycle was 24 h·day−1.The inoculation density of microalgae in each treatment group was 0.1 g·L−1, and the samples were sealed with silicone plugs. The experimental period was 11 d (the experiment ended when the biomass of microalgae reached the maximum), and three replicates were set for each treatment. Liquid samples were collected on days 0, 1, 3, 5, 7, 9, and 11 to analyse microalgal growth. Water samples were also collected on days 0, 4, 7, and 11 of the experiment; the filtrate was collected using a 0.45 μm filter membrane for the determination of various physical and chemical indicators.Physical and chemical analysesThe biomass of microalgae was measured using the optical density method17. Chlorophyll was extracted using the acetone method24, and chlorophyll concentration was calculated using Eqs. (1–3):$${c}_{\text{a}}=\frac{\left(12.70\times {A}_{663}-2.69\times {A}_{645}\right)\times {V}_{1}}{V\times \sigma }$$(1)$${c}_{\text{b}}=\frac{\left(22.90\times {A}_{645}-4.68\times {A}_{663}\right)\times {V}_{1}}{V\times \sigma }$$(2)$${c}_{\text{a}+\text{b}}=\frac{\left(20.21\times {A}_{645}+8.02\times {A}_{663}\right)\times {V}_{1}}{V\times \sigma }$$(3)where ca, cb, and ca+b represent the concentrations of chlorophyll a, chlorophyll b, and total chlorophyll concentration in µg·mL−1, respectively, A663 and A645 represent the absorbance at 663 nm and 645 nm, respectively, V refers to the volume of each sample (mL), V1 refers to the volume of acetone-based extract (mL), and σ is the optical path of the cuvette (cm).Ammonia nitrogen (NH4+-N), total nitrogen (TN), total phosphorus (TP), and chemical oxygen demand (COD) were determined using Nessler’s reagent spectrophotometry, alkaline potassium persulfate digestion UV spectrophotometry, ammonium molybdate spectrophotometry, and fast digestion spectrophotometry, respectively25. The Cu and Zn contents of the water were determined using flame atomic absorption spectrometric method (TAS-990, Purkinje General Instrument Co., Ltd., Beijing, China)26. All samples were measured thrice in parallel.DNA extraction and sequencingEach sample was taken in 50 mL using a sterile syringe. Samples were collected from each treatment group on days 1, 7, and 11 of the experiment and stored at − 80 °C. Total DNA was extracted using the E.Z.N.A.™ Soil DNA Extraction Kit (Omega Bio-tek Inc., Norcross, USA) according to the manufacturer’s protocol. DNA was quantified using a Nanodrop One spectrophotometer (Thermo Fisher Scientific Inc., Waltham, USA), and the quality of DNA extraction was checked using 1.2% agarose gel electrophoresis. After quality inspection, the samples were sequenced on an Illumina NovaSeq 6000 sequencing platform by the Shiyanjia Laboratory (http://www.shiyanjia.com) for 16S and 18S rRNA gene analyses.Data analysisData processing and charting were performed using the OriginPro software (version 10.0; OriginLab Corp., Northampton, USA). The experimental data were tested for significance of differences using the IBM SPSS Statistics software (version 26.0; IBM Corp., Armonk, USA); where p