Oxidative stress and metabolic process responses of Chlorella pyrenoidosa to nanoplastic exposure: Insights from integrated analysis of transcriptomics and metabolomics.

Oxidative stress is a universal interpretation for the toxicity mechanism of nanoplastics to microalgae. However, there is a lack of deeper insight into the regulation mechanism in microalgae response to oxidative stress, thus affecting the prevention and control for nanoplastics hazard. The integrated analysis of transcriptomics and metabolomics was employed to investigate the mechanism for the oxidative stress response of Chlorella pyrenoidosa to nanoplastics and subsequently lock the according core pathways and driver genes induced. Results indicated that the linoleic acid metabolism, glycine (Gly)-serine (Ser)-threonine (Thr) metabolism, and arginine and proline metabolism pathways of C. pyrenoidosa were collectively involved in oxidative stress. The analysis of linoleic acid metabolism suggested that nanoplastics prompted algal cells to secrete more allelochemicals, thereby leading to destroy the immune system of cells. Gly-Ser-Thr metabolism and arginine and proline metabolism pathways were core pathways involved in algal regulation of cell membrane function and antioxidant system. Key genes, such as LOX2.3, SHM1, TRPA1, and proC1, are drivers of regulating the oxidative stress of algae cells. This investigation lays the foundation for future applications of gene editing technology to limit the hazards of nanoplastics on aquatic organism.

Investigating flocculation mechanisms and ecological safety of cationic guar gum for rapid harvesting of microalgal cells.

Addressing the drawbacks of traditional flocculants on microalgae biomass harvesting is crucial for large-scale industrial applications of microalgae production. In this study, cationic bioflocculant was successfully prepared by introducing cationic groups into the side chain of guar gum, achieving in-situ algae flocculation efficiency of 83.5 % with the dosage of 18.0 mg/L under pH = 10.0. Through a harmonious integration of predictive modelling and practical experimentation, a superior cell flocculation capacity of 23.5 g/g was achieved. In addition, the environmental safety and biocompatibility of cationic guar gum was assessed, using the typical suspension quantitative bacteriostatic method and the fluorescent double-staining technique. The results showed that the inhibition efficiency of Staphylococcus aureus in the system containing 60.0 mg/L cationic guar gum was only 12.0 % and there was no inhibition against Escherichia coli colonies. These findings provide a safe and green flocculant for efficient microalgae harvesting and spent medium treatment.

Performance evaluation of typical flocculants for efficient harvesting of Chlorella sorokiniana under different carbon application modes.

In this study, the growth characteristics of microalgae cultured with different carbon sources were analyzed, and the flocculation characteristics under the influence of carbon sources were evaluated using three typical flocculants. The results showed that the organic carbon sources could significantly increase the content of extracellular proteins in microalgae. Specifically, the extracellular protein concentrations of microalgae cultured with pure BG-11, ethanol, sodium acetate and glucose were 18.2 29.2, 97.3, and 34.7 mg/g, respectively. During the flocculation process, microalgae cultured with sodium acetate exhibited a weak response to the flocculant because of excessive extracellular proteins inhibited flocculation. In addition, the flocculation efficiency was also less than 50.0% cultured with sodium acetate in all pH test ranges when alum and chitosan were used as flocculants. It could be inferred that the flocculant initially happened to charge neutralization with the negatively charged proteins in the solution and then bridged the charges with the microalgae. These findings provide insights into the effects of different carbon sources on microalgal flocculation, promising organic integration of microalgae wastewater treatment and harvesting.

Micro/nano-plastics and microalgae in aquatic environment: Influence factor, interaction, and molecular mechanisms.

Micro/nano-plastics, as emerging persistent pollutant, are frequently detected in aquatic environments together with other environmental pollutants. Microalgae are the major primary producers and bear an important responsibility for maintaining the balance of aquatic ecosystems. Numerous studies have been conducted on the influence of micro/nano-plastics on the growth, photosynthesis, oxidative stress, gene expression and metabolites of microalgae in laboratory studies. However, it is difficult to comprehensively evaluate the toxic effects of micro/nano-plastics on microalgae due to different experimental designs. Moreover, there is a lack of effective analysis of the aforementioned multi-omics data and reports on shared biological patterns. Therefore, the purpose of this review is to compare the acute, chronic, pulsed, and combined effect of micro/nano-plastics on microalgae and explore hidden rules in the molecular mechanisms of the interaction between them. Results showed that the effect of micro/nano-plastics on microalgae was related to exposure mode, exposure duration, exposure size, concentration, and type of micro/nano-plastics. Meanwhile, the phenomenon of poisoning and detoxification between micro/nano-plastics and microalgae was found. The inhibitory mechanism of micro/nano-plastics on algal growth was due to the micro/nano-plastics affected the photosynthesis, oxidative phosphorylation, and ribosome pathways of algal cells. This brought the disruption of the functions of chloroplasts, mitochondria, and ribosome, as well as impacted on energy metabolism and translation pathways, eventually leading to impairment of cell function. Besides, algae resisted this inhibitory effect by regulating the alanine, aspartate, and glutamate metabolism and purine metabolism pathways, thereby increasing the chlorophyll synthesis, inhibiting the increase of reactive oxygen species, delaying the process of lipid peroxidation, balancing the osmotic pressure of cell membrane.

Enhanced Fe(OH)2-driven reductive Dechlorination via shortened Fe-O bonds and colloidal medium.

Fe2+ is usually adsorbed to the surface of iron-bearing clay, and iron (hydr)oxide in groundwater. However, the reductive activity of Fe(OH)2, a prevalent intermediate during the transformation of Fe2+, remains unclear. In this study, high-purity Fe(OH)2 was synthesized and tested for its activity in the degradation of carbon tetrachloride (CT). XRD data confirm that the synthesized material is a pure Fe(OH)2 crystal, exhibiting sharp peaks of (001) and (100) facets. Zeta potential analysis confirms that the off-white Fe(OH)2 is a colloidal suspension with a positive charge of ∼+35-50 mV. FTIR spectra reveal the formation of a coordination compound Fe2+ with OH-/OD-, derived from NaOH/OD. SEM and HRTEM results demonstrate that the Fe(OH)2 crystal has a regular octahedral structure with a size of ∼30-70 nm and average lattice spacings of 2.58 Å. Mössbauer spectrum verifies that the Fe2+ in Fe(OH)2/Fe(OD)2 is hexacoordinated with six Fe-O bonds. XAFS data demonstrate that the Fe-O bonds become shorter as the OH-:Fe(II) ratios increase. DFT results indicate that the (100) crystal face of Fe(OH)2 more readily transfers electrons to CT. In addition to being adsorbed to iron compounds, structural Fe2+ compounds such as Fe(OH)2 could also accelerate the electron transfer from Fe2+ to CT through shortened Fe-O bonds. The rate constant of CT reduction by Fe(OH)2 is as high as 0.794 min-1 when the OH-:Fe(II) ratio is 2.5 in water. This study aims to enhance our understanding of the structure-reactivity relationship of Fe2+ compounds in groundwater, particularly in relation to electron transfer mechanisms.

Fe(II) coordination transition regulates reductive dechlorination: The overlooked abiotic role of lactate.

The coordination environment of Fe(II) significantly affect the reductive reactivity of Fe(II). Lactate is a common substrate for enhancing microbial dechlorination, but its effect on abiotic Fe(II)-driven reductive dechlorination is largely ignored. In this study, the structure-reactivity relationship of Fe(II) is investigated by regulating the ratio of lactate:Fe(II). This work shows that lactate-Fe(II) complexing enhances the abiotic Fe(II)-driven reductive dechlorination with the optimum lactate:Fe(II) ratio of 10:20. The formed hydrogen bond (Fe-OH∙∙∙∙∙∙O = C-) and Fe-O-C metal-ligand bond result in a reduced Fe(II) coordination number from six to four, which lead to the transition of Fe(II) coordination geometry from octahedron to tetrahedron/square planar. Coordinatively unsaturated Fe(II) results in the highest reductive dechlorination reactivity towards carbon tetrachloride (k1 = 0.26254 min-1). Excessive lactate concentration (> 10 mM) leads to an increased Fe(II) coordination number from four to six with a decreased reductive reactivity. Electrochemical characterization and XPS results show that lactate-Fe(II)-I (C3H5O3-:Fe(II) = 10:20) has the highest electron-donating capacity. This study reveals the abiotic effect of lactate on reductive dechlorination in a subsurface-reducing environment where Fe(II) is usually abundant.

Petroleum-contaminated soil bioremediation and microbial community succession induced by application of co-pyrolysis biochar amendment: An investigation of performances and mechanisms.

This study aimed to remediate petroleum-contaminated soil using co-pyrolysis biochar derived from rice husk and cellulose. Rice husk and cellulose were mixed in various weight ratios (0:1, 1:0, 1:1, 1:3 and 3:1) and pyrolyzed under 500 °C. These biochar variants were labeled as R0C1, R1C0, R1C1, R1C3 and R3C1, respectively. Notably, the specific surface area and carbon content of the co- pyrolysis biochar increased, potentially promoting the growth and colonization of soil microorganisms. On the 60th day, the microbial control group achieved a 46.69% removal of pollutants, while the addition of R0C1, R1C0, R1C3, R1C1 and R3C1 resulted in removals of 70.56%, 67.01%, 67.62%, 68.74% and 67.30%, respectively. In contrast, the highest efficiency observed in the abiotic treatment group was only 24.12%. This suggested that the removal of petroleum pollutants was an outcome of the collaborative influence of co-pyrolysis biochar and soil microorganisms. Furthermore, the abundance of Proteobacteria, renowned for its petroleum degradation capability, obviously increased in the treatment group with the addition of co-pyrolysis biochar. This demonstrated that co-pyrolysis biochar could notably stimulate the growth of functionally associated microorganisms. This research confirmed the promising application of co-pyrolysis biochar in the remediation of petroleum-contaminated soil.

Physiological responses and removal mechanisms of ciprofloxacin in freshwater microalgae.

Antibiotics, such as ciprofloxacin (CIP), are frequently detected in various environmental compartments, posing significant risks to ecosystems and human health. In this study, the physiological responses and elimination mechanisms of CIP in Chlorella sorokiniana and Scenedesmus dimorphus were determined. The exposure CIP had a minimal impact on the growth of microalgae, with maximum inhibit efficiency (IR) of 5.14% and 22.74 for C. sorokiniana and S. dimorphus, respectively. Notably, the photorespiration in S. dimorphus were enhanced. Both microalgae exhibited efficient CIP removal, predominantly through bioaccumulation and biodegradation processes. Intermediates involved in photolysis and biodegradation were analyzed through Liquid Chromatography High Resolution Mass Spectrometer (HPLC-MS/MS), providing insights into degradation pathways of CIP. Upregulation of key enzymes, such as dioxygenase, oxygenase and cytochrome P450, indicated their involvement in the biodegradation of CIP. These findings enhance our understanding of the physiological responses, removal mechanisms, and pathways of CIP in microalgae, facilitating the advancement of microalgae-based wastewater treatment approaches, particularly in antibiotic-contaminated environments.

Microalgae cultivation with recycled harvesting water achieved economic and sustainable production of biomass and lipid: Feasibility assessment and inhibitory factors analysis.

This study was conducted to achieve economic and sustainable production of biomass and lipids from Chlorella sorokiniana by recirculating cultivation with recycled harvesting water, to identify the major inhibitory factors in recirculating culture, and to analyze accordingly economic benefits. The results showed that recirculating microalgae cultivation (RMC) could obtain 0.20-0.32 g/L biomass and lipid content increased by 23.1 %-38.5 %. Correlation analysis showed that the extracellular polysaccharide (PSext), chemical oxygen demand (COD) and chromaticity of recirculating water inhibited photosynthesis and induced oxidative stress, thus inhibiting the growth of C. sorokiniana. In addition, the economic benefits analysis found that circulating the medium twice could save about 30 % of production cost, which is the most economical RMC solution. In conclusion, this study verified the feasibility and economy of RMC, and provided a better understanding of inhibitory factors identification in culture.

Adsorption performance of Ni(II) by KOH-modified biochar derived from different microalgae species.

Microalgae biochar is potential adsorbents to remove heavy metals from wastewater due to abundant functional groups, high porosity and wide sources, but performance is not fully developed since it depends on microalgae species attributing to distinct morphology and biomass compositions. Here, two microalgae species Chlorella Pyrenoidosa and Scenedesmus Obliquus were used for biochar preparation via KOH-modification, biochar properties and their influences on Ni(II) adsorption were investigated. Ni(II) adsorption performances responding to biochar properties and operating conditions were upgraded via progressive optimization and response surface methodology. Together, adsorption isotherms and kinetics were analyzed to obtain significant factors for Ni(II) removal. As results, 100 % of Ni(II) removal was achieved under 100 mg/L initial Ni(II) concentration as pH was higher than the biochar zero-charge point of 6.87 with low biochar dosage (0.5 g/L), which provides an efficient approach for heavy metal removal from wastewater with microalgae biochar.

Integrating transcriptomics and biochemical analysis to understand the interactive mechanisms of the coexisting exposure of nanoplastics and erythromycin on Chlorella pyrenoidosa.

Nanoplastics and antibiotics frequently co-exist in water polluted by algal blooms, but little information is available about interaction between substances. Erythromycin, as a representative of antibiotics, has been frequently detected in aquatic environments. This investigation attempted to reveal the interaction mechanism of nanoplastics and erythromycin on Chlorella pyrenoidosa. Results demonstrated that the joint toxicity of erythromycin and nanoplastics was dynamic and depended on nanoplastics concentration. Antagonistic effects of 1/2 or 1 EC50 erythromycin and nanoplastic concentration (10 mg/L) on the growth of C. pyrenoidosa was observed. The joint toxicity of 1/2 or 1 EC50 erythromycin and nanoplastic concentration (50 mg/L) was initially synergistic during 24-48 h and then turned to antagonistic during 72-96 h. Consequently, antagonistic effect was the endpoint for joint toxicity. Integration of transcriptomics and physiological biochemical analysis indicated that the co-existence of nanoplastics and erythromycin affected the signal transduction and molecular transport of algal cell membrane, induced intracellular oxidative stress, and hindered photosynthetic efficiency. Overall, this study provided a theoretical basis for evaluating the interactive mechanisms of nanoplastics and antibiotics.

Effect mechanism of metal cations on the interface interaction of cell-collector-bubble for microalgal foam flotation.

Foam flotation is generally recognized as a low-cost and efficient technology for the harvesting of microalgae for food, feed and fuel production, as well as environmental remediation. However, the harvesting efficiency of microalgae using foam flotation is restricted by the residual metal cations in the medium, and the corresponding inhibition mechanism has not yet been revealed. This study investigated the effects of metal cations in the medium on the harvesting efficiency and concentration factor during the foam flotation of Scenedesmus acuminatus. The interface interaction of cell-collector-bubble effected by metal cations was revealed by quantifying the amount of collector (cetyl trimethylammonium bromide, CTAB) between cells and bubbles, as well as the response of bubble interface characteristics. Results showed that the harvesting efficiency dropped linearly as the increase of cationic concentrations. Under the CTAB dose of 20 mg L-1, the harvesting efficiency decreased from 98.65% to 56.77% with a decrease of concentration factor from 25.41 to 9.05 in the presence of metal cations. The Na+ and Mg2+ in the medium were the major inhibitors. The inhibitory mechanisms revealed that metal cations obviously impeded the adsorption of CTAB onto the cells by competing adsorption site, resulting in a low harvesting efficiency. The presence of metal cations also inhibited the bubble coalescence and slowed down drainage velocity in the plateau channel of foam layer, forming foam with higher water content, thus reducing the concentration factor. A schematic illustration is proposed to better understand the effect mechanism of metal cations on microalgal foam flotation. This study might facilitate the process development in an effort to overcome the inhibition of cations during microalgal foam flotation.

Urea addition as an enhanced strategy for degradation of petroleum contaminants during co-composting of straw and pig manure: Evidences from microbial community and enzyme activity evaluation.

Alterations in microbial community succession patterns and enzyme activities by petroleum pollutants during co-composting of straw and swine manure with the supplementary nitrogen source are unclear. In this study, urea was added into co-composting systems, and the removal performance of petroleum, microbial enzyme activity and community changes were investigated. Results showed that the polyphenol oxidase and catalase activities which were both related to the degradation of petroleum contaminants were accordingly increased from 20.65 to 30.31 U/g and from 171.87 to 231.86 U/g due to urea addition. The removal efficiency of petroleum contaminants in composting with urea increased from 45.06% to 82.29%. The addition of urea increased the diversity and abundance of petroleum-degrading microorganisms, and enhanced microbial linkages. This study provides a novel strategy for the degradation of petroleum hydrocarbon as well as a new insight into the effect of urea on both microbial processes and composting phases.

Biochemical responses of freshwater microalgae Chlorella sorokiniana to combined exposure of Zn(â ¡) and estrone with simultaneous pollutants removal.

With the development of livestock industry, contaminants such as divalent zinc ions (Zn (Ⅱ)) and estrone are often simultaneously detected in livestock wastewater. Nevertheless, the combined toxicity of these two pollutants on microalgae is still unclear. Moreover, microalgae have the potential for biosorption and bioaccumulation of heavy metals and organic compounds. Thus, this study investigated the joint effects of Zn (Ⅱ) and estrone on microalgae Chlorella sorokiniana, in terms of growth, photosynthetic activity and biomolecules, as well as pollutants removal by algae. Interestingly, a low Zn (Ⅱ) concentration promoted C. sorokiniana growth and photosynthetic activity, while the high concentration experienced inhibition. As the increase of estrone concentration, chlorophyll a content increased continuously to resist the environmental stress. Concurrently, the secretion of extracellular polysaccharides and proteins by algae increased with exposure to Zn (Ⅱ) and estrone, reducing toxicity of pollutants to microalgae. Reactive oxygen species and superoxide dismutase activity increased as the increase of pollutant concentration after 96 h cultivation, but high pollutant concentrations resulted in damage of cells, as proved by increased MDA content. Additionally, C. sorokiniana displayed remarkable removal efficiency for Zn (Ⅱ) and estrone, reaching up to 86.14% and 84.96% respectively. The study provides insights into the biochemical responses of microalgae to pollutants and highlights the potential of microalgae in pollutants removal.

Copper regulates degradation of typical antibiotics by microalgal-fungal consortium in simulated swine wastewater: insights into metabolic routes and dissolved organic matters.

Microalgae-based biotechnology for antibiotics biodegradation in swine wastewater has been receiving an increasing attention. In this study, microalgae and fungi co-cultivation system, regulated by copper (Cu(II)), was investigated in terms of nutrients and sulfonamides degradation in simulated swine wastewater. Results showed that the removal of ammonium nitrogen (NH4+-N), total nitrogen (TN), total phosphorus (TP) and chemical oxygen demand (COD) by microalgal-fungal consortium increased under 0.1-0.5 mg/L Cu(II) with the highest removal efficiency of 79.19%, 76.18%, 93.93% and 93.46%, respectively. The addition of Cu(II) (0-0.5 mg/L) enhanced the removal of sulfamonomethoxine (SMM), sulfamethoxazole (SMX) and sulfamethazine (SMZ) from 49.05% to 58.76%, from 59.31% to 63.51%, and from 37.51% to 63.9%, respectively, and the main removal mechanism was found to be biodegradation. Biodegradation followed a pseudo-first-order model with variable half-lives (10.12 to 15.51 days for SMM, 9.01 to 10.88 days for SMX, and 8.74 to 12.85 days for SMZ). Through mass spectrometry analysis, metabolites and intermediates of sulfonamides were accordingly identified, suggesting that the degradation routes were involved with hydroxylation, deamination, oxidation, de-sulfonation and bond cleavage. Dissolved organic matters released by microalgal-fungal consortium were induced by Cu(II). Fulvic acid-like and protein-like substances were bound to Cu(II), reducing its concentration and thus mitigating the organismal damage to microorganisms. These findings drew an insightful understanding of microalgal-fungal consortium for sulfonamides remediation by Cu(II) regulation in simulated swine wastewater.

Charging and discharging of humic acid geobattery induced by green rust and oxygenation: Impact on the fate and degradation of tribromophenol in redox-alternating groundwater environments.

Humic acid (HA) and ferrous minerals (e.g. green rust, GR) are abundant in groundwater. HA acts as a geobattery that take up and release electrons in redox-alternating groundwater environments. However, the impact of this process on the fate and transformation of groundwater pollutants is not fully understood. In this work, we found that the adsorption of HA on GR inhibited the adsorption of tribromophenol (TBP) under anoxic conditions. Meanwhile, GR could donate electrons to HA, causing the electron donating capacity of HA rapidly increase from 12.7% to 27.4% in 5 min. The electron transfer process from GR to HA significantly increased the yield of hydroxyl radicals (•OH) and the degradation efficiency of TBP during GR-involved dioxygen activation process. Compared to the limited electronic selectivity (ES) of GR for •OH production (ES = 0.83%), GR-reduced HA improves the ES by an order of magnitude (ES = 8.4%). HA-involved dioxygen activation process expands the •OH generation interface from solid phase to aqueous phase, which is conducive to the degradation of TBP. This study not only deepens our understanding on the role of HA in •OH production during GR oxygenation, but also provides a promising approach for groundwater remediation under redox-fluctuating conditions.

Responses of microalgae under different physiological phases to struvite as a buffering nutrient source for biomass and lipid production.

Microalgae cultivation for biodiesel production is promising, but the high demand for nutrients, such as nitrogen and phosphorus, remains a limiting factor. This study investigated effects of struvite, a low-cost nutrient source, on microalgae production under different physiological phases. Changes in element concentrations were determined to characterize the controllable nutrient release properties of struvite. Results showed that nutrient elements could be effectively supplemented by struvite. However, responses of microalgae under different growth stages to struvite varied obviously, achieving the highest biomass (0.53 g/L) and the lowest (0.32 g/L). Moreover, the microalgal lipid production was obviously increased by adding struvite during the growth phase, providing the first evidence that struvite could serve as an alternative buffering nutrient source to culture microalgae. The integration of microalgae cultivation with struvite as a buffering nutrient source provides a novel strategy for high ammonia nitrogen wastewater treatment with microalgae for biodiesel production.

Synergistic effects of rice husk biochar and aerobic composting for heavy oil-contaminated soil remediation and microbial community succession evaluation.

Soil petroleum pollution is an urgent problem in modern society, which seriously threatens the ecological balance and environmental safety. Aerobic composting technology is considered economically acceptable and technologically feasible for the soil remediation. In this study, the combined experiment of aerobic composting with the addition of biochar materials was conducted for the remediation of heavy oil-contaminated soil, and treatments with 0, 5, 10 and 15 wt% biochar dosages were labeled as CK, C5, C10 and C15, respectively. Conventional parameters (temperature, pH, NH4+-N and NO3--N) and enzyme activities (urease, cellulase, dehydrogenase and polyphenol oxidase) during the composting process were systematically investigated. Remediation performance and functional microbial community abundance were also characterized. According to experimental consequences, removal efficiencies of CK, C5, C10 and C15 were 48.0%, 68.1%, 72.0% and 73.9%, respectively. The comparison with abiotic treatments corroborated that biostimulation rather than adsorption effect was the main removal mechanism during the biochar-assisted composting process. Noteworthy, the biochar addition regulated the succession process of microbial community and increased the abundance of microorganisms related to petroleum degradation at the genus level. This work demonstrated that aerobic composting with biochar amendment would be a fascinating technology for petroleum-contaminated soil remediation.

Towards advanced nutrient removal by microalgae-bacteria symbiosis system for wastewater treatment.

In this study, the microalgae-bacteria symbiosis (ABS) system by co-culturing Chlorella sorokiniana with activated sludge was constructed for pollutants removal, and the according interaction mechanism was investigated. The results showed that the ABS system could almost completely remove ammonia nitrogen, and the removal efficiency of total nitrogen and total phosphorus could accordingly reach up to 65.3 % and 42.6 %. Brevundimonas greatly promoted microalgal biomass growth (maximum chlorophyll-a concentration of 9.4 mg/L), and microalgae contributed to the increase in the abundance of Dokdonella and Thermomonas in ABS system, thus facilitating nitrogen removal. The extended Derjaguin-Landau-Verwey-Overbeek theory indicated a repulsive potential barrier of 561.7 KT, while tryptophan-like proteins and tyrosine-like proteins were key extracellular polymeric substances for the formation of flocs by microalgae and activated sludge. These findings provide an in-depth understanding of interaction mechanism between microalgae and activated sludge for the removal of contaminants from wastewater.

Nutrient removal and lipid production by the co-cultivation of Chlorella vulgaris and Scenedesmus dimorphus in landfill leachate diluted with recycled harvesting water.

Applying microalgae for landfill leachate (LL) treatment is promising. However, LL usually needs to be diluted with much fresh water, aggravating water shortage. In this study, mono- and co-culturing microalgae (Chlorella vulgaris and Scenedesmus dimorphus) were used to treat LL diluted with recycled harvesting water, to investigate nutrient removal and lipid production. The results showed that microalgae in co-culture treatment had more biomass and stronger superoxide dismutase activity, which might be related to humic acids contained in recycled harvesting water, according to dissolved organic matters (DOMs) analysis. In addition, the lipid content and yield of co-cultured microalgae reached 27.60 % and 66.87 mg·L-1, respectively, higher than those of mono-culture, proving the potential of co-culture for the improvement of lipid production. This study provided a freshwater-saving dilution method for LL treatment with recycled harvesting water as well as a strategy for the increase of biomass and lipid accumulation by microalgae co-cultivation.