Organics removal pathways and algae-bacteria interactions of microalgal-bacterial granular sludge treating real municipal wastewater

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This study thus attempted to investigate the removal performance and mechanisms of complex organics in real municipal wastewater in MBGS process. The results showed that complex organics could be effectively disposed during day-night cycles by MBGS, with the process performance significant impacted by the influent C/N ratio. Further metagenomic and metatranscriptomic analyses revealed that the upregulated gap2 and gpmA genes of glycolysis enhanced the conversion of complex organics to CO2 mediated by Chlorophyceae and Acidobacteriae/Sumerlaeia/Fimbriimonadia, while the upregulated petH gene of NADPH synthesis by Cyanobacteria strengthened the fixation of CO2 into biomass. Meanwhile, the functional gene of amyA in the starch metabolism by Actinobacteriota was upregulated, along with the upregulated gldA gene in the glycerolipid metabolism through Chlorophyceae and Chloroflexia/Verrucomicrobiae. Moreover, a close symbiotic relationship between Cyanobacteria and Desulfobacterota I was identified, which played a crucial role in fatty acid decomposition. This study offers new insights into degradation mechanisms of complex organics via microalgal-bacterial symbiosis, which also gains basic knowledge on the carbon cycle in natural water ecosystems mediated by microalgal-bacterial symbiosis. Biological sciences/Microbiology/Environmental microbiology/Water microbiology Biological sciences/Microbiology/Bacteria Microalgal-bacterial symbiosis Wastewater treatment Complex organics Removal pathways Functional gene Algae-bacteria interactions Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 1. Introduction Currently, with the intensification of human activities, the total amount of municipal wastewater has been increasing year by year 1 . In the era of climate change 2 , 3 and energy crisis 4 , 5 , the widely used conventional activated sludge (CAS) process is under pressure due to high energy consumption and carbon emissions 6 . As an energy-saving and environmentally-sustainable process, the microalgal-bacterial granular sludge (MBGS) can directly utilize solar energy to generate oxygen 7 , 8 , which is further used assimilate wastewater organics and nutrients into microalgae biomass, while helping to mitigate carbon emissions 9 . In fact, the MBGS process can even absorb carbon dioxide of industrial origin 10 and methane 11 , which is more conducive to achieving the carbon neutrality of wastewater treatment 12 , 13 , 14 . Understanding of the removal pathways of nutrients and organics by MBGS are of vital significance, which, nevertheless, has not been fully determined, especially for the complex organics. For nitrogen metabolism, most of the NH 4 + -N can be converted into glutamine or glutamate for microbial assimilation in non-aerated MBGS system 9 , 15 , and NO 3 − -N and NO 2 − -N can be converted to NH 4 + -N before they are metabolized 16 , while the organic nitrogen (e.g. urea, amino acids, etc.) can be hydrolyzed to NH 4 + -N through intracellular urea amidolyase 17 . For phosphate metabolism, phosphorus could be removed by MBGS through microbial assimilation and poly-phosphate accumulation 9 , and PO 4 3− -P can be transported into cells through the oxidative phosphorylation and removed through poly-phosphate accumulation under the action of poly-phosphate kinase 15 . However, it should be noted that for organics metabolism, limited knowledge is currently available since the laboratory-scale studies usually adopted the simple carbon source, e.g., acetate and glucose 15 , 18 , while the carbon source in real municipal wastewater is mainly composed of complex organics, e.g., starch 19 , but its degradation mechanisms remained unclear. In addition, the algae-bacteria symbiosis ubiquitously exist in natural aquatic ecosystems 20 . Considering the complex organics discharged into the natural water due to human activity 21 , 22 , it is also necessary to explore the degradation mechanisms for organic matter by microalgal-bacterial symbiosis. Meanwhile, the carbon metabolism is closely related to greenhouse gases emissions 23 and the organics removal is usually intimately related to nutrients removal 24 , there is an urgent need to decipher the complex organics removal mechanisms of microalgal-bacterial symbiosis, providing more information of real wastewater complex organics removal pathways. On the other hand, to date, the detailed cooperative interaction between algae and bacteria has not been depicted, and the relationships between microbial community and functional genes of MBGS in complex organics metabolism has also been unclear, which hinders its further application. It has been proven that the key role of the algae-bacteria interactions is reflected by the exchange of CO 2 and O 2 , which is a necessary prerequisite for the efficient removal of organics by MBGS 9 . However, the previous studies focused mainly on the microbial community components or the bacterial coherence, while lacking the algae-bacteria interactions in microalgal-bacterial symbiosis 25 , 26 . To discover the organics removal pathways and algae-bacteria interactions of microalgal-bacterial symbiosis in treating real municipal wastewater, this study investigated the performance and upregulated functional genes of MBGS process on real municipal wastewater under diel fluctuation. Meanwhile, the evolution of microbial community and relationships between algae and bacteria of MBGS were determined. Moreover, the relationships between microbial community and functional genes were elucidated. This study provided more information on the complex organics removal mechanisms under the algae-bacteria interactions of MBGS process for treating complex organic wastewater, which is expected to add basic knowledge for the engineering applications of MBGS process. More importantly, this study can help to understand the carbon cycling in natural aquatic systems mediated by microalgal-bacterial symbiosis. 2. Results and discussion 2.1 Performance of complex organics removal by MBGS As illustrated in Supplementary Figs. 1 to 3, the MBGS could effectively remove complex organics, evidenced by a COD removal efficiency of 82.4% in treating real municipal wastewater by the flat-plate reactor during the daytime on the day 12. It was showed that the carbon-to-nitrogen (C/N) ratio of influent could impact on the COD and TN removal during the daytime (Fig. 1 a), e.g. their removal efficiencies increased with the increase of C/N ratio from 1 to 5 and then tended to be stable when the C/N ratio exceeded 5 (Fig. 1 b). In fact, it had been reported that the C/N ratio of MBGS has been proven to be around 5 based on the empirical formula of C 100 H 158 O 38 N 17 P 9 , 27 , and that the pollutants removal mechanisms of MBGS were mainly microbial assimilation 9 , 24 . However, the C/N ratios of real municipal wastewater fluctuated significantly and even fell to around 1 on the last 10 days of the experiment (see Supplementary Table 1), which were unfavorable for pollutant removal by MBGS. Moreover, the average DO concentration during the nighttime had a positive impact on the performance of COD at night (Fig. 1 c and 1 d), while the DO concentration in real municipal wastewater could even exceed 21 mg/L at 20:00 (see Supplementary Table 2), which was beneficial for the organics removal at night 28 since the O 2 produced by microalgae during the day could be utilized by bacteria at night 24 . It should also be noted that the optimal granule size of MBGS was reported to be 1.2 to 1.3 mm at a light intensity of 200 µ mol·m − 2 ·s − 1 , thus the organics removal could be probably improved by further optimizing the granule size of MBGS 7 . In addition, Supplementary Fig. 4 showed that the MBGS had excellent settling performance in the flat-plate bioreactors, evidenced by that the lower SVI 5 values and higher density of MBGS than inclined-plate bioreactors, resulting in the better removal performance of flat-plate bioreactors, while the real municipal wastewater was more conducive to the granule growth and settling performance as compared to synthetic wastewater, which could be ascribed to the fact that the complex organics in real municipal wastewater would facilitate the bacteria growth in large granules than synthetic wastewater 28 . 2.2 Organics removal pathways According to the research 29 , the main components of organics in real municipal wastewater were carbohydrate, fat and oil composed of glycerol and fatty acid 30 , while the complex organics of carbohydrate was mainly starch 19 . As depicted in Fig. 2 a, starch, glycerol and fatty acid could be transformed into α-D-glucose-1P, glycerone-P and acetyl CoA though the starch and sucrose metabolism (ko00500), glycerolipid metabolism (ko00561) and fatty acid degradation (ko00071), respectively, while α-D-glucose-1P and glycerone-P could ultimately be converted into acetyl-CoA during the glycolysis/gluconeogenesis (ko00010), which then entered the TCA cycle 31 . Subsequently, the CO 2 generated by TCA cycle could be absorbed by microalgae ( Cyanobacteria and Chlorophyta ) though the Calvin cycle, and then converted into glyceraldehyde-3P, which eventually synthesized into biomass 32 . The relative abundance of organics metabolism pathways (i.e. ko00010, ko00071, ko00500, ko00561) became more and more evident during the experiment, and the glycolysis process was highly active since it accounted for the highest proportion and its relative abundance could reach 0.86% in the real municipal wastewater of inclined-plate reactor at the end of the experiment (see Supplementary Fig. 5a). Further analysis by CAZy annotation showed that glycosyl transferases, glycoside hydrolases and carbohydrate-binding modules related to the carbohydrate degradation 9 were abundant, while glycoside hydrolases played an important role in the decomposition of complex organics, whose relative abundance in treating real municipal wastewater was higher than that in synthetic wastewater treatment (see Supplementary Fig. 5b). The relative expression abundance of functional genes encoding 15 enzymes involved in organics metabolism and 5 enzymes related to photosynthesis were found to be upregulated in treating real municipal wastewater as compared to synthetic wastewater (see Supplementary Table 3). The expression of functional genes of gapA (encoding glyceraldehyde 3-phosphate dehydrogenase, K00134), pdhD (encoding dihydrolipoamide dehydrogenase, K00382), pps (encoding orthophosphate dikinase, K01007), petC (encoding cytochrome b6-f complex iron-sulfur subunit, K02636), petH (encoding ferredoxin- NADP + reductase, K02641), psbA (encoding photosystem II P680 reaction center D1 protein, K02703) and psbD (encoding photosystem II P680 reaction center D2 protein, K02706) were abundant (Fig. 2 b), while gldA (encoding glycerol dehydrogenase, K00005), gap2 (encoding glyceraldehyde-3-phosphate dehydrogenase (NAD(P)), K00150), amyA (encoding alpha-amylase, K01176), gpmA (encoding 2,3-bisphospho- glycerate-dependent phosphoglycerate mutase, K01834), petH , psbA and psbD showed vigorously expression activity (Fig. 2 c). It was found that the functional genes of gapA , pdhD , pps , gap2 and gpmA were involved in glycolysis process, while petH was related to photosynthetic electron transport, which could strengthen CO 2 fixation by improving the NADPH synthesis process 33 , revealing that the MBGS could achieve the decomposition of organics to CO 2 by aerobic heterotrophic bacteria and the fixation of CO 2 into microalgae biomass by upregulating the expression of functional genes of glycolysis and NADPH synthesis processes, respectively. Meanwhile, the upregulation of amyA in the starch metabolism and gldA in the glycerolipid metabolism could enhance the decomposition of starch and glycerol, respectively, while the upregulated psbA and psbD genes of photosynthesis could facilitate the generation of electrons donated by H 2 O, which were used to reduce NADP + to NADPH, resulting in the production of O 2 34 . 2.3 Algae-bacteria interactions of MBGS in treating complex organics wastewater 2.3.1 Microbial community evolution Supplementary Fig. 6 indicated that the real municipal wastewater could promote the growth of Cyanobacteria , while Cyanobacteria and Desulfobacterota I could form microalgal-bacterial symbioses situated in the outer layer of MBGS (see Supplementary Fig. 6d). As depicted in Figs. 3 a and 3 b, Proteobacteria , Bacteroidota and Cyanobacteria were the dominant phyla of MBGS with an average proportion of 51.3%, 10.6% and 6.2%, respectively, while Alphaproteobacteria , Gammaproteobacteria , Bacteroidia and Cyanobacteriia were the dominant classes with an average proportion of 28.3%, 23.0%, 10.1% and 6.3%, respectively. Meanwhile, the real municipal wastewater significantly promoted the growth of the bacterial communities of Chloroflexota ( Anaerolineae ), Acidobacteriota ( Acidobacteriae ), Verrucomimicrobiota ( Verrucomicrobiae ) and Sumerlaeota ( Sumerlaeia ) at the end of the experiment through the cluster analysis (Figs. 3 c and 3 d). It was reported that Proteobacteria could facilitate the removals of organics and nutrients through mediating polysaccharide decomposition and enzyme synthesis 15 , while Cyanobacteria could absorb nitrogen and phosphorus from wastewater 35 . Researches have shown that Bacteroidota 36 , Chloroflexota 37 , Verrucomicrobiota 38 , Actinobacteriota 39 and Acidobacteriota 40 were able to degrade polysaccharides, while the degradation of complex organics was potentially ascribed to Desulfobacterota I 41 , Armatimonadota 42 and Sumerlaeota 43 . The average relative abundance of the above-mentioned bacteria in real municipal wastewater was significantly higher than those in synthetic wastewater (see Supplementary Fig. 7), implying their adaptation to complex organics and potential contribution for the complex organics degradation, while the Alpha diversity index also showed that MBGS in real municipal wastewater had higher species richness and diversity than synthetic wastewater (see Supplementary Table 4). In addition, real wastewater could promote the genes expression of Cyanobacteria ( Cyanobacteriia ) and Chlorophyta ( Chlorophyceae ) (Fig. 4 ), which was conducive to providing sufficient O 2 for the bacteria to decompose complex organics. 2.3.2 Relationships between algae and bacteria of MBGS It had been shown that the contributions of microorganisms in wastewater treatment were not necessarily correlated with their relative abundance, but depended on their activity 44 . Therefore, the co-occurrence networks between the top 20 dominant microorganisms of MBGS at the phylum and class level based on the metetranscriptomics sequencing were shown in Fig. 5 , showing that the networks had 20 nodes with 3 modules (module 1, module 2 and module 3) at phylum level and 2 modules (module 1 and module 2) at class level, while modules were divided based on the positive correlations between microorganisms. Cyanobacteria showed a positive correlation with Desulfobacterota I 45 as the relevance degree of 0.66, while Chlorophyceae showed a close correlation with Verrucomicrobiae 46 as the relevance degree of 0.86. Meanwhile, the symbiotic relationships between Chlorophyceae with Sumerlaeia , Chloroflexia 47 , Acidobacteriae 48 and Fimbriimonadia were strengthened during real municipal wastewater treatment, evidenced by the fact that their genes expression activity in the real municipal wastewater were significantly enhanced at the end of the experiment (Fig. 4 b). In addition, competitive relationship also existed in the MBGS, such as Bacteroidota and Sumerlaeota with the relevance degree of -0.94, since these bacteria performed the same metabolism function for the decomposition of complex organics 49 . Overall, the symbiotic relationships between Chlorophyceae with Verrucomicrobiae , Sumerlaeia , Chloroflexia , Acidobacteriae and Fimbriimonadia , as well as Cyanobacteria with Desulfobacterota I were the key to the effective decomposition of complex organic matters in real municipal wastewater, as the exchange of O 2 and CO 2 between algae and bacteria was crucial for organics removal by MBGS 9 . 2.4 Relationships between microbial community and functional genes Figure 6 showed that gap2 and gpmA , as the main functional genes with high expression activity in the glycolysis process 50 , the expression of the former was closely correlated with Acidobacteriae as the relevance degree of 0.71, while the expression of the latter was closely correlated with Sumerlaeia and Fimbriimonadia as the relevance degree of 0.86 and 0.79, respectively. Meanwhile, amyA , as the functional gene with high expression activity in the starch metabolism, participated in the labile-C degradation 51 , its expression was only correlated with Actinobacteriota 52 as the relevance degree of 0.71. GldA , as the functional gene with high expression activity in the glycerolipid metabolism carried by Bacteroidota 53 , its expression was closely correlated with Chloroflexia and Verrucomicrobiae as the relevance degree of 0.86 and 0.83, respectively. In addition, the expression of fadE , a key functional gene in the fatty acid degradation 54 was closely correlated with Desulfobacterota I indicated by a relevance degree of 0.71. Overall, Acidobacteriae , Sumerlaeia and Fimbriimonadia had significantly contributed to the glycolysis process, and Actinobacteriota played a crucial role in the starch decomposition 55 . Meanwhile, Chloroflexia and Verrucomicrobiae were of vital significant for the glycerol decomposition, and Desulfobacterota I was a key bacterium for the fatty acid degradation. It appears from the above discussion that the complex organics removal mechanisms by MBGS was illustrated in Fig. 7 . The upregulated gap2 and gpmA genes of glycolysis process enhanced the conversion of complex organics into CO 2 mediated by Chlorophyceae and Acidobacteriae / Sumerlaeia / Fimbriimonadia , while the upregulated petH gene of NADPH synthesis process by Cyanobacteria strengthened the fixation of CO 2 into microalgae biomass. Meanwhile, the gene of amyA in the starch metabolism by Actinobacteriota was upregulated, along with the upregulated gldA gene in the glycerolipid metabolism with the interaction of Chlorophyceae and Chloroflexia/Verrucomicrobiae , which improved the decomposition of starch and glycerol, respectively. Moreover, fatty acid was decomposed though Cyanobacteria and Desulfobacterota I . In addition, the significantly upregulated expression of psbA and psbD carried by Chlorophyceae 56 could provide sufficient O 2 for the above aerobic heterotrophic bacteria. 3 Implication As a green process, the complex organics removal pathways of MBGS process are of vital significance, nevertheless, only the removal pathways of simple carbon source e.g., acetate and glucose were studied 15 , 18 , the complex organics removal mechanisms have not been well understood, which hindered its engineering application. Also, microalgal-bacterial symbioses ubiquitously exist in natural water, which usually containing complex organics due to human activity 57 , but the degradation mechanism has not been noticed, leading to the incomplete knowledge on the biogeochemistry cycle of carbon. In this study, the MBGS process was proved to be able to effectively dispose complex organics and transform it to biomass during day-night cycles under the interactions between algae and bacteria. This confirmed that MBGS could indeed enrich the organics from wastewater into biomass for further resource recovery. However, although MBGS showed potential to remove complex organics, the stable performance has not been achieved due to the unfavorable granule size and imbalance carbon-to -nitrogen content of wastewater. Thus future study would further optimize the reaction conditions such as setting stirring to regulate the granular size considering that stirring positively impacted the photosynthetic efficiency thus improving granule characteristics 58 , and adding inorganic carbon to increase the C/N ratio of influent as MBGS could use additional carbon dioxide to improve pollutant removal performance 10 . In addition, the future research will focus on the detailed collaborated mechanisms between algae and bacteria in microalgal-bacteria symbiosis by involving metabolite analysis such as signaling molecule. 4. Methods 4.1 Raw wastewater The raw wastewater was collected from the sewer manholes of the canteen and dormitory of university in Wuhan and the wastewater treatment plant in Hangzhou during the experiment, while synthetic wastewater was used for comparation in this experiment with the composition described as Supplementary Table 5. The pollutants concentration variations and pH values were depicted in as Supplementary Table 6. The average water temperature was approximately 24.5°C. 4.2 Experimental procedure The MBGS was derived from previous research 16 , with an average size of 4.98 mm, average density of 1.04 g/cm 3 and SVI 5 of 40.20 mL/g. The continuous-flowing bioreactors were adopted including two identical flat-plate bioreactors with vertical baffles and two identical inclined-plate bioreactors with 45° angle (see Supplementary Table 7), operating at a light intensity of 180 µ mol·m − 2 ·s − 1 (12h light/12h dark) under LED lights (MBTL-T8-18, Hangzhou Mobate Biotechnology Co., Ltd., China) (see Supplementary Fig. 8). The initial VSS concentration of MBGS in the continuous-flowing bioreactors was approximately 2.6 g/L, while the hydraulic retention time (HRT) was controlled at 12 h. Water samples were collected at 8:00 and 20:00 every day and filtered through 0.45-µm filters for further analysis. The water temperature, pH and turbidity during the experiment were shown in Supplementary Fig. 9. 4.3 Analytical methods COD, TN, NH 4 + -N, NO 3 − -N, NO 2 − -N, TP, SVI 5 and VSS were measured by standard methods 59 . The granule size and density of MBGS were determined as described 11 , 60 . The granule morphology and microbial compositions of MBGS were observed by the fluorescence microscope (Rx50, Ningbo Shunyu Instrument Co., Ltd, China) and scanning electron microscope (Gemini 500, Zeiss, Germany). The measuring instruments for light intensity, oxygen concentration, water temperature and pH value were described as previous study 28 , and the turbidity was measured by a portable turbidity meter (WGZ-1B, Hangzhou Qiwei Instrument Co., Ltd, China). The statistical significance and fitting of experimental data were described as previous research 10 . 4.4 Metagenomic and metatranscriptomic sequencing analysis 4.4.1 DNA extraction and quality inspection The total DNA was extracted using the FastDNA Spin Kit for Soil (116564384C1, MP, USA), while the Quantifluor-ST fluorometer (E6090, Promega, USA) was used to measure the absorbance value of DNA at 260 nm and 280 nm to detect the concentration of DNA with the Quant-IT PicoGreen dsDNA detection kit (P7589, Invitrogen, USA), and then the DNA quality was detected with 1% agarose gel electrophoresis. 4.4.2 RNA extraction and quality inspection The total RNA was extracted using the RNA PowerSoil® Total RNA Isolation Kit (12866-25, MoBio, USA), while 1.5% agarose gel electrophoresis was performed on the extracted RNA samples for quality judgment, and the RNA bands were required to be complete without degradation. Then, a UV spectrophotometer (NC-2000, Thermo Scientific, USA) was used to quantify RNA, requiring RNA concentration > 50 ng/µl, and a biological analyzer (2100Bioanalyzer, Agilent, USA) was used to evaluate its integrity and purity, and RIN ≥ 5.5 was required. 4.4.3 Sequence analysis The sequencing data of MBGS were analyzed by Illumina NovaSeq sequencing platform. The total metagenomic DNA of the flora extracted and the cDNA double strand synthesized by the metetranscriptomic using mRNA as template were randomly broken into short fragments by the Whole Genome Shotgun (WGS) strategy, and inserted fragment libraries of suitable length were constructed to sequence these libraries at both ends (PE). The species annotation, sequence splicing, gene prediction and functional annotation of metagenomic and metetranscriptomic sequencing were described as researches 61 , 62 . The pollutant metabolism pathways and relative abundance of CAZy class in the carbon metabolism were based on the KEGG and CAZy databases, respectively. 4.4.4 Co-occurrence networks analysis Based on the metetranscriptomics sequencing data of MBGS from four bioreactors, the Sparcc rank correlation coefficients between microorganisms, as well as microbial community and functional genes were calculated using software Mothur 63 , and then constructed the co-occurrence networks with R > 0.6 and P < 0.05. The co-occurrence networks were investigated based on the metatranscriptomic sequencing, while the concept of degree was used to describe the relationships between algae and bacteria, as well as microbial community and functional genes 64 , 65 , and finally imported into the software Gephi 0.10 ( http://gephi.guthub.io/ ) for visual display. Declarations Declaration of interests The authors declare no competing interests. Author contributions Yuting Shi : Writing - original draft, Investigation, Validation, Software, Data curation, Formal analysis. Chengxiang Xu : Supervision, Resources, Writing - review & editing. Bin Ji : Conceptualization, Funding acquisition, Writing - review & editing. Anjie Li : Writing - review & editing. Xiaoyuan Zhang : Writing - review & editing. Yu Liu : Writing - review & editing. Acknowledgements This research was funded by the National Natural Science Foundation of China (52261135627; 52270048; 51808416). We also thanked Mr. Min Lin for providing real municipal wastewater. Data availability Data will be made available on request. References Wang X, et al. Impact hotspots of reduced nutrient discharge shift across the globe with population and dietary changes. Nat. Commun. 10, 2627 (2019). Meckling J, Allan BB. The evolution of ideas in global climate policy. Nat. Clim. Change 10, 434–438 (2020). Giordano V, Tuninetti M, Laio F. 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The plastisphere of biodegradable and conventional microplastics from residues exhibit distinct microbial structure, network and function in plastic-mulching farmland. J. Hazard. Mater. 442, 130011 (2023). Li Y, Gao W, Wang C, Gao M. Distinct distribution patterns and functional potentials of rare and abundant microorganisms between plastisphere and soils. Sci. Total Environ. 873, 162413 (2023). McBride MJ, Zhu Y. Gliding Motility and Por Secretion System Genes Are Widespread among Members of the Phylum Bacteroidetes. J. Bacteriol. 195, 270–278 (2013). Qian J, et al. Revealing the mechanisms of polypyrrole (Ppy) enhancing methane production from anaerobic digestion of waste activated sludge (WAS). Water Res. 226, 119291 (2022). Nuccio EE, et al. Niche differentiation is spatially and temporally regulated in the rhizosphere. ISME J.14, 999–1014 (2020). Brouard JS, Otis C, Lemieux C, Turmel M. The Exceptionally Large Chloroplast Genome of the Green Alga Floydiella terrestris Illuminates the Evolutionary History of the Chlorophyceae. Genome Biol. Evol. 2, 240–256 (2010). Lancellotti BV, Hensley DA, Stryker R. Detection of heavy metals and VOCs in streambed sediment indicates anthropogenic impact on intermittent streams of the U.S. Virgin Islands. Sci. Rep. 13, 17238 (2023). Shen Y, Chen B, Wang S, Li A, Ji B. Necessity of stirring for outdoor microalgal-bacterial granular sludge process. J. Environ. Manage. 345, 118816 (2023). APHA. Standard methods for the examination of water and wastewater . American Public Health Association (2005). Cheng W, et al. Formation and characteristics of filamentous granular sludge. Water Sci. Technol. 82, 364–372 (2020). Yu K, Zhang T. Metagenomic and metatranscriptomic analysis of microbial community structure and gene expression of activated sludge. PLoS One 7, 38183 (2012). Huang W, et al. Metagenomic analysis reveals enhanced nutrients removal from low C/N municipal wastewater in a pilot-scale modified AAO system coupling electrolysis. Water Res. 173, 115530 (2020). Schloss PD, et al. Introducing mothur: open-source, platform-independent, community-supported software for describing and comparing microbial communities. Appl. Environ. Microbiol. 75, 7537–7541 (2009). Ma B, et al. Aerobic Denitrification Enhanced by Immobilized Slow-Released Iron/Activated Carbon Aquagel Treatment of Low C/N Micropolluted Water: Denitrification Performance, Denitrifying Bacterial Community Co-occurrence, and Implications. Environ. Sci. Technol. 57, 5252–5263 (2023). Huang S, et al. Metagenomic analysis reveals the responses of microbial communities and nitrogen metabolic pathways to polystyrene micro(nano)plastics in activated sludge systems. Water Res. 241, 120161 (2023). Additional Declarations There is NO Competing Interest. Supplementary Files 3Supplementarymaterials.docx Nine supplementary figures and seven supplementary tables. Cite Share Download PDF Status: Published Journal Publication published 24 Jun, 2024 Read the published version in Communications Earth & Environment → Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-3453507","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":267799192,"identity":"7e9e2ae1-8a8a-4a04-8907-e17539a24fba","order_by":0,"name":"Bin Ji","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAw0lEQVRIiWNgGAWjYBACA2YgTqiA8niI13IGyGIjWguIYGwjRYs5O++BgofzDifOn9/A+OBtG4O8OSEtls18CQaJ2w4nbjjGwGw4t43BcGcDIYcd5jEAarmduIGNgU2at40hweAAUVrm3E6c38bA/psELQ23ExuOMbAxE6XFshmoJeHYf+MNxxKbJeeckzDcQEiLOf8ZM8MfNWmy85sPH/zwpsxGnqAtQMBmAKEZG4CEBGH1QMD8gChlo2AUjIJRMHIBAIhSPTusOQShAAAAAElFTkSuQmCC","orcid":"","institution":"Wuhan University of Science and Technology","correspondingAuthor":true,"prefix":"","firstName":"Bin","middleName":"","lastName":"Ji","suffix":""},{"id":267799193,"identity":"0c91d3bd-ec80-4fa3-83c9-73fa0551ad35","order_by":1,"name":"Shi Shi","email":"","orcid":"","institution":"Wuhan University of Science and Technology","correspondingAuthor":false,"prefix":"","firstName":"Shi","middleName":"","lastName":"Shi","suffix":""},{"id":267799194,"identity":"db48d6b2-02fb-4e0f-940c-2c6431e4506c","order_by":2,"name":"Chengxiang Xu","email":"","orcid":"","institution":"Wuhan University of Science and Technology","correspondingAuthor":false,"prefix":"","firstName":"Chengxiang","middleName":"","lastName":"Xu","suffix":""},{"id":267799195,"identity":"6a09312a-2bb9-41aa-99f5-6dc134466dcf","order_by":3,"name":"Anjie Li","email":"","orcid":"","institution":"Beijing Normal University","correspondingAuthor":false,"prefix":"","firstName":"Anjie","middleName":"","lastName":"Li","suffix":""},{"id":267799196,"identity":"a4a1da02-cb44-46cc-b539-22e4d525abdc","order_by":4,"name":"Xiaoyuan Zhang","email":"","orcid":"","institution":"Nankai University","correspondingAuthor":false,"prefix":"","firstName":"Xiaoyuan","middleName":"","lastName":"Zhang","suffix":""},{"id":267799197,"identity":"7e9a3384-3c65-40dd-9f6d-2ed9256a6b3f","order_by":5,"name":"Yu Liu","email":"","orcid":"","institution":"Nankai University","correspondingAuthor":false,"prefix":"","firstName":"Yu","middleName":"","lastName":"Liu","suffix":""}],"badges":[],"createdAt":"2023-10-16 15:45:14","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-3453507/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-3453507/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1038/s43247-024-01499-0","type":"published","date":"2024-06-24T04:00:00+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":49892411,"identity":"bb5c1096-f5ec-4942-b662-0748b76e781b","added_by":"auto","created_at":"2024-01-19 20:55:25","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":40212,"visible":true,"origin":"","legend":"\u003cp\u003eCorrelation heat maps of the correlation between pollutants (COD, TN and TP) removal efficiencies with carbon-to-nitrogen (C/N) ratio, carbon to phosphorus (C/P) ratio, and DO concentrations during daytime (a) and nighttime (c), an exponential fitting plot of removal efficiencies of COD and TN versus C/N ratios during the day (b), a linear fitting plot of removal efficiency of COD at night versus DO concentrations (d). \u003csub\u003e*\u003c/sub\u003e：\u003cem\u003ep\u003c/em\u003e ＜ 0.05；\u003csub\u003e**\u003c/sub\u003e：\u003cem\u003ep\u003c/em\u003e ＜ 0.01; \u003csub\u003e***\u003c/sub\u003e：\u003cem\u003ep\u003c/em\u003e ＜0.001.\u003c/p\u003e","description":"","filename":"Onlinefloatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-3453507/v1/e1f71fbe4054b71963f66d47.png"},{"id":49892412,"identity":"1171247c-f2ba-4b1a-a2ce-97a96b18b44f","added_by":"auto","created_at":"2024-01-19 20:55:33","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":110449,"visible":true,"origin":"","legend":"\u003cp\u003eThe\u003cstrong\u003e \u003c/strong\u003eorganics metabolism pathways of MBGS process (a), the abundance, expression (b) and expression ratios (c) of functional genes related to organics metabolism and photosynthesis. SW1: Synthetic wastewater in the flat-plate bioreactor, RW1: Real municipal wastewater in the flat-plate bioreactor, SW2: Synthetic wastewater in the inclined-plate bioreactor, RW2: Real municipal wastewater in the inclined-plate bioreactor.\u003c/p\u003e","description":"","filename":"Onlinefloatimage3.png","url":"https://assets-eu.researchsquare.com/files/rs-3453507/v1/9330d0857cd638dfdc4d1e7b.png"},{"id":49892414,"identity":"8e3cf497-a677-453c-80ce-166d1e933a98","added_by":"auto","created_at":"2024-01-19 20:55:33","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":130331,"visible":true,"origin":"","legend":"\u003cp\u003eThe circos maps of microbial community structure of MBGS at phylum (a) and class (b) level based on the metagenomic sequencing (top 20), the clustering heatmaps of microbial community structure of MBGS at phylum (c) and class (d) level based on the metetranscriptomicssequencing (top 20). SW1: Synthetic wastewater in the flat-plate bioreactor, RW1: Real municipal wastewater in the flat-plate bioreactor, SW2: Synthetic wastewater in the inclined-plate bioreactor, RW2: Real municipal wastewater in the inclined-plate bioreactor.\u003c/p\u003e","description":"","filename":"Onlinefloatimage4.png","url":"https://assets-eu.researchsquare.com/files/rs-3453507/v1/8288064d6a2a959c8fc471c8.png"},{"id":49892417,"identity":"147a846b-09d0-4ddf-9e8a-4a8b439dc34b","added_by":"auto","created_at":"2024-01-19 20:55:33","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":53691,"visible":true,"origin":"","legend":"\u003cp\u003eThe\u003cstrong\u003e \u003c/strong\u003egene expression ratios (cDNA/DNA ratios) of dominant microorganisms at the phylum (a) and class level (b). SW1: Synthetic wastewater in the flat-plate bioreactor, RW1: Real municipal wastewater in the flat-plate bioreactor, SW2: Synthetic wastewater in the inclined-plate bioreactor, RW2: Real municipal wastewater in the inclined-plate bioreactor.\u003c/p\u003e","description":"","filename":"Onlinefloatimage5.png","url":"https://assets-eu.researchsquare.com/files/rs-3453507/v1/d4b080320b0b2b058d5e935e.png"},{"id":49892419,"identity":"a4a3afcc-3a71-40d6-9312-c7e250829585","added_by":"auto","created_at":"2024-01-19 20:55:35","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":90297,"visible":true,"origin":"","legend":"\u003cp\u003eThe co-occurrence networks between dominant microorganisms (top 20) of MBGS at phylum (a) and class (b) level. Nodes represented each dominant phylum or class, and the connections between nodes indicated a correlation between the two microorganisms. The red line indicated a positive correlation, while the green line indicated a negative correlation. The size of the node was directly proportional to the relative abundance of the microorganisms in the entire sample. The edges thickness was proportional to the relevance degree.\u003c/p\u003e","description":"","filename":"Onlinefloatimage6.png","url":"https://assets-eu.researchsquare.com/files/rs-3453507/v1/ac78ef69ae296086e24c44fe.png"},{"id":49892409,"identity":"64fedb66-5138-46b9-a685-20d6dec59f9b","added_by":"auto","created_at":"2024-01-19 20:55:23","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":77452,"visible":true,"origin":"","legend":"\u003cp\u003eThe co-occurrence networks between microbial community and functional genes\u003c/p\u003e\n\u003cp\u003eof MBGS at phylum (a) and class (b) level. Nodes represented each phylum, class or genes, and the connections between nodes indicated a correlation between microorganism and gene. The size of the node was directly proportional to the relative abundance of microorganism or function gene. The line indicated a positive correlation and the edges thickness was proportional to the relevance degree.\u003c/p\u003e","description":"","filename":"Onlinefloatimage7.png","url":"https://assets-eu.researchsquare.com/files/rs-3453507/v1/9e2c4c205f11da8275443149.png"},{"id":49892408,"identity":"5d5eb1ab-26cc-436e-8372-e2e4fabb8751","added_by":"auto","created_at":"2024-01-19 20:55:23","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":31055,"visible":true,"origin":"","legend":"\u003cp\u003eThe diagram of complex organics removal mechanisms by MBGS process.\u003c/p\u003e","description":"","filename":"Onlinefloatimage8.png","url":"https://assets-eu.researchsquare.com/files/rs-3453507/v1/10bb472e86ac7a497d0de18b.png"},{"id":49892413,"identity":"0631d990-8b7e-415e-a924-33ffe0d44f12","added_by":"auto","created_at":"2024-01-19 20:55:33","extension":"png","order_by":12,"title":"","display":"","copyAsset":false,"role":"graphical-abstract","size":67754,"visible":true,"origin":"","legend":"Algae-bacteria interactions play an essential role in the transformation of complex organics in microalgal-bacterial granular sludge (MBGS) process, but the intrinsic removal mechanisms have not been well understood. This study thus attempted to investigate the removal performance and mechanisms of complex organics in real municipal wastewater in MBGS process. The results showed that complex organics could be effectively disposed during day-night cycles by MBGS, with the process performance significant impacted by the influent C/N ratio. Further metagenomic and metatranscriptomic analyses revealed that the upregulated and genes of glycolysis enhanced the conversion of complex organics to CO mediated by and //, while the upregulated gene of NADPH synthesis by strengthened the fixation of CO into biomass. Meanwhile, the functional gene of in the starch metabolism by was upregulated, along with the upregulated gene in the glycerolipid metabolism through and . Moreover, a close symbiotic relationship between and was identified, which played a crucial role in fatty acid decomposition. This study offers new insights into removal mechanisms of complex organics via microalgal-bacterial symbiosis, which also gain basic knowledge on the carbon cycle in natural water ecosystems mediated by microalgal-bacterial symbiosis.","description":"","filename":"Onlinefloatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-3453507/v1/3b4c4b8381ccb30ac0ad2fae.png"},{"id":59000733,"identity":"689369b3-611e-4ced-98a2-4013dd4b4617","added_by":"auto","created_at":"2024-06-25 07:08:45","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1342065,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-3453507/v1/bd017c2d-2a13-4a3a-a4f9-61055bb138ab.pdf"},{"id":49892418,"identity":"af0aa590-9b01-49b9-ae73-d72169c941c0","added_by":"auto","created_at":"2024-01-19 20:55:33","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":2932931,"visible":true,"origin":"","legend":"Nine supplementary figures and seven supplementary tables.","description":"","filename":"3Supplementarymaterials.docx","url":"https://assets-eu.researchsquare.com/files/rs-3453507/v1/cf6d6279fe0de9ac7d58d8aa.docx"}],"financialInterests":"There is \u003cb\u003eNO\u003c/b\u003e Competing Interest.","formattedTitle":"Organics removal pathways and algae-bacteria interactions of microalgal-bacterial granular sludge treating real municipal wastewater","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eCurrently, with the intensification of human activities, the total amount of municipal wastewater has been increasing year by year\u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003e. In the era of climate change\u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e, \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u003c/sup\u003e and energy crisis\u003csup\u003e\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u003c/sup\u003e, the widely used conventional activated sludge (CAS) process is under pressure due to high energy consumption and carbon emissions\u003csup\u003e\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u003c/sup\u003e. As an energy-saving and environmentally-sustainable process, the microalgal-bacterial granular sludge (MBGS) can directly utilize solar energy to generate oxygen\u003csup\u003e\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e, \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u003c/sup\u003e, which is further used assimilate wastewater organics and nutrients into microalgae biomass, while helping to mitigate carbon emissions\u003csup\u003e\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u003c/sup\u003e. In fact, the MBGS process can even absorb carbon dioxide of industrial origin\u003csup\u003e\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u003c/sup\u003e and methane\u003csup\u003e\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u003c/sup\u003e, which is more conducive to achieving the carbon neutrality of wastewater treatment \u003csup\u003e\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e, \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e, \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eUnderstanding of the removal pathways of nutrients and organics by MBGS are of vital significance, which, nevertheless, has not been fully determined, especially for the complex organics. For nitrogen metabolism, most of the NH\u003csub\u003e4\u003c/sub\u003e\u003csup\u003e+\u003c/sup\u003e-N can be converted into glutamine or glutamate for microbial assimilation in non-aerated MBGS system\u003csup\u003e\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e, \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u003c/sup\u003e, and NO\u003csub\u003e3\u003c/sub\u003e\u003csup\u003e\u0026minus;\u003c/sup\u003e-N and NO\u003csub\u003e2\u003c/sub\u003e\u003csup\u003e\u0026minus;\u003c/sup\u003e-N can be converted to NH\u003csub\u003e4\u003c/sub\u003e\u003csup\u003e+\u003c/sup\u003e-N before they are metabolized\u003csup\u003e\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u003c/sup\u003e, while the organic nitrogen (e.g. urea, amino acids, etc.) can be hydrolyzed to NH\u003csub\u003e4\u003c/sub\u003e\u003csup\u003e+\u003c/sup\u003e-N through intracellular urea amidolyase\u003csup\u003e\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u003c/sup\u003e. For phosphate metabolism, phosphorus could be removed by MBGS through microbial assimilation and poly-phosphate accumulation\u003csup\u003e\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u003c/sup\u003e, and PO\u003csub\u003e4\u003c/sub\u003e\u003csup\u003e3\u0026minus;\u003c/sup\u003e-P can be transported into cells through the oxidative phosphorylation and removed through poly-phosphate accumulation under the action of poly-phosphate kinase\u003csup\u003e\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u003c/sup\u003e. However, it should be noted that for organics metabolism, limited knowledge is currently available since the laboratory-scale studies usually adopted the simple carbon source, e.g., acetate and glucose\u003csup\u003e\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e, \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/sup\u003e, while the carbon source in real municipal wastewater is mainly composed of complex organics, e.g., starch\u003csup\u003e\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u003c/sup\u003e, but its degradation mechanisms remained unclear. In addition, the algae-bacteria symbiosis ubiquitously exist in natural aquatic ecosystems\u003csup\u003e\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u003c/sup\u003e. Considering the complex organics discharged into the natural water due to human activity\u003csup\u003e\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e, \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u003c/sup\u003e, it is also necessary to explore the degradation mechanisms for organic matter by microalgal-bacterial symbiosis. Meanwhile, the carbon metabolism is closely related to greenhouse gases emissions\u003csup\u003e\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u003c/sup\u003e and the organics removal is usually intimately related to nutrients removal\u003csup\u003e\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u003c/sup\u003e, there is an urgent need to decipher the complex organics removal mechanisms of microalgal-bacterial symbiosis, providing more information of real wastewater complex organics removal pathways.\u003c/p\u003e \u003cp\u003eOn the other hand, to date, the detailed cooperative interaction between algae and bacteria has not been depicted, and the relationships between microbial community and functional genes of MBGS in complex organics metabolism has also been unclear, which hinders its further application. It has been proven that the key role of the algae-bacteria interactions is reflected by the exchange of CO\u003csub\u003e2\u003c/sub\u003e and O\u003csub\u003e2\u003c/sub\u003e, which is a necessary prerequisite for the efficient removal of organics by MBGS\u003csup\u003e\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u003c/sup\u003e. However, the previous studies focused mainly on the microbial community components or the bacterial coherence, while lacking the algae-bacteria interactions in microalgal-bacterial symbiosis\u003csup\u003e\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e, \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eTo discover the organics removal pathways and algae-bacteria interactions of microalgal-bacterial symbiosis in treating real municipal wastewater, this study investigated the performance and upregulated functional genes of MBGS process on real municipal wastewater under diel fluctuation. Meanwhile, the evolution of microbial community and relationships between algae and bacteria of MBGS were determined. Moreover, the relationships between microbial community and functional genes were elucidated. This study provided more information on the complex organics removal mechanisms under the algae-bacteria interactions of MBGS process for treating complex organic wastewater, which is expected to add basic knowledge for the engineering applications of MBGS process. More importantly, this study can help to understand the carbon cycling in natural aquatic systems mediated by microalgal-bacterial symbiosis.\u003c/p\u003e"},{"header":"2. Results and discussion","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e2.1 Performance of complex organics removal by MBGS\u003c/h2\u003e \u003cp\u003eAs illustrated in Supplementary Figs.\u0026nbsp;1 to 3, the MBGS could effectively remove complex organics, evidenced by a COD removal efficiency of 82.4% in treating real municipal wastewater by the flat-plate reactor during the daytime on the day 12. It was showed that the carbon-to-nitrogen (C/N) ratio of influent could impact on the COD and TN removal during the daytime (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ea), e.g. their removal efficiencies increased with the increase of C/N ratio from 1 to 5 and then tended to be stable when the C/N ratio exceeded 5 (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eb). In fact, it had been reported that the C/N ratio of MBGS has been proven to be around 5 based on the empirical formula of C\u003csub\u003e100\u003c/sub\u003eH\u003csub\u003e158\u003c/sub\u003eO\u003csub\u003e38\u003c/sub\u003eN\u003csub\u003e17\u003c/sub\u003eP\u003csup\u003e\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e, \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u003c/sup\u003e, and that the pollutants removal mechanisms of MBGS were mainly microbial assimilation\u003csup\u003e\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e, \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u003c/sup\u003e. However, the C/N ratios of real municipal wastewater fluctuated significantly and even fell to around 1 on the last 10 days of the experiment (see Supplementary Table\u0026nbsp;1), which were unfavorable for pollutant removal by MBGS. Moreover, the average DO concentration during the nighttime had a positive impact on the performance of COD at night (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ec and \u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ed), while the DO concentration in real municipal wastewater could even exceed 21 mg/L at 20:00 (see Supplementary Table\u0026nbsp;2), which was beneficial for the organics removal at night\u003csup\u003e\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e\u003c/sup\u003e since the O\u003csub\u003e2\u003c/sub\u003e produced by microalgae during the day could be utilized by bacteria at night\u003csup\u003e\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u003c/sup\u003e. It should also be noted that the optimal granule size of MBGS was reported to be 1.2 to 1.3 mm at a light intensity of 200 \u0026micro; mol\u0026middot;m\u003csup\u003e\u0026minus;\u0026thinsp;2\u003c/sup\u003e\u0026middot;s\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, thus the organics removal could be probably improved by further optimizing the granule size of MBGS\u003csup\u003e\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eIn addition, Supplementary Fig.\u0026nbsp;4 showed that the MBGS had excellent settling performance in the flat-plate bioreactors, evidenced by that the lower SVI\u003csub\u003e5\u003c/sub\u003e values and higher density of MBGS than inclined-plate bioreactors, resulting in the better removal performance of flat-plate bioreactors, while the real municipal wastewater was more conducive to the granule growth and settling performance as compared to synthetic wastewater, which could be ascribed to the fact that the complex organics in real municipal wastewater would facilitate the bacteria growth in large granules than synthetic wastewater\u003csup\u003e\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003e2.2 Organics removal pathways\u003c/h2\u003e \u003cp\u003eAccording to the research\u003csup\u003e\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e\u003c/sup\u003e, the main components of organics in real municipal wastewater were carbohydrate, fat and oil composed of glycerol and fatty acid\u003csup\u003e\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e\u003c/sup\u003e, while the complex organics of carbohydrate was mainly starch\u003csup\u003e\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u003c/sup\u003e. As depicted in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ea, starch, glycerol and fatty acid could be transformed into α-D-glucose-1P, glycerone-P and acetyl CoA though the starch and sucrose metabolism (ko00500), glycerolipid metabolism (ko00561) and fatty acid degradation (ko00071), respectively, while α-D-glucose-1P and glycerone-P could ultimately be converted into acetyl-CoA during the glycolysis/gluconeogenesis (ko00010), which then entered the TCA cycle\u003csup\u003e\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e\u003c/sup\u003e. Subsequently, the CO\u003csub\u003e2\u003c/sub\u003e generated by TCA cycle could be absorbed by microalgae (\u003cem\u003eCyanobacteria\u003c/em\u003e and \u003cem\u003eChlorophyta\u003c/em\u003e) though the Calvin cycle, and then converted into glyceraldehyde-3P, which eventually synthesized into biomass\u003csup\u003e\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e\u003c/sup\u003e. The relative abundance of organics metabolism pathways (i.e. ko00010, ko00071, ko00500, ko00561) became more and more evident during the experiment, and the glycolysis process was highly active since it accounted for the highest proportion and its relative abundance could reach 0.86% in the real municipal wastewater of inclined-plate reactor at the end of the experiment (see Supplementary Fig.\u0026nbsp;5a). Further analysis by CAZy annotation showed that glycosyl transferases, glycoside hydrolases and carbohydrate-binding modules related to the carbohydrate degradation\u003csup\u003e\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u003c/sup\u003e were abundant, while glycoside hydrolases played an important role in the decomposition of complex organics, whose relative abundance in treating real municipal wastewater was higher than that in synthetic wastewater treatment (see Supplementary Fig.\u0026nbsp;5b).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThe relative expression abundance of functional genes encoding 15 enzymes involved in organics metabolism and 5 enzymes related to photosynthesis were found to be upregulated in treating real municipal wastewater as compared to synthetic wastewater (see Supplementary Table\u0026nbsp;3). The expression of functional genes of \u003cem\u003egapA\u003c/em\u003e (encoding glyceraldehyde 3-phosphate dehydrogenase, K00134), \u003cem\u003epdhD\u003c/em\u003e (encoding dihydrolipoamide dehydrogenase, K00382), \u003cem\u003epps\u003c/em\u003e (encoding orthophosphate dikinase, K01007), \u003cem\u003epetC\u003c/em\u003e (encoding cytochrome b6-f complex iron-sulfur subunit, K02636), \u003cem\u003epetH\u003c/em\u003e (encoding ferredoxin- NADP\u0026thinsp;+\u0026thinsp;reductase, K02641), \u003cem\u003epsbA\u003c/em\u003e (encoding photosystem II P680 reaction center D1 protein, K02703) and \u003cem\u003epsbD\u003c/em\u003e (encoding photosystem II P680 reaction center D2 protein, K02706) were abundant (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eb), while \u003cem\u003egldA\u003c/em\u003e (encoding glycerol dehydrogenase, K00005), \u003cem\u003egap2\u003c/em\u003e (encoding glyceraldehyde-3-phosphate dehydrogenase (NAD(P)), K00150), \u003cem\u003eamyA\u003c/em\u003e (encoding alpha-amylase, K01176), \u003cem\u003egpmA\u003c/em\u003e (encoding 2,3-bisphospho- glycerate-dependent phosphoglycerate mutase, K01834), \u003cem\u003epetH\u003c/em\u003e, \u003cem\u003epsbA\u003c/em\u003e and \u003cem\u003epsbD\u003c/em\u003e showed vigorously expression activity (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ec). It was found that the functional genes of \u003cem\u003egapA\u003c/em\u003e, \u003cem\u003epdhD\u003c/em\u003e, \u003cem\u003epps\u003c/em\u003e, \u003cem\u003egap2\u003c/em\u003e and \u003cem\u003egpmA\u003c/em\u003e were involved in glycolysis process, while \u003cem\u003epetH\u003c/em\u003e was related to photosynthetic electron transport, which could strengthen CO\u003csub\u003e2\u003c/sub\u003e fixation by improving the NADPH synthesis process\u003csup\u003e\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e\u003c/sup\u003e, revealing that the MBGS could achieve the decomposition of organics to CO\u003csub\u003e2\u003c/sub\u003e by aerobic heterotrophic bacteria and the fixation of CO\u003csub\u003e2\u003c/sub\u003e into microalgae biomass by upregulating the expression of functional genes of glycolysis and NADPH synthesis processes, respectively. Meanwhile, the upregulation of \u003cem\u003eamyA\u003c/em\u003e in the starch metabolism and \u003cem\u003egldA\u003c/em\u003e in the glycerolipid metabolism could enhance the decomposition of starch and glycerol, respectively, while the upregulated \u003cem\u003epsbA\u003c/em\u003e and \u003cem\u003epsbD\u003c/em\u003e genes of photosynthesis could facilitate the generation of electrons donated by H\u003csub\u003e2\u003c/sub\u003eO, which were used to reduce NADP\u0026thinsp;+\u0026thinsp;to NADPH, resulting in the production of O\u003csub\u003e2\u003c/sub\u003e\u003csup\u003e34\u003c/sup\u003e.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003e2.3 Algae-bacteria interactions of MBGS in treating complex organics wastewater\u003c/h2\u003e \u003cdiv id=\"Sec6\" class=\"Section3\"\u003e \u003ch2\u003e2.3.1 Microbial community evolution\u003c/h2\u003e \u003cp\u003eSupplementary Fig.\u0026nbsp;6 indicated that the real municipal wastewater could promote the growth of \u003cem\u003eCyanobacteria\u003c/em\u003e, while \u003cem\u003eCyanobacteria\u003c/em\u003e and \u003cem\u003eDesulfobacterota I\u003c/em\u003e could form microalgal-bacterial symbioses situated in the outer layer of MBGS (see Supplementary Fig.\u0026nbsp;6d). As depicted in Figs.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ea and \u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eb, \u003cem\u003eProteobacteria\u003c/em\u003e, \u003cem\u003eBacteroidota\u003c/em\u003e and \u003cem\u003eCyanobacteria\u003c/em\u003e were the dominant phyla of MBGS with an average proportion of 51.3%, 10.6% and 6.2%, respectively, while \u003cem\u003eAlphaproteobacteria\u003c/em\u003e, \u003cem\u003eGammaproteobacteria\u003c/em\u003e, \u003cem\u003eBacteroidia\u003c/em\u003e and \u003cem\u003eCyanobacteriia\u003c/em\u003e were the dominant classes with an average proportion of 28.3%, 23.0%, 10.1% and 6.3%, respectively. Meanwhile, the real municipal wastewater significantly promoted the growth of the bacterial communities of \u003cem\u003eChloroflexota\u003c/em\u003e (\u003cem\u003eAnaerolineae\u003c/em\u003e), \u003cem\u003eAcidobacteriota\u003c/em\u003e (\u003cem\u003eAcidobacteriae\u003c/em\u003e), \u003cem\u003eVerrucomimicrobiota\u003c/em\u003e (\u003cem\u003eVerrucomicrobiae\u003c/em\u003e) and \u003cem\u003eSumerlaeota\u003c/em\u003e (\u003cem\u003eSumerlaeia\u003c/em\u003e) at the end of the experiment through the cluster analysis (Figs.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ec and \u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ed). It was reported that \u003cem\u003eProteobacteria\u003c/em\u003e could facilitate the removals of organics and nutrients through mediating polysaccharide decomposition and enzyme synthesis\u003csup\u003e\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u003c/sup\u003e, while \u003cem\u003eCyanobacteria\u003c/em\u003e could absorb nitrogen and phosphorus from wastewater\u003csup\u003e\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e\u003c/sup\u003e. Researches have shown that \u003cem\u003eBacteroidota\u003c/em\u003e\u003csup\u003e\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e\u003c/sup\u003e, \u003cem\u003eChloroflexota\u003c/em\u003e\u003csup\u003e\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e\u003c/sup\u003e, \u003cem\u003eVerrucomicrobiota\u003c/em\u003e\u003csup\u003e\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e\u003c/sup\u003e, \u003cem\u003eActinobacteriota\u003c/em\u003e\u003csup\u003e\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e\u003c/sup\u003e and \u003cem\u003eAcidobacteriota\u003c/em\u003e\u003csup\u003e\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e\u003c/sup\u003e were able to degrade polysaccharides, while the degradation of complex organics was potentially ascribed to \u003cem\u003eDesulfobacterota I\u003c/em\u003e\u003csup\u003e\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e\u003c/sup\u003e, \u003cem\u003eArmatimonadota\u003c/em\u003e\u003csup\u003e\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e\u003c/sup\u003e and \u003cem\u003eSumerlaeota\u003c/em\u003e\u003csup\u003e\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e\u003c/sup\u003e. The average relative abundance of the above-mentioned bacteria in real municipal wastewater was significantly higher than those in synthetic wastewater (see Supplementary Fig.\u0026nbsp;7), implying their adaptation to complex organics and potential contribution for the complex organics degradation, while the Alpha diversity index also showed that MBGS in real municipal wastewater had higher species richness and diversity than synthetic wastewater (see Supplementary Table\u0026nbsp;4). In addition, real wastewater could promote the genes expression of \u003cem\u003eCyanobacteria\u003c/em\u003e (\u003cem\u003eCyanobacteriia\u003c/em\u003e) and \u003cem\u003eChlorophyta\u003c/em\u003e (\u003cem\u003eChlorophyceae\u003c/em\u003e) (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e), which was conducive to providing sufficient O\u003csub\u003e2\u003c/sub\u003e for the bacteria to decompose complex organics.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section3\"\u003e \u003ch2\u003e2.3.2 Relationships between algae and bacteria of MBGS\u003c/h2\u003e \u003cp\u003eIt had been shown that the contributions of microorganisms in wastewater treatment were not necessarily correlated with their relative abundance, but depended on their activity\u003csup\u003e\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e\u003c/sup\u003e. Therefore, the co-occurrence networks between the top 20 dominant microorganisms of MBGS at the phylum and class level based on the metetranscriptomics sequencing were shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e, showing that the networks had 20 nodes with 3 modules (module 1, module 2 and module 3) at phylum level and 2 modules (module 1 and module 2) at class level, while modules were divided based on the positive correlations between microorganisms. \u003cem\u003eCyanobacteria\u003c/em\u003e showed a positive correlation with \u003cem\u003eDesulfobacterota I\u003c/em\u003e\u003csup\u003e\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e\u003c/sup\u003e as the relevance degree of 0.66, while \u003cem\u003eChlorophyceae\u003c/em\u003e showed a close correlation with \u003cem\u003eVerrucomicrobiae\u003c/em\u003e\u003csup\u003e\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e\u003c/sup\u003e as the relevance degree of 0.86. Meanwhile, the symbiotic relationships between \u003cem\u003eChlorophyceae\u003c/em\u003e with \u003cem\u003eSumerlaeia\u003c/em\u003e, \u003cem\u003eChloroflexia\u003c/em\u003e\u003csup\u003e\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e\u003c/sup\u003e, \u003cem\u003eAcidobacteriae\u003c/em\u003e\u003csup\u003e\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e\u003c/sup\u003e and \u003cem\u003eFimbriimonadia\u003c/em\u003e were strengthened during real municipal wastewater treatment, evidenced by the fact that their genes expression activity in the real municipal wastewater were significantly enhanced at the end of the experiment (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eb). In addition, competitive relationship also existed in the MBGS, such as \u003cem\u003eBacteroidota\u003c/em\u003e and \u003cem\u003eSumerlaeota\u003c/em\u003e with the relevance degree of -0.94, since these bacteria performed the same metabolism function for the decomposition of complex organics\u003csup\u003e\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e\u003c/sup\u003e. Overall, the symbiotic relationships between \u003cem\u003eChlorophyceae\u003c/em\u003e with \u003cem\u003eVerrucomicrobiae\u003c/em\u003e, \u003cem\u003eSumerlaeia\u003c/em\u003e, \u003cem\u003eChloroflexia\u003c/em\u003e, \u003cem\u003eAcidobacteriae\u003c/em\u003e and \u003cem\u003eFimbriimonadia\u003c/em\u003e, as well as \u003cem\u003eCyanobacteria\u003c/em\u003e with \u003cem\u003eDesulfobacterota I\u003c/em\u003e were the key to the effective decomposition of complex organic matters in real municipal wastewater, as the exchange of O\u003csub\u003e2\u003c/sub\u003e and CO\u003csub\u003e2\u003c/sub\u003e between algae and bacteria was crucial for organics removal by MBGS\u003csup\u003e\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003e2.4 Relationships between microbial community and functional genes\u003c/h2\u003e \u003cp\u003eFigure\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e showed that \u003cem\u003egap2\u003c/em\u003e and \u003cem\u003egpmA\u003c/em\u003e, as the main functional genes with high expression activity in the glycolysis process\u003csup\u003e\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e\u003c/sup\u003e, the expression of the former was closely correlated with \u003cem\u003eAcidobacteriae\u003c/em\u003e as the relevance degree of 0.71, while the expression of the latter was closely correlated with \u003cem\u003eSumerlaeia\u003c/em\u003e and \u003cem\u003eFimbriimonadia\u003c/em\u003e as the relevance degree of 0.86 and 0.79, respectively. Meanwhile, \u003cem\u003eamyA\u003c/em\u003e, as the functional gene with high expression activity in the starch metabolism, participated in the labile-C degradation\u003csup\u003e\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e\u003c/sup\u003e, its expression was only correlated with \u003cem\u003eActinobacteriota\u003c/em\u003e\u003csup\u003e\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e\u003c/sup\u003e as the relevance degree of 0.71. \u003cem\u003eGldA\u003c/em\u003e, as the functional gene with high expression activity in the glycerolipid metabolism carried by \u003cem\u003eBacteroidota\u003c/em\u003e\u003csup\u003e\u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e\u003c/sup\u003e, its expression was closely correlated with \u003cem\u003eChloroflexia\u003c/em\u003e and \u003cem\u003eVerrucomicrobiae\u003c/em\u003e as the relevance degree of 0.86 and 0.83, respectively. In addition, the expression of \u003cem\u003efadE\u003c/em\u003e, a key functional gene in the fatty acid degradation\u003csup\u003e\u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e\u003c/sup\u003e was closely correlated with \u003cem\u003eDesulfobacterota I\u003c/em\u003e indicated by a relevance degree of 0.71. Overall, \u003cem\u003eAcidobacteriae\u003c/em\u003e, \u003cem\u003eSumerlaeia\u003c/em\u003e and \u003cem\u003eFimbriimonadia\u003c/em\u003e had significantly contributed to the glycolysis process, and \u003cem\u003eActinobacteriota\u003c/em\u003e played a crucial role in the starch decomposition\u003csup\u003e\u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e\u003c/sup\u003e. Meanwhile, \u003cem\u003eChloroflexia\u003c/em\u003e and \u003cem\u003eVerrucomicrobiae\u003c/em\u003e were of vital significant for the glycerol decomposition, and \u003cem\u003eDesulfobacterota I\u003c/em\u003e was a key bacterium for the fatty acid degradation.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eIt appears from the above discussion that the complex organics removal mechanisms by MBGS was illustrated in Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e. The upregulated \u003cem\u003egap2\u003c/em\u003e and \u003cem\u003egpmA\u003c/em\u003e genes of glycolysis process enhanced the conversion of complex organics into CO\u003csub\u003e2\u003c/sub\u003e mediated by \u003cem\u003eChlorophyceae\u003c/em\u003e and \u003cem\u003eAcidobacteriae\u003c/em\u003e/\u003cem\u003eSumerlaeia\u003c/em\u003e/\u003cem\u003eFimbriimonadia\u003c/em\u003e, while the upregulated \u003cem\u003epetH\u003c/em\u003e gene of NADPH synthesis process by \u003cem\u003eCyanobacteria\u003c/em\u003e strengthened the fixation of CO\u003csub\u003e2\u003c/sub\u003e into microalgae biomass. Meanwhile, the gene of \u003cem\u003eamyA\u003c/em\u003e in the starch metabolism by \u003cem\u003eActinobacteriota\u003c/em\u003e was upregulated, along with the upregulated \u003cem\u003egldA\u003c/em\u003e gene in the glycerolipid metabolism with the interaction of \u003cem\u003eChlorophyceae\u003c/em\u003e and \u003cem\u003eChloroflexia/Verrucomicrobiae\u003c/em\u003e, which improved the decomposition of starch and glycerol, respectively. Moreover, fatty acid was decomposed though \u003cem\u003eCyanobacteria\u003c/em\u003e and \u003cem\u003eDesulfobacterota I\u003c/em\u003e. In addition, the significantly upregulated expression of \u003cem\u003epsbA\u003c/em\u003e and \u003cem\u003epsbD\u003c/em\u003e carried by \u003cem\u003eChlorophyceae\u003c/em\u003e\u003csup\u003e\u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e56\u003c/span\u003e\u003c/sup\u003e could provide sufficient O\u003csub\u003e2\u003c/sub\u003e for the above aerobic heterotrophic bacteria.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"3 Implication","content":"\u003cp\u003eAs a green process, the complex organics removal pathways of MBGS process are of vital significance, nevertheless, only the removal pathways of simple carbon source e.g., acetate and glucose were studied\u003csup\u003e\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e, \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/sup\u003e, the complex organics removal mechanisms have not been well understood, which hindered its engineering application. Also, microalgal-bacterial symbioses ubiquitously exist in natural water, which usually containing complex organics due to human activity\u003csup\u003e\u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e57\u003c/span\u003e\u003c/sup\u003e, but the degradation mechanism has not been noticed, leading to the incomplete knowledge on the biogeochemistry cycle of carbon. In this study, the MBGS process was proved to be able to effectively dispose complex organics and transform it to biomass during day-night cycles under the interactions between algae and bacteria. This confirmed that MBGS could indeed enrich the organics from wastewater into biomass for further resource recovery. However, although MBGS showed potential to remove complex organics, the stable performance has not been achieved due to the unfavorable granule size and imbalance carbon-to -nitrogen content of wastewater. Thus future study would further optimize the reaction conditions such as setting stirring to regulate the granular size considering that stirring positively impacted the photosynthetic efficiency thus improving granule characteristics\u003csup\u003e\u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e58\u003c/span\u003e\u003c/sup\u003e, and adding inorganic carbon to increase the C/N ratio of influent as MBGS could use additional carbon dioxide to improve pollutant removal performance\u003csup\u003e\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u003c/sup\u003e. In addition, the future research will focus on the detailed collaborated mechanisms between algae and bacteria in microalgal-bacteria symbiosis by involving metabolite analysis such as signaling molecule.\u003c/p\u003e"},{"header":"4. Methods","content":"\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003e4.1 Raw wastewater\u003c/h2\u003e \u003cp\u003eThe raw wastewater was collected from the sewer manholes of the canteen and dormitory of university in Wuhan and the wastewater treatment plant in Hangzhou during the experiment, while synthetic wastewater was used for comparation in this experiment with the composition described as Supplementary Table\u0026nbsp;5. The pollutants concentration variations and pH values were depicted in as Supplementary Table\u0026nbsp;6. The average water temperature was approximately 24.5\u0026deg;C.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003e4.2 Experimental procedure\u003c/h2\u003e \u003cp\u003eThe MBGS was derived from previous research\u003csup\u003e\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u003c/sup\u003e, with an average size of 4.98 mm, average density of 1.04 g/cm\u003csup\u003e3\u003c/sup\u003e and SVI\u003csub\u003e5\u003c/sub\u003e of 40.20 mL/g. The continuous-flowing bioreactors were adopted including two identical flat-plate bioreactors with vertical baffles and two identical inclined-plate bioreactors with 45\u0026deg; angle (see Supplementary Table\u0026nbsp;7), operating at a light intensity of 180 \u0026micro; mol\u0026middot;m\u003csup\u003e\u0026minus;\u0026thinsp;2\u003c/sup\u003e\u0026middot;s\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e (12h light/12h dark) under LED lights (MBTL-T8-18, Hangzhou Mobate Biotechnology Co., Ltd., China) (see Supplementary Fig.\u0026nbsp;8). The initial VSS concentration of MBGS in the continuous-flowing bioreactors was approximately 2.6 g/L, while the hydraulic retention time (HRT) was controlled at 12 h. Water samples were collected at 8:00 and 20:00 every day and filtered through 0.45-\u0026micro;m filters for further analysis. The water temperature, pH and turbidity during the experiment were shown in Supplementary Fig.\u0026nbsp;9.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003e4.3 Analytical methods\u003c/h2\u003e \u003cp\u003eCOD, TN, NH\u003csub\u003e4\u003c/sub\u003e\u003csup\u003e+\u003c/sup\u003e-N, NO\u003csub\u003e3\u003c/sub\u003e\u003csup\u003e\u0026minus;\u003c/sup\u003e-N, NO\u003csub\u003e2\u003c/sub\u003e\u003csup\u003e\u0026minus;\u003c/sup\u003e-N, TP, SVI\u003csub\u003e5\u003c/sub\u003e and VSS were measured by standard methods\u003csup\u003e\u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e59\u003c/span\u003e\u003c/sup\u003e. The granule size and density of MBGS were determined as described\u003csup\u003e\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e60\u003c/span\u003e\u003c/sup\u003e. The granule morphology and microbial compositions of MBGS were observed by the fluorescence microscope (Rx50, Ningbo Shunyu Instrument Co., Ltd, China) and scanning electron microscope (Gemini 500, Zeiss, Germany). The measuring instruments for light intensity, oxygen concentration, water temperature and pH value were described as previous study\u003csup\u003e\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e\u003c/sup\u003e, and the turbidity was measured by a portable turbidity meter (WGZ-1B, Hangzhou Qiwei Instrument Co., Ltd, China). The statistical significance and fitting of experimental data were described as previous research\u003csup\u003e\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003e4.4 Metagenomic and metatranscriptomic sequencing analysis\u003c/h2\u003e \u003cdiv id=\"Sec15\" class=\"Section3\"\u003e \u003ch2\u003e4.4.1 DNA extraction and quality inspection\u003c/h2\u003e \u003cp\u003eThe total DNA was extracted using the FastDNA Spin Kit for Soil (116564384C1, MP, USA), while the Quantifluor-ST fluorometer (E6090, Promega, USA) was used to measure the absorbance value of DNA at 260 nm and 280 nm to detect the concentration of DNA with the Quant-IT PicoGreen dsDNA detection kit (P7589, Invitrogen, USA), and then the DNA quality was detected with 1% agarose gel electrophoresis.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section3\"\u003e \u003ch2\u003e4.4.2 RNA extraction and quality inspection\u003c/h2\u003e \u003cp\u003eThe total RNA was extracted using the RNA PowerSoil\u0026reg; Total RNA Isolation Kit (12866-25, MoBio, USA), while 1.5% agarose gel electrophoresis was performed on the extracted RNA samples for quality judgment, and the RNA bands were required to be complete without degradation. Then, a UV spectrophotometer (NC-2000, Thermo Scientific, USA) was used to quantify RNA, requiring RNA concentration\u0026thinsp;\u0026gt;\u0026thinsp;50 ng/\u0026micro;l, and a biological analyzer (2100Bioanalyzer, Agilent, USA) was used to evaluate its integrity and purity, and RIN\u0026thinsp;\u0026ge;\u0026thinsp;5.5 was required.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section3\"\u003e \u003ch2\u003e4.4.3 Sequence analysis\u003c/h2\u003e \u003cp\u003eThe sequencing data of MBGS were analyzed by Illumina NovaSeq sequencing platform. The total metagenomic DNA of the flora extracted and the cDNA double strand synthesized by the metetranscriptomic using mRNA as template were randomly broken into short fragments by the Whole Genome Shotgun (WGS) strategy, and inserted fragment libraries of suitable length were constructed to sequence these libraries at both ends (PE). The species annotation, sequence splicing, gene prediction and functional annotation of metagenomic and metetranscriptomic sequencing were described as researches\u003csup\u003e\u003cspan citationid=\"CR61\" class=\"CitationRef\"\u003e61\u003c/span\u003e, \u003cspan citationid=\"CR62\" class=\"CitationRef\"\u003e62\u003c/span\u003e\u003c/sup\u003e. The pollutant metabolism pathways and relative abundance of CAZy class in the carbon metabolism were based on the KEGG and CAZy databases, respectively.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec18\" class=\"Section3\"\u003e \u003ch2\u003e4.4.4 Co-occurrence networks analysis\u003c/h2\u003e \u003cp\u003eBased on the metetranscriptomics sequencing data of MBGS from four bioreactors, the Sparcc rank correlation coefficients between microorganisms, as well as microbial community and functional genes were calculated using software Mothur\u003csup\u003e\u003cspan citationid=\"CR63\" class=\"CitationRef\"\u003e63\u003c/span\u003e\u003c/sup\u003e, and then constructed the co-occurrence networks with R\u0026thinsp;\u0026gt;\u0026thinsp;0.6 and \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05. The co-occurrence networks were investigated based on the metatranscriptomic sequencing, while the concept of degree was used to describe the relationships between algae and bacteria, as well as microbial community and functional genes\u003csup\u003e\u003cspan citationid=\"CR64\" class=\"CitationRef\"\u003e64\u003c/span\u003e, \u003cspan citationid=\"CR65\" class=\"CitationRef\"\u003e65\u003c/span\u003e\u003c/sup\u003e, and finally imported into the software Gephi 0.10 (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://gephi.guthub.io/\u003c/span\u003e\u003cspan address=\"http://gephi.guthub.io/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) for visual display.\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e"},{"header":"Declarations","content":"\u003cp\u003e \u003ch2\u003eDeclaration of interests\u003c/h2\u003e \u003cp\u003eThe authors declare no competing interests.\u003c/p\u003e \u003c/p\u003e\u003ch2\u003eAuthor contributions\u003c/h2\u003e \u003cp\u003e \u003cb\u003eYuting Shi\u003c/b\u003e: Writing - original draft, Investigation, Validation, Software, Data curation, Formal analysis. \u003cb\u003eChengxiang Xu\u003c/b\u003e: Supervision, Resources, Writing - review \u0026amp; editing. \u003cb\u003eBin Ji\u003c/b\u003e: Conceptualization, Funding acquisition, Writing - review \u0026amp; editing. \u003cb\u003eAnjie Li\u003c/b\u003e: Writing - review \u0026amp; editing. \u003cb\u003eXiaoyuan Zhang\u003c/b\u003e: Writing - review \u0026amp; editing. \u003cb\u003eYu Liu\u003c/b\u003e: Writing - review \u0026amp; editing.\u003c/p\u003e\u003ch2\u003eAcknowledgements\u003c/h2\u003e \u003cp\u003eThis research was funded by the National Natural Science Foundation of China (52261135627; 52270048; 51808416). We also thanked Mr. Min Lin for providing real municipal wastewater.\u003c/p\u003e\u003ch2\u003eData availability\u003c/h2\u003e \u003cp\u003eData will be made available on request.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eWang X, et al. Impact hotspots of reduced nutrient discharge shift across the globe with population and dietary changes. Nat. Commun. 10, 2627 (2019).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMeckling J, Allan BB. The evolution of ideas in global climate policy. Nat. Clim. Change 10, 434\u0026ndash;438 (2020).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGiordano V, Tuninetti M, Laio F. Efficient agricultural practices in Africa reduce crop water footprint despite climate change, but rely on blue water resources. Commun. Earth Environ. 4, 475 (2023).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eTong D, et al. 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Water Res. 241, 120161 (2023).\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"nature-portfolio","isNatureJournal":true,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"","title":"Nature Portfolio","twitterHandle":"","acdcEnabled":false,"dfaEnabled":false,"editorialSystem":"ejp","reportingPortfolio":"","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"Microalgal-bacterial symbiosis, Wastewater treatment, Complex organics, Removal pathways, Functional gene, Algae-bacteria interactions","lastPublishedDoi":"10.21203/rs.3.rs-3453507/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-3453507/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"Algae-bacteria interactions play an essential role in the transformation of complex organics in microalgal-bacterial granular sludge (MBGS), but the intrinsic removal mechanisms have not been well understood. This study thus attempted to investigate the removal performance and mechanisms of complex organics in real municipal wastewater in MBGS process. The results showed that complex organics could be effectively disposed during day-night cycles by MBGS, with the process performance significant impacted by the influent C/N ratio. Further metagenomic and metatranscriptomic analyses revealed that the upregulated gap2 and gpmA genes of glycolysis enhanced the conversion of complex organics to CO2 mediated by Chlorophyceae and Acidobacteriae/Sumerlaeia/Fimbriimonadia, while the upregulated petH gene of NADPH synthesis by Cyanobacteria strengthened the fixation of CO2 into biomass. Meanwhile, the functional gene of amyA in the starch metabolism by Actinobacteriota was upregulated, along with the upregulated gldA gene in the glycerolipid metabolism through Chlorophyceae and Chloroflexia/Verrucomicrobiae. Moreover, a close symbiotic relationship between Cyanobacteria and Desulfobacterota I was identified, which played a crucial role in fatty acid decomposition. This study offers new insights into degradation mechanisms of complex organics via microalgal-bacterial symbiosis, which also gains basic knowledge on the carbon cycle in natural water ecosystems mediated by microalgal-bacterial symbiosis.","manuscriptTitle":"Organics removal pathways and algae-bacteria interactions of microalgal-bacterial granular sludge treating real municipal wastewater","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-01-19 20:55:17","doi":"10.21203/rs.3.rs-3453507/v1","editorialEvents":[],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"communications-earth-and-environment","isNatureJournal":true,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"commsenv","sideBox":"Learn more about [Communications Earth and Environment](https://www.nature.com/commsenv/)","snPcode":"","submissionUrl":"","title":"Communications Earth \u0026 Environment","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"ejp","reportingPortfolio":"Communications Series","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"6482f25f-a3fa-40be-b015-d72b38ff9fe5","owner":[],"postedDate":"January 19th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[{"id":28216962,"name":"Biological sciences/Microbiology/Environmental microbiology/Water microbiology"},{"id":28216963,"name":"Biological sciences/Microbiology/Bacteria"}],"tags":[],"updatedAt":"2024-06-25T07:08:39+00:00","versionOfRecord":{"articleIdentity":"rs-3453507","link":"https://doi.org/10.1038/s43247-024-01499-0","journal":{"identity":"communications-earth-and-environment","isVorOnly":false,"title":"Communications Earth \u0026 Environment"},"publishedOn":"2024-06-24 04:00:00","publishedOnDateReadable":"June 24th, 2024"},"versionCreatedAt":"2024-01-19 20:55:17","video":"","vorDoi":"10.1038/s43247-024-01499-0","vorDoiUrl":"https://doi.org/10.1038/s43247-024-01499-0","workflowStages":[]},"version":"v1","identity":"rs-3453507","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-3453507","identity":"rs-3453507","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}