Soil microalga-derived L-glutamic acid enhances the growth and yield of pepper by recruiting nitrogen-fixing bacteria

Soil microalga-derived L-glutamic acid enhances the growth and yield of pepper by recruiting nitrogen-fixing bacteria | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Soil microalga-derived L-glutamic acid enhances the growth and yield of pepper by recruiting nitrogen-fixing bacteria Cong Liu, Hongqing Wei, Yuwei Xing, Zhonghua Shen, Jie Cao, Jun Wang This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-9526545/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 4 You are reading this latest preprint version Abstract Background and aims Reducing chemical fertilizer dependence while sustaining crop productivity is a key challenge for sustainable agriculture. Soil microalgae are promising biofertilizers, yet their growth-promoting mechanisms remain largely unclear. This study aimed to evaluate the effects of soil microalgae on pepper growth and rhizosphere processes, and to clarify the mechanisms underlying yield enhancement. Methods This study isolated two soil microalgae Anabaena azotica and Scenedesmus sp., which were applied in pot and field experiments, with three groups: chemical fertilizer alone, chemical fertilizer plus Anabaena azotica (A), and chemical fertilizer plus a mixed inoculum of A. azotica and Scenedesmus sp. (AS). Transcriptomic profiling of root and third-generation amplicon sequencing of rhizosphere soil were conducted to explore the underlying mechanism. Results The results showed that A and AS application increased pepper yield by 36.42% and 107.87%, respectively. Especially, AS application increased soil available nitrogen content by 11.98%. Moreover, Scenedesmus sp. enriched nitrogen-fixing bacteria, and thereby promoted nitrogen uptake and utilization in the roots, which consequently increased pepper yield. Furthermore, L-glutamic acid was found to be the key metabolite through which Scenedesmus sp. exerted its yield-enhancing effects. Conclusions Microalga-derived L-glutamic acid could recruit nitrogen-fixing bacteria, strengthened rhizosphere nitrogen cycling, increased plant nitrogen acquisition, and enhanced pepper yield. This study provided important scientific evidence for the development and utilization of microalgae-based biofertilizer to improve crop yields in the sustainable agriculture. Scenedesmus sp. Yield improvement nitrogen-fixing bacteria L-glutamic acid Pepper Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Introduction By 2050, the global population is expected to reach 9 billion, necessitating a 50% increase in agricultural output to meet the food demand (Muller et al., 2017 ; Van et al., 2021). To achieve this food production enhancement, the amount of fertilizer usage needs to increase by at least 4-fold, which would considerably increase agricultural production costs (Hartmann et al., 2015 ; Martre et al., 2024 ). The excessive fertilizer application would decrease soil pH, reduce soil organic matter (SOM) and ultimately diminish the effectiveness of yield increases (Nosengo 2003 ; Spohn et al., 2023 ). Thus, the enhancement of agricultural productivity should not rely solely on the increasing use of fertilizer, and other efficient approaches should be explored. Soil microalgae have attracted increasing attention in recent years due to their excellent plant growth-promoting properties (Weissberg et al., 2023 ). Microalgae are rich in macronutrients and micronutrients that are vital for plant growth and development and produce a variety of bioactive substances to drive soil biogeochemical cycles (Vasconcelos et al., 2015; Deepika et al., 2020). For example, nitrogen-fixing cyanobacteria could form biocrusts on desert surfaces, increase soil moisture and nitrogen content, and thereby promote plant growth (Wu et al., 2013 ). Microalgae from the genus Anabaena increased plant height and fresh weight of peppers ( Capsicum annuum L.) by 8.7–17.5% and 22.8–51.8%, respectively (Bello et al., 2021 ). Spirulina platensis and Chlorella vulgaris increased the yield of maize ( Zea mays L.) by 16.9% and 14.9%, respectively (Dineshkumar et al., 2019 ; Uysal et al., 2015 ). Thus, soil microalgae might serve as an important approach to enhance crop yields. Soil ecosystem health and biodiversity are crucial for stable agricultural productivity (Feng et al., 2024 ). As primary producers, soil microalgae safeguard the viability of rhizosphere soil microecosystem by providing oxygen and carbon sources (Kapoore et al., 2021 ; Solomon et al., 2023 ). Moreover, soil microalgae secrete extracellular polymeric substances (EPS) including proteins and polysaccharides to support microbial adhesion, metabolism, and reproduction (Thompson et al., 2012 ; Deepika et al., 2020). In environments where microalgae and bacteria coexist, nitrogen-fixing bacteria supply nitrogen to microalgae and utilize the dissolved organic matter (DOM) released by microalgae as a source of carbon, sulfur, and phosphorus (Thompson et al., 2012 ). We hypothesized that the supplementation of microalgae might enhance soil biodiversity by producing certain substances and subsequently promote plant crop yield. To verify our hypothesis, soil microalgae were firstly isolated from the pepper plantation and then used in pot experiments and field trials to evaluate their effects on the growth and yield of peppers in both uncultivated and cultivated soils. Rhizosphere soil physicochemical properties of pepper plants at different growth stages (seedling, flowering, and maturity) were measured to analyze the impacts of the microalgae on soil properties. During the maturity stage, the nitrogen, phosphorus, and potassium contents in the roots, stems, and leaves of peppers were measued, and transcriptome sequencing of pepper roots were conducted to investigate the effects of microalgae on the absorption and utilization of nutrients by the roots. Moreover, 16S rRNA, 18S rRNA and metagenomic sequencing were performed on the rhizosphere soil of pepper plants to investigate the impacts of microalgae on microbial community structure and function. Furthermore, the main nitrogen-fixing bacteria enriched by the microalgae were isolated and used to investigate the potential mechanism through which substance microalga Scenedesmus sp. exerted the growth-promoting effects. This study would provide a novel approach for improving crop productivity and offer new insights into the application of soil microalgae in modern sustainable agriculture. Materials and methods Soil microalgae isolation and identification The A. azotica and Scenedesmus sp. used in this study were isolated from the soil in a pepper field in Bohu County, Bayingolin Mongol Autonomous Prefecture, Xinjiang, China (41°55′42″ N, 86°37′10″ E). Briefly, 10 g soil samples were inoculated into 50 mL of BG11 liquid medium (Tab. S2) in 100 mL flasks, which were incubated in an incubator (25 ± 1°C, light intensity of 2700 ± 50 lx, and a photoperiod of 16 h light: 8 h dark). After 3 weeks, the enriched microalgal liquid was homogenized, serially diluted from 10 − 1 to 10 − 4 , and spread onto BG11 solid medium. The plates were incubated in the incubator until single microalgal colonies were clearly visible. The microalgal cells were observed under the Nikon Eclipse Ti2 microscope (Fig. S1 ), and genomic DNA was extracted for PCR amplification using 16S primers 27F-1492R (5'-AGRGTTYGATYMTGGCTCAG-3' and 5'-RGYTACCTTGTTACGACTT-3') and 18S primers EukA-EukB (5'-AACCTGGTTGCTGCCAGTGCAGT-3' and 5'-TGATCCTTGCAGGTTCACCTAC-3'). The PCR products were sequenced using Sanger sequencing (Lingen Biotechnology Co., Ltd., Shanghai, China), and the taxonomic classification of the microalgae was established through a BLASTn search of the NCBI GenBank database. Microalgae cultivation Both A. azotica and Scenedesmus sp. were cultured in the BG11 medium until the stationary phase to mueaure the biomass and contents of nitrogen, phosphorus, and potassium in the microalgae using the standard methods, elemental analyzer (Flash 2000, Thermo Scientific, MA, USA) and automatic nutrient analyzer (QuAAtro, SEAL, Germany), respectively. The microalgal suspension was centrifuged at 4,000 g for 3 min, resuspended in distilled water, centrifuged again to revmove the culture medium, and then used in the following experiments. Pot experiments using uncultivated and cultivated soils The uncultivated and cultivated soils were collected from Baguan Mountain and experimental field of Ocean University of China, respectively. The soil properties are presented in Tab. S4. The pepper variety Capsicum annuum L. ‘Honglong 23’ was provided by the Bazhou Green Collar Vocational Skills Training School. Pepper seedlings were raised following the protocol described by Rodrigues et al. ( 2024 ) and transplanted into the pots at the three-true-leaf stage. Pot experiments using uncultivated soil and field experiment using cultivated soil were conducted to assess the effects of microalgae on pepper growth and yield (Fig. S2). Both experiments comprised three groups: CK, chemical fertilizer only; A, chemical fertilizer and A. azotica at 10 g/667 m 2 ; AS, chemical fertilizer and a mixed microalgae consisting of A. azotica and Scenedesmus sp. at 1.92 g/667 m 2 at a 1:1 weight ratio. The application amount of chemical fertilizers in above three groups were 60 kg/667 m² urea and 45 kg/667 m² potassium dihydrogen phosphate. Each group included three replicates, and each replicate included six pots, with three plants per pot. Both chemical fertilizers and microalgae were applied in liquid form and split into four equal doses on the 1st, 7th, 14th, and 21st day after transplantation. Measurement of pepper growth and yield On the 26th, 58th, and 112th day post-transplantation, pepper plant height, leaf length, and leaf width (n = 6) were measured using a measuring tape, and the stem diameter was measured using vernier caliper. Pepper yield was determined on the 112th day using the weighing method. Physicochemical characterization of pepper rhizosphere soil On the 112th day, 50 g rhizosphere soil tightly adhered to the roots was collected using a sterile brush and stored at ‒80°C for 18S rRNA, 16S rRNA and metagenomic sequencing (Xu et al., 2022 ). The remaining soil was air-dried in a cool place and ground for further analysis. The pH, AP content, and AK content of rhizosphere soil were measured following the methods of Hartmann et al. ( 2015 ), and the contents of SOM and AN were determined according to Reichert et al. (2012). The soil acid phosphatase and urease activities were measured using the methods of Lebrun et al. ( 2012 ), and the activities of sucrase and peroxidase were measured using the 3,5-dinitrosalicylic acid colorimetric and colorimetric methods, respectively (Zhou et al., 2022 ). Nutrient composition of pepper plants and root transcriptome Considering that the yield-enhancing effects of microalgae were more pronounced in the uncultivated soil, the pepper roots, stems, and leaves from the uncultivated soil experiment were collected for the determination of nitrogen, phosphorus, and potassium contents. For transcriptome analysis, root samples were thoroughly ground in liquid nitrogen, and total RNA was extracted using the GenElute™ Total RNA Purification Kit (Sigma-Aldrich, USA). RNA quality was assessed using a 5300 Bioanalyzer (Agilent Clara, Technologies, CA, USA) and quantified using an ND-2000 spectrophotometer (NanoDrop Technologies). High-quality RNA samples were selected for library construction and sequencing by Shanghai Majorbio Bio-Pharm Technology Co., Ltd. Rhizosphere soil microbial and metagenomic sequencing Total DNA was extracted from rhizosphere soil samples using E. Z. N. A.® Soil DNA Kits, assessed using 1% agarose gel electrophoresis and quantified using a QuantiFluor TM -ST Blue Fluorescence Quantification System (Promega Corporation). PCR amplification was performed using the 16S primers 27F-1492R and 18S primers EukA-EukB (Liu et al., 2023b ; Kalu et al., 2023). The PCR products were detected using 2% agarose gel electrophoresis, recovered using the AxyPrep DNA Gel Extraction Kit, eluted with Tris-HCl, and detected using 2% agarose gel electrophoresis. For microbial sequencing, paired-end sequencing was performed using a third-generation sequencing platform (PacBio Sequel II, Pacific Biosciences, CA, USA). Metagenomic sequencing was performed using the Illumina NovaSeq sequencing platform, and each sample was analyzed using the MajorBio platform. Isolation and identification of microalgae-enriched bacteria After pot experiments, rhizosphere soil from the AS group was homogenized, and the supernatant was serially diluted with MS buffer (10 mM MgSO₄, 100 mM NaCl, 50 mM Tris-HCl, 0.01% gelatin). The diluted suspension (150 µL) was then spread onto Ashby’s Medium (Tab. S5) and incubated at 30°C for 48 h. Bacterial isolates were randomly selected and separately transferred to new Ashby’s Medium plates using a sterile toothpick. Each isolated bacterium was streaked onto a fresh Ashby’s Medium plate for colony purification, and the 16S rRNA gene of each bacterial strain was amplified using primers 27F-1492R for Sanger sequencing. The 16S rRNA gene sequences of the bacterial isolates were subjected to BLAST searches using the NCBI GenBank database to obtain detailed taxonomic information. The isolated Methylobacterium sp., Bradyrhizobium japonicum , and Sphingomonas sp. were cultured in yeast extract mannitol agar (YMA; Tab. S6), Reasoner's 2A agar (R2A; Tab. S7), and Luria–Bertani Agar (LB; Tab. S8), respectively. After centrifugation at 6,000 g for 10 min, the bacterial cultures at OD 600 of 1.0 were used in the following experiments. Growth-promoting effects of Scenedesmus sp. EPS on nitrogen-fixing bacteria A total of 24 groups were set up to determine whether the EPS and filtrate of Scenedesmus sp. and A. azotica promoted the growth of three isolated nitrogen-fixing bacteria (Tab. S9). Scenedesmus sp. and A. azotica at stationary phase were filtered through 0.22-µm membranes to obtain the filtrate, and EPS were extracted using the heating method (Zhou et al., 2024 ). Cultures of Methylobacterium sp., B. japonicum , and Sphingomonas sp. at the mid-exponential phase were adjusted to an OD 600 of 0.1–0.3 using R2A, YMA, and LB media, respectively. Then, the corresponding bacterial cultures (200 mL) were dispensed into each group, and 2 mL of BG11 medium was added as the control. Subsequently, 2 mL of Scenedesmus sp. EPS, Scenedesmus sp. filtrate, A. azotica EPS, A. azotica filtrate, Scenedesmus sp. EPS and A. azotica EPS combiniation (1:1 v/v), and Scenedesmus sp. filtrate and 1 mL A. azotica filtrate combiniation (1:1 v/v) were separately added into the above bacterial cultures. OD 600 values of each group were measured every 3 h to plot the growth curves. Composition analysis of Scenedesmus sp. EPS and filtrate EPS (500 µL) and filtrate (500 µL) of Scenedesmus sp. were separately mixed with methanol (500 µL), vortexed, and centrifugated at 10,000 g for 10 min at 4°C. The supernatant was then filtered through 0.22-µm membranes, and 2-chlorophenylalanine (100 µg/mL) was added as the internal standard to a final concentration of 1 mg/L prior to analysis. Metabolites in the EPS and filtrate were profiled by HPLC–MS/MS, which was performed on a C18 column (Zorbax Eclipse C18, 1.8 µm × 2.1 mm × 100 mm) at 30°C with a flow rate of 0.3 mL/min using 0.1% formic acid in water (A) and acetonitrile (B) as the mobile phases (Wang et al., 2019 ). Effect of Scenedesmus sp. EPS components on the growth of nitrogen-fixing bacteria The three most abundant compounds L-glutamic acid (LGA), 2,5-anhydro-1-deoxy-1-[(1-hydroxy-2-butanyl)amino]-D-mannitol (DM), and V-pyrro/NO (VP) were selected to identify EPS components that stimulate the growth of three nitrogen-fixing bacteria. Twelve groups were established (Tab. S10), and each compound was dosed to match its concentration in Scenedesmus sp. EPS. LGA, DM, and VP were individually added into Methylobacterium sp. , B. japonicum , and Sphingomonas sp. suspensions, which were prepared by diluting cultures with the corresponding medium to OD 600 = 0.3. During the experiment, OD 600 values of each group were measured every 3 h to plot growth curves. To further validate the growth-promoting effect of LGA on nitrogen-fixing bacteria, assays were conducted on solid media (Tab. S11). Prior to plate pouring, LGA was added into the media at doses corresponding to their concentrations in Scenedesmus sp. EPS. Mid-exponential-phase cultures of Methylobacterium sp. , B. japonicum , and Sphingomonas sp. were diluted to OD 600 = 0.1, and 2.5 µL of each suspension was separately spot-inoculated at the center of the plates. Plates were incubated at 28°C for 36 h, and the colonies were photographed for analysis. Growth-promoting effects of microalga-derived L-glutamic acid and nitrogen-fixing bacteria To test whether Scenedesmus sp. enriched nitrogen-fixing bacteria and promoted plant growth via LGA secretion, a pot experiment was conducted with six groups (Tab. S12). All pots received the same chemical fertilizer as basal fertilization, and N, P, and K inputs were kept constant across groups. The control group only received the chemical fertilizer, and the other groups received the basal fertilizer and either a nitrogen-fixing bacterial inoculum, LGA, or Scenedesmus sp. EPS alone, or bacterial inoculum supplemented with LGA, or the bacterial inoculum supplemented with Scenedesmus sp . EPS. The soil was the same as that used in the uncultivated-soil experiment, sterilized by gamma irradiation (60 kGy, Guo et al., 2024 ), and filled into plastic pots (7 × 7 × 7.3 cm, L × W × H). Pepper seedlings at the three-true-leaf stage were transplanted one week after fertilization to initiate the experiment (day 0), and the designated additives were applied on the 1st, 7th, and 14th day. Plants were sampled on the 30th day to measure the growth indicators. The effects of combined application of Scenedesmus sp. and nitrogen-fixing bacteria A pot experiment was conducted to evaluate the effects of Scenedesmus sp. co-application with nitrogen-fixing bacteria (Tab. S13). All groups received the same chemical fertilizer as a uniform basal application, and N, P, and K inputs were kept constant across treatments. The control group only received the basalfertilizer, and the other groups received the basal fertilizer and one of the following supplemental inputs: A. azotica , Scenedesmus sp., a nitrogen-fixing bacterial inoculum, the bacterial inoculum supplemented with A. azotica , and the bacterial inoculum supplemented with Scenedesmus sp . Soil was sterilized by gamma irradiation (60 kGy, Guo et al., 2024 ), transferred to pots (7 × 7 × 7.3 cm, L × W × H), and amended according to the assigned groups. All inputs were applied once as aqueous preparations. Pepper seedlings at the three-true-leaf stage were transplanted one week after fertilization to initiate the experiment (day 0), and plants were harvested on the 30th day for growth measurements. Statistical analyses Gene expression levels were quantified with RSEM, and differential expression was assessed using DESeq2 (Love et al., 2014 ). GO enrichment and KEGG pathway analyses were performed using gotools and Python SciPy, respectively(Shen et al., 2024 ). For 16S rRNA data, functional profiles were predicted with Phylogenetic Investigation of Communities by Reconstruction of Unobserved States version 2 (PICRUSt2). Metagenomic functions were annotated by BLASTP against the KEGG database ( http://www.genome.jp/kegg/ ) with an e-value cutoff of ≤ 1 × 10 − 5 , and KEGG pathways related to nitrogen metabolism were extracted for downstream analysis(Gao et al., 2022 ). Paired-end reads were merged with FLASH (v1.2.7) and quality-filtered with fastp to obtain clean Tags (Bokulich et al., 2013 ). Chimeras were identified and removed by aligning tags to a reference annotation database, yielding Effective Tags (Haas et al., 2011 ). OTUs were clustered from all effective Tags using UPARSE (v7.0.1001, Edgar 2013 ). Species annotation analysis was performed using RDP Classifier (version 2.2) against the Silva132 database (Wang et al., 2007 ; Quast et al., 2012 ). QIIME was used to compute α-diversity indices and β-diversity distance matrices. A co-occurrence network was constructed using Spearman correlations (r > 0.7, P < 0.05), and correlations were calculated in RStudio (4.1.0) and visualized in Gephi (v0.9.2, Wei et al., 2020 ). Differences among groups were tested by one-way ANOVA (LSD, P 3.5, P < 0.05) and STAMP. Additional statistical analyses were conducted in IBM SPSS Statistics. A partial least squares path model (PLS-PM) was built with the R package “plspm” (v0.5.0), and model fit was evaluated using the goodness-of-fit (GOF) index (Li et al., 2023 b). Other data visualizations were created using Origin 2018 software. Results Microalgae enhance pepper growth and yield in both uncultivated and cultivated soils Compared with the control group, the A and AS application resulted in larger pepper plants and fruits in the uncultivated soil (Fig. 1 a,b). Specifically, the A application increased pepper plant height, stem diameter, leaf length, and leaf width by 6.64%, 4.89%, 5.49%, and 11.76%, respectively ( P < 0.05; Fig. 1 c–f). By contrast, the AS application increased pepper plant height, stem diameter, leaf length, and leaf width by 14.83%, 12.79%, 10.44%, and 16.47%, respectively ( P < 0.05). Additionally, the A and AS application increased pepper yields by 36.42% and 107.87%, respectively ( P < 0.05; Fig. 1 g). In the cultivated soil, the A and AS applications exhibited similar growth-promoting effects and increased pepper yields by 26.91% and 48.05%, respectively (Fig. S3 and Fig. S4). Thus, the application of microalgae A. azotica and Scenedesmus sp. significantly increased the growth and yield of peppers. Microalgae improve rhizosphere soil physicochemical properties Both A and AS application significantly increased soil pH and SOM content in the uncultivated soil at 28 and 56 d post-transplantation, and the AS application also significantly enhanced the available soil nutrient content and soil enzyme activities (Fig. 1 h-n and Fig. S5). At 112 d, the A application increased the contents of available nitrogen (AN), available phosphorus (AP), available potassium (AK), SOM and sucrase activity by 7.57%, 8.30%, 8.02%, 65.84%, and 5.36%, respectively ( P < 0.05; Fig. 1 h-j, l, n). By contrast, the AS application improved these indices by 11.98%, 18.24%, 12.54%, 65.53%, and 9.63%, respectively. Moreover, the AS application increased soil urease, acid phosphatase, and peroxidase activities by 2.73%, 28.79%, and 8.49%, respectively ( P < 0.05). In the cultivated soil, both A and AS application also enhanced soil pH and SOM content, and the AS application significantly improved available soil nutrient contents and enzyme activities (Fig. S6). In the uncultivated soil, the RDA results indicated that acid phosphatase activity (r² = 0.95, P = 0.00005), AP content (r² = 0.91, P = 0.0005), sucrase activity (r² = 0.885, P = 0.0015), AK content (r² = 0.85, P = 0.0035), and AN content (r² = 0.84, P = 0.0050) significantly affected pepper growth and yield (Fig. 1 o). The Mantel test showed that the AN content, sucrase activity, and urease activity were the major factors affecting pepper growth and yield (Fig. 1 p). In the cultivated soil, the RDA results showed that AK content, AP content, urease activity, peroxidase activity, sucrase activity, and AN content significantly affected pepper growth and yield (Fig. S7a). The Mantel test revealed that sucrase activity, urease activity, pH, AN content, AP content, and AK content were the primary factors influencing pepper growth and yield (Fig. S7b). Overall, both uncultivated and cultivated soil experiments demonstrated that pepper growth and yield were significantly affected by the AN content, urease activity, and sucrase activity. Scenedesmus sp. promotes the absorption and utilization of nitrogen by pepper roots Compared with the control group, the A and AS application increased nitrogen content in pepper roots in the uncultivated soil by 26.95% and 64.99% ( P < 0.05; Fig. 2 a), nitrogen content in pepper stems by 25.58% and 48.84% ( P < 0.05; Fig. 2 b), and nitrogen content in pepper leaves by 12.54% and 29.15%, respectively ( P < 0.05; Fig. 2 c). The A and AS application also increased phosphorus content in pepper roots by 44.80% and 66.52% ( P < 0.05; Fig. 2 d), phosphorus content in pepper stems by 15.76% and 25.62% ( P < 0.05; Fig. 2 e), and phosphorus content in pepper leaves by 27.40% and 57.88%, respectively ( P < 0.05; Fig. 2 f). The potassium content in pepper plants did not differ significantly between the control and microalgae application groups (Fig. S8). In the cultivated soil, plant nitrogen content in the A and AS application group exhibited the similar results, but plant phosphorus content showed no significant difference compared to that of the control group ( P > 0.05; Fig. S9). Compared with the control group, the AS application had a greater impact on the transcriptome of pepper roots (Fig. 2 g). After the A application, 4,820 differentially expressed genes (DEGs) were identified, of which 2,111 were upregulated and 2,709 were downregulated (Fig. S10a). The AS application induced 5,725 DEGs, including 3,313 upregulated and 2,412 downregulated genes (Fig. 2 h). Clustering analysis of the DEGs revealed that the contrl, A application, and AS application all clustered within their groups, with high reproducibility between parallel samples (Fig. S10b). Thus, the data could be used for further analysis. Gene ontology (GO) analysis revealed that the DEGs between the A application and control groups were primarily enriched in biological processes (BPs), including the regulation of protein serine/threonine phosphatase activity, abscisic acid-activated signaling pathway, primary cell wall biogenesis in plants, glutathione metabolic process, and ethylene-activated signaling pathway (Fig. S10c). The DEGs between the AS application and control group were primarily enriched in BPs and cellular components (CCs), including the glutathione metabolic process, light harvesting in photosystem I, and the photosystem I reaction center (Fig. 2 i). Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis showed that the A application mainly affected brassinosteroid biosynthesis, monoterpenoid biosynthesis, isoquinoline alkaloid biosynthesis, and nitrogen metabolism (Fig. S10d), while the AS application predominantly affected brassinosteroid biosynthesis, photosynthesis, and nitrogen metabolism (Fig. 2 j). We further analyzed the effects of the A and AS application on nitrogen metabolism in pepper roots. Compared with the A application, the AS application further upregulated the expression of genes ( GS1 and GS2 ) involved in the synthesis of L-glutamine from ammonium nitrogen in pepper roots and the expression of genes ( NR , NIR1 , and NIR2 ) responsible for converting nitrate to nitrite and nitrite to ammonium nitrogen. These results indicated that Scenedesmus sp. effectively promotes nitrogen uptake and utilization in pepper roots. Scenedesmus sp. enriches nitrogen-fixing bacteria The A and AS application had no significant effect on α-diversity of prokaryotic and eukaryotic microbial communities in the rhizosphere soil (Fig. S11). However, both A and AS application altered the community structures of prokaryotic and eukaryotic microorganisms (Fig. 3 a and Fig. S12a). Among the top 20 prokaryotic microorganisms in the relative abundance, Acidobacteriales_norank and RB41 were exclusively in the control group, with relative abundances of 5.94% and 5.12%, respectively (Fig. 3 b). The A and AS applications reduced the relative abundance of Candidatus Udaeobacter by 41.87% and 43.95%, respectively ( P < 0.05). The AS application also decreased the relative abundance of RB41 by 48.60% and increased the relative abundances of Flavisolibacter and Sphingomonas by 27.78% and 57.98%, respectively ( P < 0.05). The statistical analysis of metagenomic profiles (STAMP) analysis further confirmed that the AS application significantly increased the relative abundances of Flavisolibacter and Sphingomonas ( P < 0.05; Fig. 3 c). For eukaryotes, Halobiotus was the dominant genus exclusively in the AS application, with a relative abundance of 6.23% (Fig. S12b). The A application increased the relative abundances of Polymyxa , Chlorosarcinopsis , and Heterochlamydomonas by 527.75%, 51.10%, and 419.35%, respectively ( P < 0.05). The AS application decreased the relative abundance of Hyphochytrium by 61.69% ( P < 0.05). The STAMP analysis showed that the A application significantly increased the relative abundances of Polymyxa and Heterochlamydomonas ( P < 0.05; Fig. S12c), and the AS application significantly increased the relative abundance of Halobiotus ( P < 0.05). LEfSe analysis showed that the A application significantly enriched Chthoniobacter , whereas the AS application significantly enriched Methylobacterium , Tolypothrix , and Microcoleus (Fig. 3 d). For eukaryotic microorganisms, the AS application significantly enriched Palmellopsis and Scenedesmus (Fig. S12d), which confirmed that Scenedesmus sp. could survive well in pepper rhizosphere soil. Co-occurrence network analysis showed that the A and AS application reduced the network diameter and average path length, increased the average clustering coefficient, and enhanced community stability. The AS application increased the modularity of co-occurrence network, in which microorganisms with higher degree values were located in highly interconnected modules and thereby exhibited stronger resistance to external disturbances (Fig. S13 and Tab. S14). Moreover, the AS application promoted prokaryotic microorganisms Microcoleus and Gemmatimonas and eukaryotic microorganisms Heterochlamydomonas , Amphisiella , and Filamoeba , establishing them as core microorganisms (Fig. 3 e and Fig. S12e). Scenedesmus sp. affects soil nitrogen cycling by enriching nitrogen-fixing bacteria The A and AS application significantly upregulated genes associated with ammonia uptake, nitrification (conversion of hydroxylamine to ammonia), and nitrogen fixation in rhizosphere soil of pepper plants. Besides the genes linked to nitrogen fixation, the AS application also significantly upregulated genes associated with assimilatory nitrate reduction, nitrate uptake, and nitrite uptake (Fig. 3 a). The 16S rRNA sequencing analysis showed that the A application significantly increased the OTU number and relative abundance of bacteria involved in the nitrate reduction, whereas the AS application significantly increased the OTU number and relative abundance of bacteria involved in nitrogen fixation (Fig. S14). Metagenomic analysis showed that the AS application increased the abundances of Bradyrhizobium sp., Sphingomonas panaciterrae , Azospirillum sp., and Trichormus azotica by 144.20%, 712.03%, 116.76%, and 170.96%, respectively ( P < 0.05; Fig. 4 b). Partial least squares path modeling (PLS-PM) analysis revealed that Scenedesmus sp. positively affected soil nitrogen cycling by influencing prokaryotic microbial community in the rhizosphere, thereby promoting pepper plant growth and increasing pepper yield (Fig. 4 c). Scenedesmus sp. enriches nitrogen-fixing bacteria by secreting L-glutamic acid Three nitrogen-fixing bacteria were isolated from the rhizosphere soil after AS application and identified as Methylobacterium sp., Bradyrhizobium japonicum , and Sphingomonas sp. (Fig. S15). Pot experiments showed that the A application increased plant height, stem diameter, leaf length, and leaf width of peppers by 19.34%, 9.66%, 26.14%, and 23.08%, respectively ( P < 0.05; Fig. S16). The AS application increased plant height, stem diameter, leaf length, and leaf width of peppers by 71.70%, 37.28%, 71.59%, and 59.61%, respectively ( P < 0.05). By contrast, the combined application of Scenedesmus sp. and three nitrogen-fixing bacteria increased plant height, stem diameter, leaf length, and leaf width by 117.45%, 52.27%, 111.36%, and 115.38%, displaying the strongest growth-promoting effects ( P < 0.05). The filtrate and EPS of A. azotica and the filtrate of Scenedesmus sp. exhibited no effect on the growth of nitrogen-fixing bacteria. However, the addition of Scenedesmus sp. EPS advanced the stationary phases of MS, BJ, and SS by 6, 3, and 3 h, respectively (Fig. S17a-c). To identify which substances promoted the growth of nitrogen-fixing bacteria, a comprehensive analysis of the EPS and filtrate of Scenedesmus sp. was conducted. A total of 16 components were identified in the EPS of Scenedesmus sp. and absent in the filtrate (Fig. 5 a, Tab. S15). The top three substances (V-pyrro/NO, L-glutamic acid, and 2,5-Anhydro-1-deoxy-1-[(1-hydroxy-2-butanyl)amino]-D-mannitol) with the highest concentrations were selected to test their ability to promote nitrogen-fixing bacteria growth. The results showed that L-glutamic acid advanced thee stationary phase of three nitrogen-fixing bacteria by 3–6 h, significantly increased the colony diameters of three bacteria on solid media, and markedly accelerated their growth rates (Fig. 5 b-e). These results confirmed that Scenedesmus sp. secreted L-glutamic acid to enrich nitrogen-fixing bacteria. The single application of nitrogen-fixing bacteria increased the plant height, stem diameter, leaf length, and leaf width of pepper plants by 26.06%, 28.86%, 15.76%, and 29.88%, respectively ( P < 0.05; Fig. 6 b-c). The additional supplementation with L-glutamic acid or Scenedesmus sp. EPS further increased pepper plant height, stem diameter, leaf length, and leaf width by 14.77–17.72%, 15.06–17.18%, 6.43–7.30%, and 11.60–11.79%, respectively ( P < 0.05). The single application of nitrogen-fixing bacteria increased the chlorophyll content, maximum photosynthetic efficiency, and actual photosynthetic efficiency of pepper leaves by 65.34%, 2.26% and 27.23%, respectively ( P < 0.05; Fig. 6 g-i). The additional supplementation with L-glutamic acid or Scenedesmus sp. EPS further increased the chlorophyll content, maximum photosynthetic efficiency, and actual photosynthetic efficiency of pepper leaves by 12.63–17.00%, 4.67–6.20%, and 3.13–4.03%, respectively ( P < 0.05). Discussion This study found that the single application of A. azotica increased the yield of pepper in uncultivated and cultivated soils by 36.42% and 26.91%, respectively. Conversely, the combined application of A. azotica and Scenedesmus sp. increased the pepper yield by 107.87% and 48.05%, respectively. Thus, introducing Scenedesmus sp. further enhanced pepper yield. Previous study reported that A. azotica promoted rice growth in barren and fertile soils and increased the yield by 38.00% and 107.00%, respectively (Li et al., 2025 ). Scenedesmus sp. also enhanced the growth of tomatoes ( Solanum lycopersicum ) and increased the yield by 32.05% (Silambarasan et al., 2021 ). It was reported that the combined application of cyanobacteria and green algae exerted strong growth-promoting and yield-enhancing effects (Gonçalves 2021 ), and the combined application of A. azotica and Chlorella pyrenoidosa increased wheat yield by 21.4%, which was 7.2–9.8% higher than that in treatments with single microalgal species (Do et al., 2019). Therefore, the combined application of A. azotica and Scenedesmus sp. might be an effective approach for enhancing crop yield. Soil physicochemical properties could directly affect plant growth and yield. The RDA and Mantel test results revealed that pepper yield was primarily influenced by soil AN content, urease activity, and sucrase activity. The A application increased soil AN content and sucrase activity by 7.57% and 5.36%, respectively, with no significant impact on soil urease activity. The AS application increased soil AN content, urease activity, and sucrase activity by 11.98%, 2.73%, and 9.63%, respectively. The improvement of microalgae on soil physicochemical properties might be linked to their nitrogen-fixing and phosphate-solubilizing functions (Bhardwaj et al., 2014 ; Singh et al., 2016 ). Nitrogen-fixing microalgae were significantly positively correlated with the soil AN content (Li et al., 2024 ; Zhang et al., 2024 ), and A. azotica increased soil AN content by 16.50–51.00% through nitrogen fixation (Alvarez et al., 2021; Liu et al., 2025 ). However, studies on the function of Scenedesmus sp. in increasing soil AN content have not yet been reported. Microalgae could stimulate the growth and metabolic activity of soil microbial communities by releasing various extracellular metabolites and providing easily degradable organic matter. This process would considerably enhance the activity of key enzymes and promote soil nutrient cycling and transformation efficiency (Dai et al., 2018 ; Alvarez et al., 2021). For example, green algae could increase urease activity by 2.4 times (Barone et al., 2019 ), and inoculating Scenedesmus sp. into the soil significantly enhanced soil sucrase and urease activities (Zhang et al., 2024 ). In this study, both Scenedesmus sp. and A. azotica significantly reduced the relative abundance of Candidatus Udaeobacter , while introducing Scenedesmus sp. also significantly decreased the relative abundance of RB41 . C. Udaeobacter is an acidophilic microorganism that exhibits a significant negative correlation with soil pH (Willms et al., 2021 ), and RB41 expression is significantly negatively correlated with SOM and AN content (Liu et al., 2023; Shi et al., 2023 ). Therefore, the decrease in the relative abundances of C. Udaeobacter and RB41 were likely attributed to the capacity of A. azotica and Scenedesmus sp. to increase SOM content and pH, with Scenedesmus sp. addition leading to a substantial increase in AN content. The A. azotica application made eukaryotic microorganisms Hormotilopsis , Chlorosarcinopsis , and Cryothecomonas become the core microbial constituents. Hormotilopsis and Chlorosarcinopsis are soil green algae (Flechtner et al., 2008 ; Novakovskaya et al., 2023 ), and the increased abundance of these green algae might be attributed to the alterations in the soil environment conditions induced by A. azotica (Suleiman et al., 2020 ). In addition, introducing Scenedesmus sp. enriched Flavisolibacter, Sphingomonas , Methylobacterium , Tolypothrix , and Microcoleus , establishing Microcoleus and Gemmatimonas as the core microorganisms. Flavisolibacter , Sphingomonas , Methylobacterium , and Gemmatimonas are plant growth-promoting bacteria with nitrogen-fixing function (Desnoues et al., 2003 ; Jiao et al., 2024 ; Sultana et al., 2024 ) and Tolypothrix and Microcoleus are nitrogen-fixing cyanobacteria (Alvarez et al., 2021). Introducing Scenedesmus sp. also significantly upregulated the genes associated with nitrogen fixation and increased the abundances of Bradyrhizobium sp., Sphingomonas panaciterrae , Azospirillum sp., and Trichormus azotica by 144.20%, 712.03%, 116.76%, and 170.96%, respectively. However, the A. azotica application did not affect the abundance of these nitrogen-fixing bacteria. It was reported that Bradyrhizobium sp. fixed nitrogen in crop roots and increase the nitrogen content in the rhizosphere soil of crops by 13.36–16.82% (Terakado-Tonooka et al., 2013 )d panaciterrae promoted plant growth by enhancing the AN content in plant roots by 18.65–46.15% (Sultana et al., 2024 ). Azospirillum sp. and T. azotica also had nitrogen-fixing functions and increased soil AN content by 12.25% and 10.20–14.30%, respectively (Gladkikh et al., 2008 ; Cassán et al., 2016). SEM model also showed that Scenedesmus sp. promoted nitrogen cycling in the soil by influencing rhizosphere prokaryotic microbial communities, thereby promoting plant growth and pepper yields. Thus, Scenedesmus sp. increased soil AN content by enriching nitrogen-fixing bacteria, promoted the absorption and utilization of soil nitrogen by the roots, and ultimately enhanced pepper growth and yield. Studies have shown that microalgae could enrich microorganisms with specific functions by producing certain substances (Thompson et al., 2012 ). In this study, three nitrogen-fixing bacteria were isolated from the rhizosphere soil in the AS application group, and the enrichment effect of Scenedesmus sp. EPS on these bacteria was further verified. To identify the specific components of EPS responsible for this effect remain unclear, a comprehensive spectral analysis of the EPS produced by Scenedesmus sp. were conducted. The results revealed high levels of L-glutamic acid, 2,5-Anhydro-1-deoxy-1-[(1-hydroxy-2-butanyl)amino]-D-mannitol, and V-pyrro/NO. Previous studies found that introducing trace amounts of L-glutamic acid could reshape the plant microbiome (Kim et al., 2021 ; Wang et al., 2020 ). This study also found that introducing Scenedesmus sp. EPS and L-glutamic acid at concentrations equivalent to those of EPS significantly promoted the growth of these three nitrogen-fixing bacteria, with no significant differences between the two treatments. L-glutamic acid served as a nitrogen source for nitrogen-fixing bacteria and might act as a precursor for synthesizing essential amino acids, enzymes and other proteins, thereby promoting their growth (Rehm et al., 2010 ). These findings suggest that Scenedesmus sp. enriched nitrogen-fixing bacteria through L-glutamic acid secretion. Soil elements must be absorbed and utilized by plants to manifest their effects. The A. azotica application increased nitrogen content in the roots, stems, and leaves of pepper plants by 26.95%, 25.58%, and 12.54%, respectively. The combined application of A. azotica and Scenedesmus sp. increased nitrogen content in the roots, stems, and leaves by 64.99%, 48.84%, and 29.15%, respectively. Studies have shown that microalgal biofertilizers derived from high-rate microalgal ponds dominated by green algae significantly increased the nitrogen content of maize plants, and nitrogen-fixing cyanobacteria enhanced nitrogen content in crop roots (De et al., 2021). Further analysis revealed that the A. azotica application upregulated the expression of genes related to nitrate and nitrite absorption (gene-LOC107848172, gene-LOC107875603, and gene-LOC107875600) and the gene (gene-LOC107870135) responsible for synthesizing L-glutamine from ammonium nitrogen during nitrogen metabolism in pepper roots. The expression of genes (gene-LOC107841845 and gene-LOC107870135) involved in synthesizing L-glutamine from ammonium nitrogen was further upregulated after introducing Scenedesmus sp. Moreover, the expression of the genes responsible for converting nitrate to nitrite and nitrite to ammonium nitrogen (gene-LOC107867964, gene-LOC107853280, and gene-LOC107840295) was significantly upregulated. Nitrogen absorbed by plants from the soil is initially converted to ammonium nitrogen, which is subsequently assimilated into glutamine and glutamate via the glutamine synthetase/glutamate synthase pathway. Glutamine and glutamate are essential precursors for synthesizing various plant proteins, promoting cell division, and accelerating plant growth (Ruiz et al., 2000 ; Tabuchi et al., 2007). The increase in available soil nitrogen content might significantly upregulate the expression of key genes in crop roots, including transporter protein genes (e.g., NRT1 and NRT2 families) responsible for nitrate and nitrite absorption, nitrate reductase (NR), nitrite reductase (NiR) genes involved in nitrate reduction, and glutamine synthetase (GS) and glutamate synthase (GOGAT) genes that mediate ammonium assimilation pathways (Wang et al., 2021 ). This coordinated regulatory mechanism of gene expression enabled crops to absorb, convert and assimilate nitrogen from the soil more efficiently, thereby improving nitrogen-use efficiency and promoting plant growth and crop yields. Conclusions In summary, Scenedesmus sp. significantly increased the abundance of nitrogen-fixing bacteria in the rhizosphere soil of pepper plants by secreting L-glutamic acid. These nitrogen-fixing bacteria further increased the soil AN content, which subsequently upregulated the expression of genes in pepper roots responsible for converting nitrate to nitrite, nitrite to ammonium nitrogen, and ammonium nitrogen to L-glutamine. Finally, the improved absorption and utilization of nitrogen by the roots boosted pepper growth and yield. This study provided important scientific evidence for the development and utilization of microalgae-based biofertilizer to improve crop yields in the sustainable agriculture. Declarations Competing interests The authors declare no competing interests. Author contributions L.C. and W.H.Q.: conceived and designed the experiments, performed software analysis, formal analysis, data curation, methodology, and wrote the first version of the manuscript. X.Y.W. and S.Z.H.: performed methodology, project administration, visualization, validation, and conducted the experiments. C.J.: performed investigation and project administration. W.J.: provided resources, supervised the project, administered the study, validated the research, and contributed to writing—review and editing. All authors discussed the data, contributed to the writing and reviewing of manuscript drafts. All authors reviewed and approved the submitted version. Acknowledgements We acknowledge the financial support from the Scientific Research Project of China Metallurgical Geology Bureau (CMGBKY202501). Availability of data and materials The raw sequencing data of 16S rRNA and 18S rRNA in this study have been deposited in the NCBI Sequence Read Archive (SRA) under accession number PRJNA1268602. The raw metagenomic data have been deposited in the NCBI SRA under accession number PRJNA1273704, and the raw transcriptome data have been deposited in the NCBI SRA under accession number PRJNA1273509. References Barone V et al (2019) Effect of living cells of microalgae or their extracts on soil enzyme activities. Arch Agron Soil Sci 65:712–726. https://doi.org/10.1080/03650340.2018.1521513 Bello AS et al (2021) Enhancement in bell pepper ( Capsicum annuum L.) plants with application of Roholtiella sp. ( Nostocales ) under soilless cultivation. Agronomy 11:1624. https://doi.org/10.3390/agronomy11081624 Bhardwaj D et al (2014) Biofertilizers function as key player in sustainable agriculture by improving soil fertility, plant tolerance and crop productivity. Microb Cell Fact 13:1–10. https://doi.org/10.1186/1475-2859-13-66 Bokulich NA et al (2013) Quality-filtering vastly improves diversity estimates from Illumina amplicon sequencing. Nat Methods 10:57–59. https://doi.org/10.1038/nmeth.2276 Cassán F, Diaz-Zorita M (2016) Azospirillum sp. in current agriculture: From the laboratory to the field. Soil Biol Biochem 103:117–130. https://doi.org/10.1016/j.soilbio.2016.08.020 Dai Z et al (2018) Long-term nitrogen fertilization decreases bacterial diversity and favors the growth of Actinobacteria and Proteobacteria in agro-ecosystems across the globe. Glob. Change Biol. 24, 3452–3461. https://doi.org/10.1111/gcb.14163 De Paula Pereira ASA et al (2021) Organomineral fertilizers pastilles from microalgae grown in wastewater: Ammonia volatilization and plant growth. Sci Total Environ 779:146205. https://doi.org/10.1016/j.scitotenv.2021.146205 Deepika P, MubarakAli D (2020) Production and assessment of microalgal liquid fertilizer for the enhanced growth of four crop plants. Biocatal Agric Biotechnol 28:101701. https://doi.org/10.1016/j.bcab.2020.101701 Desnoues N et al (2003) Nitrogen fixation genetics and regulation in a Pseudomonas stutzeri strain associated with rice. Microbiology 149:2251–2262. https://doi.org/10.1099/mic.0.26270-0 Dineshkumar R et al (2019) The impact of using microalgae as biofertilizer in maize ( Zea mays L). Waste Biomass Valoriz 10:1101–1110. https://doi.org/10.1007/s12649-017-0123-7 Edgar RC (2013) UPARSE: highly accurate OTU sequences from microbial amplicon reads. Nat Methods 10:996–998. https://doi.org/10.1038/nmeth.2604 Feng J et al (2024) Geologically younger ecosystems are more dependent on soil biodiversity for supporting function. Nat Commun 15:4141. https://doi.org/10.1038/s41467-024-48289-y Flechtner VR, Johansen JR, Belnap J (2008) The biological soil crusts of the San Nicolas Island: Enigmatic algae from a geographically isolated ecosystem. West North Am Naturalist 68:405–436. https://doi.org/10.3398/1527-0904-68.4.405 Gao Y et al (2022) Metagenomics and network analysis elucidating the coordination between fermentative bacteria and microalgae in a novel bacterial-algal coupling reactor (BACR) for mariculture wastewater treatment. Water Res 215:118256. https://doi.org/10.1016/j.watres.2022.118256 Gladkikh A et al (2008) Nitrogen-fixing cyanobacterium Trichormus variabilis of the Lake Baikal phytoplankton. Microbiology 77:726–733. https://doi.org/10.1134/S0026261708060118 Gonçalves AL (2021) The use of microalgae and cyanobacteria in the improvement of agricultural practices: a review on their biofertilising, biostimulating and biopesticide roles. Appl Sci 11:871. https://doi.org/10.3390/app11020871 Guo S et al (2024) Predatory protists impact plant performance by promoting plant growth-promoting rhizobacterial consortia. ISME J 18:wrae180. https://doi.org/10.1093/ismejo/wrae180 Haas BJ et al (2011) Chimeric 16S rRNA sequence formation and detection in Sanger and 454-pyrosequenced PCR amplicons. Genome Res 21:494–504. https://doi.org/10.1101/gr.112730.110 Hartmann M et al (2015) Distinct soil microbial diversity under long-term organic and conventional farming. ISME J 9:1177–1194. https://doi.org/10.1038/ismej.2014.210 Jiao L et al (2024) Dynamic microbial regulation of triiron tetrairon phosphate nanomaterials in the tomato rhizosphere. Environ Sci -Nano 11:1157–1169. https://doi.org/10.1039/D3EN00797A Kapoore RV, Wood EE, Llewellyn CA (2021) Algae biostimulants: A critical look at microalgal biostimulants for sustainable agricultural practices. Biotechnol Adv 49:107754. https://doi.org/10.1016/j.biotechadv.2021.107754 Kim DR et al (2021) Glutamic acid reshapes the plant microbiota to protect plants against pathogens. Microbiome 9:1–18. https://doi.org/10.1186/s40168-021-01186-8 Lebrun JD et al (2012) Assessing impacts of copper on soil enzyme activities in regard to their natural spatiotemporal variation under long-term different land uses. Soil Biol Biochem 49:150–156. https://doi.org/10.1016/j.soilbio.2012.02.027 Li C et al (2024) The influence of microalgae fertilizer on soil water conservation and soil improvement: Yield and quality of potted tomatoes. Agronomy 14:2102. https://doi.org/10.3390/agronomy14092102 Li S et al (2025) Enhancement of rice production and soil carbon sequestration utilizing nitrogen-fixing cyanobacteria. Appl Soil Ecol 207:105940. https://doi.org/10.1016/j.apsoil.2025.105940 Li Z et al (2023) Marine toxin domoic acid alters nitrogen cycling in sediments. Nat Commun 14:7873. https://doi.org/10.1038/s41467-023-43265-4 Liu G et al (2023b) Effects of thaw slump on soil bacterial communities on the Qinghai-Tibet Plateau. CATENA 232:107342. https://doi.org/10.1016/j.catena.2023.107342 Liu J et al (2025) Application of microalgae in remediation of heavy metal-contaminated soils and its stimulatory effect on wheat growth. Algal Res 88:103995. https://doi.org/10.1016/j.algal.2025.103995 Love MI, Huber W, Anders S (2014) Moderated estimation of fold change and dispersion for RNA-seq data with DESeq2. Genome Biol 15:550. https://doi.org/10.1186/s13059-014-0550-8 Martre P et al (2024) Global needs for nitrogen fertilizer to improve wheat yield under climate change. Nat Plants 10:1081–1090. https://doi.org/10.1038/s41477-024-01739-3 Muller A et al (2017) Strategies for feeding the world more sustainably with organic agriculture. Nat commun 8:1–13. https://doi.org/10.1038/s41467-017-01410-w Nosengo N (2003) Fertilized to death. Nature 425:894–896. https://doi.org/10.1038/425894a Novakovskaya IV et al (2023) Heterochlamydomonas uralensis sp. Nov.( Chlorophyta , Chlamydomonadaceae ), new species described from the mountain tundra community in the Subpolar Urals (Russia). Diversity 15:673. https://doi.org/10.3390/d15050673 Quast C et al (2012) The SILVA ribosomal RNA gene database project: improved data processing and web-based tools. Nucleic Acids Res 41:D590–D596. https://doi.org/10.1093/nar/gks1219 Rodrigues S et al (2024) Effect of a zinc phosphate shell on the uptake and translocation of foliarly applied ZnO nanoparticles in pepper plants ( Capsicum annuum ). Environ Sci Technol 58:3213–3223. https://doi.org/10.1021/acs.est.3c08723 Reichert JM et al (2023) Soil morphological, physical and chemical properties affecting Eucalyptus spp. productivity on Entisols and Ultisols[J]. Soil Tillage Res 226:105563. https://doi.org/10.1016/j.still.2022.105563 Rehm N et al (2010) L-Glutamine as a nitrogen source for Corynebacterium glutamicum : derepression of the AmtR regulon and implications for nitrogen sensing. Microbiology 156:3180–3193. https://doi.org/10.1099/mic.0.040667-0 Ruiz JM, Castilla N, Romero L (2000) Nitrogen metabolism in pepper plants applied with different bioregulators. J Agric Food Chem 48:2925–2929. https://doi.org/10.1021/jf990394h Shen C et al (2024) Comparative transcriptome analysis and Arabidopsis thaliana overexpression reveal key genes associated with cadmium transport and distribution in root of two Capsicum annuum cultivars. J Hazard Mater 465:133365. https://doi.org/10.1016/j.jhazmat.2023.133365 Shi B et al (2023) Effects of the polyhalogenated carbazoles 3-bromocarbazole and 1, 3, 6, 8-tetrabromocarbazole on soil microbial communities. Environ Res 239:117379. https://doi.org/10.1016/j.envres.2023.117379 Singh JS et al (2016) Cyanobacteria: a precious bio-resource in agriculture, ecosystem, and environmental sustainability. Front Microbiol 7:529. https://doi.org/10.3389/fmicb.2016.00529 Silambarasan S et al (2021) Removal of nutrients from domestic wastewater by microalgae coupled to lipid augmentation for biodiesel production and influence of deoiled algal biomass as biofertilizer for Solanum lycopersicum cultivation. Chemosphere 268:129323. https://doi.org/10.1016/j.chemosphere.2020.129323 Spohn M, Braun S, Sierra CA (2023) Continuous decrease in soil organic matter despite increased plant productivity in an 80-years-old phosphorus-addition experiment. Commun Earth Environ 4:251. https://doi.org/10.1038/s43247-023-00915-1 Solomon W et al (2023) Potential benefit of microalgae and their interaction with bacteria to sustainable crop production. Plant Growth Regul 101:53–65. https://doi.org/10.1007/s10725-023-01019-8 Suleiman AKA et al (2020) From toilet to agriculture: Fertilization with microalgal biomass from wastewater impacts the soil and rhizosphere active microbiomes, greenhouse gas emissions and plant growth. Resour Conserv Recycl 161:104924. https://doi.org/10.1016/j.resconrec.2020.104924 Sultana R et al (2024) Sphingomonas panaciterrae PB20 increases growth, photosynthetic pigments, antioxidants, and mineral nutrient contents in spinach ( Spinacia oleracea L). Heliyon 10:25596. https://doi.org/10.1016/j.heliyon.2024.e25596 Terakado-Tonooka J, Fujihara S, Ohwaki Y (2013) Possible contribution of Bradyrhizobium on nitrogen fixation in sweet potatoes. Plant Soil 367:639–650. https://doi.org/10.1007/s11104-012-1495-x Thompson AW et al (2012) Unicellular cyanobacterium symbiotic with a single-celled eukaryotic alga. Science 337:1546–1550. https://doi.org/10.1126/science.1222700 Uysal O, Uysal FO, Ekinci K (2015) Evaluation of microalgae as microbial fertilizer. Eur J Sustain Dev 4:77. https://doi.org/10.14207/ejsd.2015.v4n2p77 Van Dijk M et al (2021) A meta-analysis of projected global food demand and population at risk of hunger for the period 2010–2050. Nat Food 2:494–501. https://doi.org/10.1038/s43016-021-00322-9 Vasconcelos Fernandes T et al (2015) Closing domestic nutrient cycles using microalgae. Environ Sci Technol 49:12450–12456. https://doi.org/10.1021/acs.est.5b02858 Wang Q et al (2007) Naive Bayesian classifier for rapid assignment of rRNA sequences into the new bacterial taxonomy. Appl Environ Microbiol 73:5261–5267. https://doi.org/10.1128/AEM.00062-07 Wang C et al (2021) Comparative transcriptome analysis reveals different low-nitrogen-responsive genes in pepper cultivars. Horticulturae 7:110. https://doi.org/10.3390/horticulturae7050110 Wang X et al (2019) An integrated approach to characterize intestinal metabolites of four phenylethanoid glycosides and intestinal microbe-mediated antioxidant activity evaluation in vitro using UHPLC-Q-Exactive High-Resolution Mass Spectrometry and a 1, 1-Diphenyl-2-picrylhydrazyl-based assay. Front Pharmacol 10:826. https://doi.org/10.3389/fphar.2019.00826 Wang YF et al (2020) L-glutamic acid induced the colonization of high-efficiency nitrogen-fixing strain Ac63 ( Azotobacter chroococcum ) in roots of Amaranthus tricolor. Plant Soil 451:357–370. https://doi.org/10.1007/s11104-020-04531-2 Wei G et al (2020) Similar drivers but different effects lead to distinct ecological patterns of soil bacterial and archaeal communities. Soil Biol Biochem 144:107759. https://doi.org/10.1016/j.soilbio.2020.107759 Weissberg O, Aharonovich D, Sher D (2023) Phototroph-heterotroph interactions during growth and long-term starvation across Prochlorococcus and Alteromonas diversity. ISME J 17:227–237. https://doi.org/10.1038/s41396-022-01330-8 Willms IM et al (2021) The ubiquitous soil verrucomicrobial clade ‘ Candidatus Udaeobacter ’shows preferences for acidic pH. Environ Microbiol Rep 13:878–883. https://doi.org/10.1111/1758-2229.13006 Wu Y et al (2013) Development of artificially induced biological soil crusts in fields and their effects on top soil. Plant Soil 370:115–124. https://doi.org/10.1007/s11104-013-1611-6 Xu F et al (2022) Coordination of root auxin with the fungus Piriformospora indica and bacterium Bacillus cereus enhances rice rhizosheath formation under soil drying. ISME J 16:801–811. https://doi.org/10.1038/s41396-021-01133-3 Zhang C et al (2024) Soil health improvement by inoculation of indigenous microalgae in saline soil. Environ Geochem Health 46:23. https://doi.org/10.1007/s10653-023-01790-7 Zhou Y et al (2022) Long-term fertilizer postponing promotes soil organic carbon sequestration in paddy soils by accelerating lignin degradation and increasing microbial necromass. Soil Biol Biochem 175:108839. https://doi.org/10.1016/j.soilbio.2022.108839 Zhou Y et al (2024) Microalgal extracellular polymeric substances (EPS) and their roles in cultivation, biomass harvesting, and bioproducts extraction. Bioresour Technol 406:131054. https://doi.org/10.1016/j.biortech.2024.131054 Supplementary Files Supplementarymaterial.docx Cite Share Download PDF Status: Under Review Version 1 posted Reviewers invited by journal 03 May, 2026 Editor invited by journal 28 Apr, 2026 Editor assigned by journal 28 Apr, 2026 First submitted to journal 27 Apr, 2026 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-9526545","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":633706613,"identity":"2d4a9248-ed93-4a05-b7b3-da76a853a97c","order_by":0,"name":"Cong Liu","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Cong","middleName":"","lastName":"Liu","suffix":""},{"id":633706614,"identity":"fde5c45c-af59-4d23-969a-205d3ec0692b","order_by":1,"name":"Hongqing Wei","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Hongqing","middleName":"","lastName":"Wei","suffix":""},{"id":633706615,"identity":"3f709a46-3ecd-4f43-92fb-fee99b71aa94","order_by":2,"name":"Yuwei Xing","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Yuwei","middleName":"","lastName":"Xing","suffix":""},{"id":633706616,"identity":"e41d5945-fbb8-471a-8c34-8067de7f3a3e","order_by":3,"name":"Zhonghua Shen","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Zhonghua","middleName":"","lastName":"Shen","suffix":""},{"id":633706617,"identity":"4bee07ec-4a84-46b4-805a-58388914dace","order_by":4,"name":"Jie Cao","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Jie","middleName":"","lastName":"Cao","suffix":""},{"id":633706618,"identity":"f3bec55f-5b2c-4891-90bf-08aeecf91c7f","order_by":5,"name":"Jun Wang","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAzElEQVRIiWNgGAWjYLACCQYGOQiLjQQtxiRqAYLEBqK1GBw/e/iFZdud9H6J5AcMH8oOM/DPbiCg5UxemoVk27PcmTPSDBhnnDvMIHHnAH4tZgdyzAwk2w7nbriRw8DM23aYwUAigYCW82/AWtINQFr+EqXlRo7xA6CWBLAWRmK02N94Y8Ygce6w4cyeZwYHe86l80jcIKBFsj/H+LNE2WF5fvbkhw9+lFnL8c8goAUI2KQloKwDQMxDUD0QMH/8QIyyUTAKRsEoGLkAAG3NQ1JUflIWAAAAAElFTkSuQmCC","orcid":"","institution":"Ocean University of China","correspondingAuthor":true,"prefix":"","firstName":"Jun","middleName":"","lastName":"Wang","suffix":""}],"badges":[],"createdAt":"2026-04-25 14:53:06","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-9526545/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-9526545/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":109067827,"identity":"123068cf-737c-4b44-a890-2c9c4a701bc4","added_by":"auto","created_at":"2026-05-12 10:01:25","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":586824,"visible":true,"origin":"","legend":"\u003cp\u003eEffects of microalgae \u003cem\u003eAnabaena azotica\u003c/em\u003e alone (A) and in combination with \u003cem\u003eScenedesmus\u003c/em\u003esp. (AS) on pepper growth, yield, and rhizosphere soil properties (\u003cem\u003en\u003c/em\u003e= 6). (a) Pepper plants and (b) fruitsfrom different groups. Comparison of plant height (c), stem diameter (d), leaf width (e), leaf length (f), and fruit yield(g) of pepper plants at different time points, and available nitrogen (AN; (h), available phosphorus (AP; i), available potassium (AK; j), urease activity (UE; k), sucrase activity (SC; l), acid phosphatase activity (ACP; m), and organic matter (OM; n) content in the rhizosphere soil. (o) Redundancy analysis (RDA) of pepper growth and yield with soil physicochemical properties. Colored dots and triangles represented pepper growth and yield, while vectors represented soil physicochemical properties. (p) Mantel test analysis of pepper growth and yield with soil physicochemical properties (Spearman’s correlation coefficients). Mantel’s r statistics were indicated by line width, and line color representedstatistical significance. Solid lines indicated positive correlations, while dashed lines indicated negative correlations. POD, Peroxidase activity. Data in figures (c-n) were presented as mean ± SD, and different letters indicated significant differences at \u003cem\u003eP\u003c/em\u003e\u0026lt; 0.05 (one-way ANOVA corrected by Duncan's test).\u003c/p\u003e","description":"","filename":"image1.png","url":"https://assets-eu.researchsquare.com/files/rs-9526545/v1/6a2352c3a12e6c2bdf279e00.png"},{"id":109009760,"identity":"1b04b5c0-81f2-4a73-a25a-00641ac02343","added_by":"auto","created_at":"2026-05-11 16:16:51","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":493617,"visible":true,"origin":"","legend":"\u003cp\u003eEffects of microalgae \u003cem\u003eAnabaena azotica\u003c/em\u003e alone (A) and in combination with \u003cem\u003eScenedesmus\u003c/em\u003esp. (AS) on nutrient uptake and gene expression in pepper roots. (a–c) Nitrogen and (d–f) phosphorus contents in roots, stems, and leaves at different time points (\u003cem\u003en\u003c/em\u003e = 6). Data were presented as mean ±SD, and different letters indicate significant differences at \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05 (one-way ANOVA corrected by Duncan's test). (g) Venn diagram showing overlaps of differentially expressed genes (DEGs) among groups. (h) Volcano plot,(i) GO and (j) KEGG enrichment analyses of differentially expressed genes between the control groupand the AS group. Each point represented a specific gene, with red points indicating significantly upregulated genes, green points indicating significantly downregulated genes, and gray points representing non-significant genes. The dots size represented the number of genes/transcripts in the GO Term, and the color of the dots corresponded to different Padjust ranges (only the top 20 enrichment results are shown). (k) Differentially expressed genes in the nitrogen metabolism process of pepper roots between the control group and the AS group. The color intensity indicated the degree of change relative to the control group, and deeper color indicated greater fold-change(g-k, \u003cem\u003en\u003c/em\u003e = 6).\u003c/p\u003e","description":"","filename":"image2.png","url":"https://assets-eu.researchsquare.com/files/rs-9526545/v1/c2405284dc7a8568071dc774.png"},{"id":109009757,"identity":"6e1d04f5-9660-49b5-853b-50a7e7df1940","added_by":"auto","created_at":"2026-05-11 16:16:51","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":472696,"visible":true,"origin":"","legend":"\u003cp\u003eEffectsof microalgae \u003cem\u003eAnabaena azotica\u003c/em\u003e alone (A) and in combination with \u003cem\u003eScenedesmus\u003c/em\u003esp. (AS) on pepper rhizosphere prokaryotic microbial communities.(a) Principal Coordinate Analysis (PCoA) basedBray-Curtis dissimilarity matrix (OTU level); (b) Relative abundance percentages; (c) Statistical Analysis of Metagenomic Profiles (STAMP) analysis of the top 20 genera of prokaryotic microorganisms. The \u003cem\u003eP\u003c/em\u003e values were calculated using a t-test, with a significance threshold of \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05 and a confidence interval of 95%. (d) Linear Discriminant Analysis Effect Size (LEfSe) analysis of prokaryotic microorganisms (genus level, Kruskal-Wallis test, \u003cem\u003eP\u003c/em\u003e\u0026lt; 0.05 and LDA ≥ 3). (e) Core microbial network analysis of the top 20 genera of prokaryotic microorganisms.The nodes size indicated absolute abundance, and the nodes color represented the degree. Red lines indicated positive correlations, while green lines indicated negative correlations. The network structure pattern was concentric, and the connection mode was haystack.\u003c/p\u003e","description":"","filename":"image3.png","url":"https://assets-eu.researchsquare.com/files/rs-9526545/v1/e7c21b07f02638ca687d88fb.png"},{"id":109009763,"identity":"6d1a977c-bc67-4573-8113-b36416dd042a","added_by":"auto","created_at":"2026-05-11 16:16:51","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":198458,"visible":true,"origin":"","legend":"\u003cp\u003eMechanism of \u003cem\u003eScenedesmus \u003c/em\u003esp.-enhanced nitrogen cycling via microbial recruitment. (a) Gene abundance of nitrogen cycling processes in the rhizosphere soil of pepper under the different treatment groups. The color gradient represented the fold change of genes. (b) Changes in the abundance of microorganisms involved in the nitrogen fixation process in the rhizosphere soil of pepper under the different treatment groups. The blue indicated downregulation, red indicated upregulation, and the intensity of the color represented the degree of change relative to the control group; the size of the circles represented the number of OTUs. (c) Structural equation model (SEM) testing the effects of microalgae on pepper yield. The thickness of the lines was proportional to the coefficient size, and GOF represented the overall explanatory power of the model.\u003c/p\u003e","description":"","filename":"image4.png","url":"https://assets-eu.researchsquare.com/files/rs-9526545/v1/6d2d31b59ca65c1642551cb6.png"},{"id":109009762,"identity":"98e9e8b0-b7c0-4a68-b008-ad1a410505ce","added_by":"auto","created_at":"2026-05-11 16:16:51","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":986736,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003eScenedesmus\u003c/em\u003e sp. promoted the growth of three nitrogen-fixing bacteria through the secretion of L-glutamic acid. (a) Components of EPS and filtrate of \u003cem\u003eScenedesmus\u003c/em\u003esp.. Growth curves of (b) \u003cem\u003eMethylobacterium\u003c/em\u003e sp., (c) \u003cem\u003eSphingomonas\u003c/em\u003e sp., and (d) \u003cem\u003eB. japonicum\u003c/em\u003e after adding EPS components L-glutamic acid (LGA), 2,5-Anhydro-1-deoxy-1-[(1-hydroxy-2-butanyl)amino]-D-mannitol (DM), and V-pyrro/NO (VP). (e) Growth-promoting effects of L-glutamic acid and EPS of \u003cem\u003eScenedesmus\u003c/em\u003e sp. on \u003cem\u003eMethylobacterium\u003c/em\u003e sp., \u003cem\u003eSphingomonas\u003c/em\u003esp., and\u003cem\u003e B. japonicum\u003c/em\u003e.\u003c/p\u003e","description":"","filename":"image5.png","url":"https://assets-eu.researchsquare.com/files/rs-9526545/v1/d7795fe6ce2ce5fbc7702380.png"},{"id":109009758,"identity":"1cb67bb3-cfec-40ba-8394-f2f61335385a","added_by":"auto","created_at":"2026-05-11 16:16:51","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":603515,"visible":true,"origin":"","legend":"\u003cp\u003eEffects of L-glutamic acid and \u003cem\u003eScenedesmus\u003c/em\u003esp. EPS alone and in combination with nitrogen-fixing bacteria on pepper growth. (a) Pepper plants under different treatments. (b) Plant height, (c) stem diameter, (d) leaf length, (e) feaf width, and (f) fresh weight of peppers under different groups. (g) Chlorophyll content, (h) maximum photosynthetic efficiency, and (i) actual photosynthetic efficiency in pepper leaves under different groups. LGA and EPS in Figure (a) represented L-glutamic acid and extracellular polymeric substances, respectively. Data in figures (b-e) were presented as mean ±SD (n = 6), and different letters indicated significant differences at \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05 (one-way ANOVA corrected by Duncan's test).\u003c/p\u003e","description":"","filename":"image6.png","url":"https://assets-eu.researchsquare.com/files/rs-9526545/v1/b5996dc91a8524c032f58d55.png"},{"id":109069247,"identity":"9a7a8c24-1a44-4baf-8b66-bd3a349f67ba","added_by":"auto","created_at":"2026-05-12 10:21:56","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":3571369,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-9526545/v1/bedaae84-731b-46cc-b695-589fb409337d.pdf"},{"id":109009759,"identity":"7f46cfe3-019c-48cf-a25c-74fba2cdc71d","added_by":"auto","created_at":"2026-05-11 16:16:51","extension":"docx","order_by":11,"title":"","display":"","copyAsset":false,"role":"supplement","size":30648764,"visible":true,"origin":"","legend":"","description":"","filename":"Supplementarymaterial.docx","url":"https://assets-eu.researchsquare.com/files/rs-9526545/v1/aad697b101f937ef59590989.docx"}],"financialInterests":"","formattedTitle":"Soil microalga-derived L-glutamic acid enhances the growth and yield of pepper by recruiting nitrogen-fixing bacteria","fulltext":[{"header":"Introduction","content":"\u003cp\u003eBy 2050, the global population is expected to reach 9\u0026nbsp;billion, necessitating a 50% increase in agricultural output to meet the food demand (Muller et al., \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e2017\u003c/span\u003e; Van et al., 2021). To achieve this food production enhancement, the amount of fertilizer usage needs to increase by at least 4-fold, which would considerably increase agricultural production costs (Hartmann et al., \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2015\u003c/span\u003e; Martre et al., \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). The excessive fertilizer application would decrease soil pH, reduce soil organic matter (SOM) and ultimately diminish the effectiveness of yield increases (Nosengo \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2003\u003c/span\u003e; Spohn et al., \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). Thus, the enhancement of agricultural productivity should not rely solely on the increasing use of fertilizer, and other efficient approaches should be explored.\u003c/p\u003e \u003cp\u003eSoil microalgae have attracted increasing attention in recent years due to their excellent plant growth-promoting properties (Weissberg et al., \u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). Microalgae are rich in macronutrients and micronutrients that are vital for plant growth and development and produce a variety of bioactive substances to drive soil biogeochemical cycles (Vasconcelos et al., 2015; Deepika et al., 2020). For example, nitrogen-fixing cyanobacteria could form biocrusts on desert surfaces, increase soil moisture and nitrogen content, and thereby promote plant growth (Wu et al., \u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e2013\u003c/span\u003e). Microalgae from the genus \u003cem\u003eAnabaena\u003c/em\u003e increased plant height and fresh weight of peppers (\u003cem\u003eCapsicum annuum\u003c/em\u003e L.) by 8.7\u0026ndash;17.5% and 22.8\u0026ndash;51.8%, respectively (Bello et al., \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). \u003cem\u003eSpirulina platensis\u003c/em\u003e and \u003cem\u003eChlorella vulgaris\u003c/em\u003e increased the yield of maize (\u003cem\u003eZea mays\u003c/em\u003e L.) by 16.9% and 14.9%, respectively (Dineshkumar et al., \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Uysal et al., \u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). Thus, soil microalgae might serve as an important approach to enhance crop yields.\u003c/p\u003e \u003cp\u003eSoil ecosystem health and biodiversity are crucial for stable agricultural productivity (Feng et al., \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). As primary producers, soil microalgae safeguard the viability of rhizosphere soil microecosystem by providing oxygen and carbon sources (Kapoore et al., \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Solomon et al., \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). Moreover, soil microalgae secrete extracellular polymeric substances (EPS) including proteins and polysaccharides to support microbial adhesion, metabolism, and reproduction (Thompson et al., \u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e2012\u003c/span\u003e; Deepika et al., 2020). In environments where microalgae and bacteria coexist, nitrogen-fixing bacteria supply nitrogen to microalgae and utilize the dissolved organic matter (DOM) released by microalgae as a source of carbon, sulfur, and phosphorus (Thompson et al., \u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e2012\u003c/span\u003e). We hypothesized that the supplementation of microalgae might enhance soil biodiversity by producing certain substances and subsequently promote plant crop yield.\u003c/p\u003e \u003cp\u003eTo verify our hypothesis, soil microalgae were firstly isolated from the pepper plantation and then used in pot experiments and field trials to evaluate their effects on the growth and yield of peppers in both uncultivated and cultivated soils. Rhizosphere soil physicochemical properties of pepper plants at different growth stages (seedling, flowering, and maturity) were measured to analyze the impacts of the microalgae on soil properties. During the maturity stage, the nitrogen, phosphorus, and potassium contents in the roots, stems, and leaves of peppers were measued, and transcriptome sequencing of pepper roots were conducted to investigate the effects of microalgae on the absorption and utilization of nutrients by the roots. Moreover, 16S rRNA, 18S rRNA and metagenomic sequencing were performed on the rhizosphere soil of pepper plants to investigate the impacts of microalgae on microbial community structure and function. Furthermore, the main nitrogen-fixing bacteria enriched by the microalgae were isolated and used to investigate the potential mechanism through which substance microalga \u003cem\u003eScenedesmus\u003c/em\u003e sp. exerted the growth-promoting effects. This study would provide a novel approach for improving crop productivity and offer new insights into the application of soil microalgae in modern sustainable agriculture.\u003c/p\u003e"},{"header":"Materials and methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eSoil microalgae isolation and identification\u003c/h2\u003e \u003cp\u003eThe \u003cem\u003eA. azotica\u003c/em\u003e and \u003cem\u003eScenedesmus\u003c/em\u003e sp. used in this study were isolated from the soil in a pepper field in Bohu County, Bayingolin Mongol Autonomous Prefecture, Xinjiang, China (41\u0026deg;55\u0026prime;42\u0026Prime; N, 86\u0026deg;37\u0026prime;10\u0026Prime; E). Briefly, 10 g soil samples were inoculated into 50 mL of BG11 liquid medium (Tab. S2) in 100 mL flasks, which were incubated in an incubator (25\u0026thinsp;\u0026plusmn;\u0026thinsp;1\u0026deg;C, light intensity of 2700\u0026thinsp;\u0026plusmn;\u0026thinsp;50 lx, and a photoperiod of 16 h light: 8 h dark). After 3 weeks, the enriched microalgal liquid was homogenized, serially diluted from 10\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e to 10\u003csup\u003e\u0026minus;\u0026thinsp;4\u003c/sup\u003e, and spread onto BG11 solid medium. The plates were incubated in the incubator until single microalgal colonies were clearly visible. The microalgal cells were observed under the Nikon Eclipse Ti2 microscope (Fig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e), and genomic DNA was extracted for PCR amplification using 16S primers 27F-1492R (5'-AGRGTTYGATYMTGGCTCAG-3' and 5'-RGYTACCTTGTTACGACTT-3') and 18S primers EukA-EukB (5'-AACCTGGTTGCTGCCAGTGCAGT-3' and 5'-TGATCCTTGCAGGTTCACCTAC-3'). The PCR products were sequenced using Sanger sequencing (Lingen Biotechnology Co., Ltd., Shanghai, China), and the taxonomic classification of the microalgae was established through a BLASTn search of the NCBI GenBank database.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eMicroalgae cultivation\u003c/h3\u003e\n\u003cp\u003eBoth \u003cem\u003eA. azotica\u003c/em\u003e and \u003cem\u003eScenedesmus\u003c/em\u003e sp. were cultured in the BG11 medium until the stationary phase to mueaure the biomass and contents of nitrogen, phosphorus, and potassium in the microalgae using the standard methods, elemental analyzer (Flash 2000, Thermo Scientific, MA, USA) and automatic nutrient analyzer (QuAAtro, SEAL, Germany), respectively. The microalgal suspension was centrifuged at 4,000 \u003cem\u003eg\u003c/em\u003e for 3 min, resuspended in distilled water, centrifuged again to revmove the culture medium, and then used in the following experiments.\u003c/p\u003e\n\u003ch3\u003ePot experiments using uncultivated and cultivated soils\u003c/h3\u003e\n\u003cp\u003eThe uncultivated and cultivated soils were collected from Baguan Mountain and experimental field of Ocean University of China, respectively. The soil properties are presented in Tab. S4. The pepper variety \u003cem\u003eCapsicum annuum\u003c/em\u003e L. \u0026lsquo;Honglong 23\u0026rsquo; was provided by the Bazhou Green Collar Vocational Skills Training School. Pepper seedlings were raised following the protocol described by Rodrigues et al. (\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e2024\u003c/span\u003e) and transplanted into the pots at the three-true-leaf stage.\u003c/p\u003e \u003cp\u003ePot experiments using uncultivated soil and field experiment using cultivated soil were conducted to assess the effects of microalgae on pepper growth and yield (Fig. S2). Both experiments comprised three groups: CK, chemical fertilizer only; A, chemical fertilizer and \u003cem\u003eA. azotica\u003c/em\u003e at 10 g/667 m\u003csup\u003e2\u003c/sup\u003e; AS, chemical fertilizer and a mixed microalgae consisting of \u003cem\u003eA. azotica\u003c/em\u003e and \u003cem\u003eScenedesmus\u003c/em\u003e sp. at 1.92 g/667 m\u003csup\u003e2\u003c/sup\u003e at a 1:1 weight ratio. The application amount of chemical fertilizers in above three groups were 60 kg/667 m\u0026sup2; urea and 45 kg/667 m\u0026sup2; potassium dihydrogen phosphate. Each group included three replicates, and each replicate included six pots, with three plants per pot. Both chemical fertilizers and microalgae were applied in liquid form and split into four equal doses on the 1st, 7th, 14th, and 21st day after transplantation.\u003c/p\u003e\n\u003ch3\u003eMeasurement of pepper growth and yield\u003c/h3\u003e\n\u003cp\u003eOn the 26th, 58th, and 112th day post-transplantation, pepper plant height, leaf length, and leaf width (n\u0026thinsp;=\u0026thinsp;6) were measured using a measuring tape, and the stem diameter was measured using vernier caliper. Pepper yield was determined on the 112th day using the weighing method.\u003c/p\u003e\n\u003ch3\u003ePhysicochemical characterization of pepper rhizosphere soil\u003c/h3\u003e\n\u003cp\u003eOn the 112th day, 50 g rhizosphere soil tightly adhered to the roots was collected using a sterile brush and stored at ‒80\u0026deg;C for 18S rRNA, 16S rRNA and metagenomic sequencing (Xu et al., \u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). The remaining soil was air-dried in a cool place and ground for further analysis. The pH, AP content, and AK content of rhizosphere soil were measured following the methods of Hartmann et al. (\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2015\u003c/span\u003e), and the contents of SOM and AN were determined according to Reichert et al. (2012). The soil acid phosphatase and urease activities were measured using the methods of Lebrun et al. (\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2012\u003c/span\u003e), and the activities of sucrase and peroxidase were measured using the 3,5-dinitrosalicylic acid colorimetric and colorimetric methods, respectively (Zhou et al., \u003cspan citationid=\"CR62\" class=\"CitationRef\"\u003e2022\u003c/span\u003e).\u003c/p\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003e\u003cb\u003eNutrient composition of pepper plants and root transcriptome\u003c/b\u003e\u003c/h2\u003e \u003cp\u003eConsidering that the yield-enhancing effects of microalgae were more pronounced in the uncultivated soil, the pepper roots, stems, and leaves from the uncultivated soil experiment were collected for the determination of nitrogen, phosphorus, and potassium contents. For transcriptome analysis, root samples were thoroughly ground in liquid nitrogen, and total RNA was extracted using the GenElute\u0026trade; Total RNA Purification Kit (Sigma-Aldrich, USA). RNA quality was assessed using a 5300 Bioanalyzer (Agilent Clara, Technologies, CA, USA) and quantified using an ND-2000 spectrophotometer (NanoDrop Technologies). High-quality RNA samples were selected for library construction and sequencing by Shanghai Majorbio Bio-Pharm Technology Co., Ltd.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eRhizosphere soil microbial and metagenomic sequencing\u003c/h3\u003e\n\u003cp\u003eTotal DNA was extracted from rhizosphere soil samples using E. Z. N. A.\u0026reg; Soil DNA Kits, assessed using 1% agarose gel electrophoresis and quantified using a QuantiFluor\u003csup\u003eTM\u003c/sup\u003e-ST Blue Fluorescence Quantification System (Promega Corporation). PCR amplification was performed using the 16S primers 27F-1492R and 18S primers EukA-EukB (Liu et al., \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2023b\u003c/span\u003e; Kalu et al., 2023). The PCR products were detected using 2% agarose gel electrophoresis, recovered using the AxyPrep DNA Gel Extraction Kit, eluted with Tris-HCl, and detected using 2% agarose gel electrophoresis. For microbial sequencing, paired-end sequencing was performed using a third-generation sequencing platform (PacBio Sequel II, Pacific Biosciences, CA, USA). Metagenomic sequencing was performed using the Illumina NovaSeq sequencing platform, and each sample was analyzed using the MajorBio platform.\u003c/p\u003e\n\u003ch3\u003eIsolation and identification of microalgae-enriched bacteria\u003c/h3\u003e\n\u003cp\u003eAfter pot experiments, rhizosphere soil from the AS group was homogenized, and the supernatant was serially diluted with MS buffer (10 mM MgSO₄, 100 mM NaCl, 50 mM Tris-HCl, 0.01% gelatin). The diluted suspension (150 \u0026micro;L) was then spread onto Ashby\u0026rsquo;s Medium (Tab. S5) and incubated at 30\u0026deg;C for 48 h. Bacterial isolates were randomly selected and separately transferred to new Ashby\u0026rsquo;s Medium plates using a sterile toothpick. Each isolated bacterium was streaked onto a fresh Ashby\u0026rsquo;s Medium plate for colony purification, and the 16S rRNA gene of each bacterial strain was amplified using primers 27F-1492R for Sanger sequencing. The 16S rRNA gene sequences of the bacterial isolates were subjected to BLAST searches using the NCBI GenBank database to obtain detailed taxonomic information. The isolated \u003cem\u003eMethylobacterium\u003c/em\u003e sp., \u003cem\u003eBradyrhizobium japonicum\u003c/em\u003e, and \u003cem\u003eSphingomonas\u003c/em\u003e sp. were cultured in yeast extract mannitol agar (YMA; Tab. S6), Reasoner's 2A agar (R2A; Tab. S7), and Luria\u0026ndash;Bertani Agar (LB; Tab. S8), respectively. After centrifugation at 6,000 \u003cem\u003eg\u003c/em\u003e for 10 min, the bacterial cultures at OD\u003csub\u003e600\u003c/sub\u003e of 1.0 were used in the following experiments.\u003c/p\u003e \u003cp\u003e \u003cb\u003eGrowth-promoting effects of\u003c/b\u003e \u003cb\u003eScenedesmus\u003c/b\u003e \u003cb\u003esp. EPS on nitrogen-fixing bacteria\u003c/b\u003e\u003c/p\u003e \u003cp\u003eA total of 24 groups were set up to determine whether the EPS and filtrate of \u003cem\u003eScenedesmus\u003c/em\u003e sp. and \u003cem\u003eA. azotica\u003c/em\u003e promoted the growth of three isolated nitrogen-fixing bacteria (Tab. S9). \u003cem\u003eScenedesmus\u003c/em\u003e sp. and \u003cem\u003eA. azotica\u003c/em\u003e at stationary phase were filtered through 0.22-\u0026micro;m membranes to obtain the filtrate, and EPS were extracted using the heating method (Zhou et al., \u003cspan citationid=\"CR63\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). Cultures of \u003cem\u003eMethylobacterium\u003c/em\u003e sp., \u003cem\u003eB. japonicum\u003c/em\u003e, and \u003cem\u003eSphingomonas\u003c/em\u003e sp. at the mid-exponential phase were adjusted to an OD\u003csub\u003e600\u003c/sub\u003e of 0.1\u0026ndash;0.3 using R2A, YMA, and LB media, respectively. Then, the corresponding bacterial cultures (200 mL) were dispensed into each group, and 2 mL of BG11 medium was added as the control. Subsequently, 2 mL of \u003cem\u003eScenedesmus\u003c/em\u003e sp. EPS, \u003cem\u003eScenedesmus\u003c/em\u003e sp. filtrate, \u003cem\u003eA. azotica\u003c/em\u003e EPS, \u003cem\u003eA. azotica\u003c/em\u003e filtrate, \u003cem\u003eScenedesmus\u003c/em\u003e sp. EPS and \u003cem\u003eA. azotica\u003c/em\u003e EPS combiniation (1:1 v/v), and \u003cem\u003eScenedesmus\u003c/em\u003e sp. filtrate and 1 mL \u003cem\u003eA. azotica\u003c/em\u003e filtrate combiniation (1:1 v/v) were separately added into the above bacterial cultures. OD\u003csub\u003e600\u003c/sub\u003e values of each group were measured every 3 h to plot the growth curves.\u003c/p\u003e \u003cp\u003e \u003cb\u003eComposition analysis of\u003c/b\u003e \u003cb\u003eScenedesmus\u003c/b\u003e \u003cb\u003esp. EPS and filtrate\u003c/b\u003e\u003c/p\u003e \u003cp\u003eEPS (500 \u0026micro;L) and filtrate (500 \u0026micro;L) of \u003cem\u003eScenedesmus\u003c/em\u003e sp. were separately mixed with methanol (500 \u0026micro;L), vortexed, and centrifugated at 10,000 \u003cem\u003eg\u003c/em\u003e for 10 min at 4\u0026deg;C. The supernatant was then filtered through 0.22-\u0026micro;m membranes, and 2-chlorophenylalanine (100 \u0026micro;g/mL) was added as the internal standard to a final concentration of 1 mg/L prior to analysis. Metabolites in the EPS and filtrate were profiled by HPLC\u0026ndash;MS/MS, which was performed on a C18 column (Zorbax Eclipse C18, 1.8 \u0026micro;m \u0026times; 2.1 mm \u0026times; 100 mm) at 30\u0026deg;C with a flow rate of 0.3 mL/min using 0.1% formic acid in water (A) and acetonitrile (B) as the mobile phases (Wang et al., \u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e2019\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003cb\u003eEffect of\u003c/b\u003e \u003cb\u003eScenedesmus\u003c/b\u003e \u003cb\u003esp. EPS components on the growth of nitrogen-fixing bacteria\u003c/b\u003e\u003c/p\u003e \u003cp\u003eThe three most abundant compounds L-glutamic acid (LGA), 2,5-anhydro-1-deoxy-1-[(1-hydroxy-2-butanyl)amino]-D-mannitol (DM), and V-pyrro/NO (VP) were selected to identify EPS components that stimulate the growth of three nitrogen-fixing bacteria. Twelve groups were established (Tab. S10), and each compound was dosed to match its concentration in \u003cem\u003eScenedesmus\u003c/em\u003e sp. EPS. LGA, DM, and VP were individually added into \u003cem\u003eMethylobacterium sp.\u003c/em\u003e, \u003cem\u003eB. japonicum\u003c/em\u003e, and \u003cem\u003eSphingomonas\u003c/em\u003e sp. suspensions, which were prepared by diluting cultures with the corresponding medium to OD\u003csub\u003e600\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;0.3. During the experiment, OD\u003csub\u003e600\u003c/sub\u003e values of each group were measured every 3 h to plot growth curves.\u003c/p\u003e \u003cp\u003eTo further validate the growth-promoting effect of LGA on nitrogen-fixing bacteria, assays were conducted on solid media (Tab. S11). Prior to plate pouring, LGA was added into the media at doses corresponding to their concentrations in \u003cem\u003eScenedesmus\u003c/em\u003e sp. EPS. Mid-exponential-phase cultures of \u003cem\u003eMethylobacterium sp.\u003c/em\u003e, \u003cem\u003eB. japonicum\u003c/em\u003e, and \u003cem\u003eSphingomonas sp.\u003c/em\u003e were diluted to OD\u003csub\u003e600\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;0.1, and 2.5 \u0026micro;L of each suspension was separately spot-inoculated at the center of the plates. Plates were incubated at 28\u0026deg;C for 36 h, and the colonies were photographed for analysis.\u003c/p\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eGrowth-promoting effects of microalga-derived L-glutamic acid and nitrogen-fixing bacteria\u003c/h2\u003e \u003cp\u003eTo test whether \u003cem\u003eScenedesmus sp.\u003c/em\u003e enriched nitrogen-fixing bacteria and promoted plant growth via LGA secretion, a pot experiment was conducted with six groups (Tab. S12). All pots received the same chemical fertilizer as basal fertilization, and N, P, and K inputs were kept constant across groups. The control group only received the chemical fertilizer, and the other groups received the basal fertilizer and either a nitrogen-fixing bacterial inoculum, LGA, or \u003cem\u003eScenedesmus\u003c/em\u003e sp. EPS alone, or bacterial inoculum supplemented with LGA, or the bacterial inoculum supplemented with \u003cem\u003eScenedesmus sp\u003c/em\u003e. EPS. The soil was the same as that used in the uncultivated-soil experiment, sterilized by gamma irradiation (60 kGy, Guo et al., \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2024\u003c/span\u003e), and filled into plastic pots (7 \u0026times; 7 \u0026times; 7.3 cm, L \u0026times; W \u0026times; H). Pepper seedlings at the three-true-leaf stage were transplanted one week after fertilization to initiate the experiment (day 0), and the designated additives were applied on the 1st, 7th, and 14th day. Plants were sampled on the 30th day to measure the growth indicators.\u003c/p\u003e \u003cp\u003e \u003cb\u003eThe effects of combined application of\u003c/b\u003e \u003cb\u003eScenedesmus\u003c/b\u003e \u003cb\u003esp. and nitrogen-fixing bacteria\u003c/b\u003e\u003c/p\u003e \u003cp\u003eA pot experiment was conducted to evaluate the effects of \u003cem\u003eScenedesmus\u003c/em\u003e sp. co-application with nitrogen-fixing bacteria (Tab. S13). All groups received the same chemical fertilizer as a uniform basal application, and N, P, and K inputs were kept constant across treatments. The control group only received the basalfertilizer, and the other groups received the basal fertilizer and one of the following supplemental inputs: \u003cem\u003eA. azotica\u003c/em\u003e, \u003cem\u003eScenedesmus\u003c/em\u003e sp., a nitrogen-fixing bacterial inoculum, the bacterial inoculum supplemented with \u003cem\u003eA. azotica\u003c/em\u003e, and the bacterial inoculum supplemented with \u003cem\u003eScenedesmus sp\u003c/em\u003e. Soil was sterilized by gamma irradiation (60 kGy, Guo et al., \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2024\u003c/span\u003e), transferred to pots (7 \u0026times; 7 \u0026times; 7.3 cm, L \u0026times; W \u0026times; H), and amended according to the assigned groups. All inputs were applied once as aqueous preparations. Pepper seedlings at the three-true-leaf stage were transplanted one week after fertilization to initiate the experiment (day 0), and plants were harvested on the 30th day for growth measurements.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eStatistical analyses\u003c/h2\u003e \u003cp\u003eGene expression levels were quantified with RSEM, and differential expression was assessed using DESeq2 (Love et al., \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2014\u003c/span\u003e). GO enrichment and KEGG pathway analyses were performed using gotools and Python SciPy, respectively(Shen et al., \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). For 16S rRNA data, functional profiles were predicted with Phylogenetic Investigation of Communities by Reconstruction of Unobserved States version 2 (PICRUSt2). Metagenomic functions were annotated by BLASTP against the KEGG database (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://www.genome.jp/kegg/\u003c/span\u003e\u003cspan address=\"http://www.genome.jp/kegg/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) with an e-value cutoff of \u0026le;\u0026thinsp;1 \u0026times; 10\u003csup\u003e\u0026minus;\u0026thinsp;5\u003c/sup\u003e, and KEGG pathways related to nitrogen metabolism were extracted for downstream analysis(Gao et al., \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). Paired-end reads were merged with FLASH (v1.2.7) and quality-filtered with fastp to obtain clean Tags (Bokulich et al., \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2013\u003c/span\u003e). Chimeras were identified and removed by aligning tags to a reference annotation database, yielding Effective Tags (Haas et al., \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2011\u003c/span\u003e). OTUs were clustered from all effective Tags using UPARSE (v7.0.1001, Edgar \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2013\u003c/span\u003e). Species annotation analysis was performed using RDP Classifier (version 2.2) against the Silva132 database (Wang et al., \u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e2007\u003c/span\u003e; Quast et al., \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e2012\u003c/span\u003e). QIIME was used to compute α-diversity indices and β-diversity distance matrices. A co-occurrence network was constructed using Spearman correlations (r\u0026thinsp;\u0026gt;\u0026thinsp;0.7, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05), and correlations were calculated in RStudio (4.1.0) and visualized in Gephi (v0.9.2, Wei et al., \u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). Differences among groups were tested by one-way ANOVA (LSD, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05) and t-tests in SPSS Statistics. Differentially abundant taxa were identified using LEfSe (LDA\u0026thinsp;\u0026gt;\u0026thinsp;3.5, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05) and STAMP. Additional statistical analyses were conducted in IBM SPSS Statistics. A partial least squares path model (PLS-PM) was built with the R package \u0026ldquo;plspm\u0026rdquo; (v0.5.0), and model fit was evaluated using the goodness-of-fit (GOF) index (Li et al., \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2023\u003c/span\u003eb). Other data visualizations were created using Origin 2018 software.\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eMicroalgae enhance pepper growth and yield in both uncultivated and cultivated soils\u003c/h2\u003e \u003cp\u003eCompared with the control group, the A and AS application resulted in larger pepper plants and fruits in the uncultivated soil (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ea,b). Specifically, the A application increased pepper plant height, stem diameter, leaf length, and leaf width by 6.64%, 4.89%, 5.49%, and 11.76%, respectively (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05; Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ec\u0026ndash;f). By contrast, the AS application increased pepper plant height, stem diameter, leaf length, and leaf width by 14.83%, 12.79%, 10.44%, and 16.47%, respectively (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05). Additionally, the A and AS application increased pepper yields by 36.42% and 107.87%, respectively (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05; Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eg). In the cultivated soil, the A and AS applications exhibited similar growth-promoting effects and increased pepper yields by 26.91% and 48.05%, respectively (Fig. S3 and Fig. S4). Thus, the application of microalgae \u003cem\u003eA. azotica\u003c/em\u003e and \u003cem\u003eScenedesmus\u003c/em\u003e sp. significantly increased the growth and yield of peppers.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003eMicroalgae improve rhizosphere soil physicochemical properties\u003c/h2\u003e \u003cp\u003eBoth A and AS application significantly increased soil pH and SOM content in the uncultivated soil at 28 and 56 d post-transplantation, and the AS application also significantly enhanced the available soil nutrient content and soil enzyme activities (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eh-n and Fig. S5). At 112 d, the A application increased the contents of available nitrogen (AN), available phosphorus (AP), available potassium (AK), SOM and sucrase activity by 7.57%, 8.30%, 8.02%, 65.84%, and 5.36%, respectively (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05; Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eh-j, l, n). By contrast, the AS application improved these indices by 11.98%, 18.24%, 12.54%, 65.53%, and 9.63%, respectively. Moreover, the AS application increased soil urease, acid phosphatase, and peroxidase activities by 2.73%, 28.79%, and 8.49%, respectively (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05). In the cultivated soil, both A and AS application also enhanced soil pH and SOM content, and the AS application significantly improved available soil nutrient contents and enzyme activities (Fig. S6).\u003c/p\u003e \u003cp\u003eIn the uncultivated soil, the RDA results indicated that acid phosphatase activity (r\u0026sup2; = 0.95, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.00005), AP content (r\u0026sup2; = 0.91, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.0005), sucrase activity (r\u0026sup2; = 0.885, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.0015), AK content (r\u0026sup2; = 0.85, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.0035), and AN content (r\u0026sup2; = 0.84, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.0050) significantly affected pepper growth and yield (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eo). The Mantel test showed that the AN content, sucrase activity, and urease activity were the major factors affecting pepper growth and yield (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ep). In the cultivated soil, the RDA results showed that AK content, AP content, urease activity, peroxidase activity, sucrase activity, and AN content significantly affected pepper growth and yield (Fig. S7a). The Mantel test revealed that sucrase activity, urease activity, pH, AN content, AP content, and AK content were the primary factors influencing pepper growth and yield (Fig. S7b). Overall, both uncultivated and cultivated soil experiments demonstrated that pepper growth and yield were significantly affected by the AN content, urease activity, and sucrase activity.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eScenedesmus\u003c/b\u003e \u003cb\u003esp. promotes the absorption and utilization of nitrogen by pepper roots\u003c/b\u003e\u003c/p\u003e \u003cp\u003eCompared with the control group, the A and AS application increased nitrogen content in pepper roots in the uncultivated soil by 26.95% and 64.99% (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05; Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ea), nitrogen content in pepper stems by 25.58% and 48.84% (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05; Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eb), and nitrogen content in pepper leaves by 12.54% and 29.15%, respectively (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05; Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ec). The A and AS application also increased phosphorus content in pepper roots by 44.80% and 66.52% (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05; Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ed), phosphorus content in pepper stems by 15.76% and 25.62% (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05; Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ee), and phosphorus content in pepper leaves by 27.40% and 57.88%, respectively (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05; Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ef). The potassium content in pepper plants did not differ significantly between the control and microalgae application groups (Fig. S8). In the cultivated soil, plant nitrogen content in the A and AS application group exhibited the similar results, but plant phosphorus content showed no significant difference compared to that of the control group (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026gt;\u0026thinsp;0.05; Fig. S9).\u003c/p\u003e \u003cp\u003eCompared with the control group, the AS application had a greater impact on the transcriptome of pepper roots (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eg). After the A application, 4,820 differentially expressed genes (DEGs) were identified, of which 2,111 were upregulated and 2,709 were downregulated (Fig. S10a). The AS application induced 5,725 DEGs, including 3,313 upregulated and 2,412 downregulated genes (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eh). Clustering analysis of the DEGs revealed that the contrl, A application, and AS application all clustered within their groups, with high reproducibility between parallel samples (Fig. S10b). Thus, the data could be used for further analysis.\u003c/p\u003e \u003cp\u003eGene ontology (GO) analysis revealed that the DEGs between the A application and control groups were primarily enriched in biological processes (BPs), including the regulation of protein serine/threonine phosphatase activity, abscisic acid-activated signaling pathway, primary cell wall biogenesis in plants, glutathione metabolic process, and ethylene-activated signaling pathway (Fig. S10c). The DEGs between the AS application and control group were primarily enriched in BPs and cellular components (CCs), including the glutathione metabolic process, light harvesting in photosystem I, and the photosystem I reaction center (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ei). Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis showed that the A application mainly affected brassinosteroid biosynthesis, monoterpenoid biosynthesis, isoquinoline alkaloid biosynthesis, and nitrogen metabolism (Fig. S10d), while the AS application predominantly affected brassinosteroid biosynthesis, photosynthesis, and nitrogen metabolism (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ej). We further analyzed the effects of the A and AS application on nitrogen metabolism in pepper roots. Compared with the A application, the AS application further upregulated the expression of genes (\u003cem\u003eGS1\u003c/em\u003e and \u003cem\u003eGS2\u003c/em\u003e) involved in the synthesis of L-glutamine from ammonium nitrogen in pepper roots and the expression of genes (\u003cem\u003eNR\u003c/em\u003e, \u003cem\u003eNIR1\u003c/em\u003e, and \u003cem\u003eNIR2\u003c/em\u003e) responsible for converting nitrate to nitrite and nitrite to ammonium nitrogen. These results indicated that \u003cem\u003eScenedesmus\u003c/em\u003e sp. effectively promotes nitrogen uptake and utilization in pepper roots.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eScenedesmus\u003c/b\u003e \u003cb\u003esp. enriches nitrogen-fixing bacteria\u003c/b\u003e\u003c/p\u003e \u003cp\u003eThe A and AS application had no significant effect on α-diversity of prokaryotic and eukaryotic microbial communities in the rhizosphere soil (Fig. S11). However, both A and AS application altered the community structures of prokaryotic and eukaryotic microorganisms (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ea and Fig. S12a). Among the top 20 prokaryotic microorganisms in the relative abundance, \u003cem\u003eAcidobacteriales_norank\u003c/em\u003e and \u003cem\u003eRB41\u003c/em\u003e were exclusively in the control group, with relative abundances of 5.94% and 5.12%, respectively (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eb). The A and AS applications reduced the relative abundance of \u003cem\u003eCandidatus Udaeobacter\u003c/em\u003e by 41.87% and 43.95%, respectively (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05). The AS application also decreased the relative abundance of \u003cem\u003eRB41\u003c/em\u003e by 48.60% and increased the relative abundances of \u003cem\u003eFlavisolibacter\u003c/em\u003e and \u003cem\u003eSphingomonas\u003c/em\u003e by 27.78% and 57.98%, respectively (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05). The statistical analysis of metagenomic profiles (STAMP) analysis further confirmed that the AS application significantly increased the relative abundances of \u003cem\u003eFlavisolibacter\u003c/em\u003e and \u003cem\u003eSphingomonas\u003c/em\u003e (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05; Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ec).\u003c/p\u003e \u003cp\u003eFor eukaryotes, \u003cem\u003eHalobiotus\u003c/em\u003e was the dominant genus exclusively in the AS application, with a relative abundance of 6.23% (Fig. S12b). The A application increased the relative abundances of \u003cem\u003ePolymyxa\u003c/em\u003e, \u003cem\u003eChlorosarcinopsis\u003c/em\u003e, and \u003cem\u003eHeterochlamydomonas\u003c/em\u003e by 527.75%, 51.10%, and 419.35%, respectively (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05). The AS application decreased the relative abundance of \u003cem\u003eHyphochytrium\u003c/em\u003e by 61.69% (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05). The STAMP analysis showed that the A application significantly increased the relative abundances of \u003cem\u003ePolymyxa\u003c/em\u003e and \u003cem\u003eHeterochlamydomonas\u003c/em\u003e (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05; Fig. S12c), and the AS application significantly increased the relative abundance of \u003cem\u003eHalobiotus\u003c/em\u003e (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05).\u003c/p\u003e \u003cp\u003eLEfSe analysis showed that the A application significantly enriched \u003cem\u003eChthoniobacter\u003c/em\u003e, whereas the AS application significantly enriched \u003cem\u003eMethylobacterium\u003c/em\u003e, \u003cem\u003eTolypothrix\u003c/em\u003e, and \u003cem\u003eMicrocoleus\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ed). For eukaryotic microorganisms, the AS application significantly enriched \u003cem\u003ePalmellopsis\u003c/em\u003e and \u003cem\u003eScenedesmus\u003c/em\u003e (Fig. S12d), which confirmed that \u003cem\u003eScenedesmus\u003c/em\u003e sp. could survive well in pepper rhizosphere soil.\u003c/p\u003e \u003cp\u003eCo-occurrence network analysis showed that the A and AS application reduced the network diameter and average path length, increased the average clustering coefficient, and enhanced community stability. The AS application increased the modularity of co-occurrence network, in which microorganisms with higher degree values were located in highly interconnected modules and thereby exhibited stronger resistance to external disturbances (Fig. S13 and Tab. S14). Moreover, the AS application promoted prokaryotic microorganisms \u003cem\u003eMicrocoleus\u003c/em\u003e and \u003cem\u003eGemmatimonas\u003c/em\u003e and eukaryotic microorganisms \u003cem\u003eHeterochlamydomonas\u003c/em\u003e, \u003cem\u003eAmphisiella\u003c/em\u003e, and \u003cem\u003eFilamoeba\u003c/em\u003e, establishing them as core microorganisms (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ee and Fig. S12e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eScenedesmus\u003c/b\u003e \u003cb\u003esp. affects soil nitrogen cycling by enriching nitrogen-fixing bacteria\u003c/b\u003e\u003c/p\u003e \u003cp\u003eThe A and AS application significantly upregulated genes associated with ammonia uptake, nitrification (conversion of hydroxylamine to ammonia), and nitrogen fixation in rhizosphere soil of pepper plants. Besides the genes linked to nitrogen fixation, the AS application also significantly upregulated genes associated with assimilatory nitrate reduction, nitrate uptake, and nitrite uptake (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ea). The 16S rRNA sequencing analysis showed that the A application significantly increased the OTU number and relative abundance of bacteria involved in the nitrate reduction, whereas the AS application significantly increased the OTU number and relative abundance of bacteria involved in nitrogen fixation (Fig. S14). Metagenomic analysis showed that the AS application increased the abundances of \u003cem\u003eBradyrhizobium\u003c/em\u003e sp., \u003cem\u003eSphingomonas panaciterrae\u003c/em\u003e, \u003cem\u003eAzospirillum\u003c/em\u003e sp., and \u003cem\u003eTrichormus azotica\u003c/em\u003e by 144.20%, 712.03%, 116.76%, and 170.96%, respectively (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05; Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eb). Partial least squares path modeling (PLS-PM) analysis revealed that \u003cem\u003eScenedesmus\u003c/em\u003e sp. positively affected soil nitrogen cycling by influencing prokaryotic microbial community in the rhizosphere, thereby promoting pepper plant growth and increasing pepper yield (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ec).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eScenedesmus\u003c/b\u003e \u003cb\u003esp. enriches nitrogen-fixing bacteria by secreting L-glutamic acid\u003c/b\u003e\u003c/p\u003e \u003cp\u003eThree nitrogen-fixing bacteria were isolated from the rhizosphere soil after AS application and identified as \u003cem\u003eMethylobacterium\u003c/em\u003e sp., \u003cem\u003eBradyrhizobium japonicum\u003c/em\u003e, and \u003cem\u003eSphingomonas\u003c/em\u003e sp. (Fig. S15). Pot experiments showed that the A application increased plant height, stem diameter, leaf length, and leaf width of peppers by 19.34%, 9.66%, 26.14%, and 23.08%, respectively (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05; Fig. S16). The AS application increased plant height, stem diameter, leaf length, and leaf width of peppers by 71.70%, 37.28%, 71.59%, and 59.61%, respectively (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05). By contrast, the combined application of \u003cem\u003eScenedesmus\u003c/em\u003e sp. and three nitrogen-fixing bacteria increased plant height, stem diameter, leaf length, and leaf width by 117.45%, 52.27%, 111.36%, and 115.38%, displaying the strongest growth-promoting effects (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05).\u003c/p\u003e \u003cp\u003eThe filtrate and EPS of \u003cem\u003eA. azotica\u003c/em\u003e and the filtrate of \u003cem\u003eScenedesmus\u003c/em\u003e sp. exhibited no effect on the growth of nitrogen-fixing bacteria. However, the addition of \u003cem\u003eScenedesmus\u003c/em\u003e sp. EPS advanced the stationary phases of MS, BJ, and SS by 6, 3, and 3 h, respectively (Fig. S17a-c). To identify which substances promoted the growth of nitrogen-fixing bacteria, a comprehensive analysis of the EPS and filtrate of \u003cem\u003eScenedesmus\u003c/em\u003e sp. was conducted. A total of 16 components were identified in the EPS of \u003cem\u003eScenedesmus\u003c/em\u003e sp. and absent in the filtrate (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ea, Tab. S15). The top three substances (V-pyrro/NO, L-glutamic acid, and 2,5-Anhydro-1-deoxy-1-[(1-hydroxy-2-butanyl)amino]-D-mannitol) with the highest concentrations were selected to test their ability to promote nitrogen-fixing bacteria growth. The results showed that L-glutamic acid advanced thee stationary phase of three nitrogen-fixing bacteria by 3\u0026ndash;6 h, significantly increased the colony diameters of three bacteria on solid media, and markedly accelerated their growth rates (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eb-e). These results confirmed that \u003cem\u003eScenedesmus\u003c/em\u003e sp. secreted L-glutamic acid to enrich nitrogen-fixing bacteria.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThe single application of nitrogen-fixing bacteria increased the plant height, stem diameter, leaf length, and leaf width of pepper plants by 26.06%, 28.86%, 15.76%, and 29.88%, respectively (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05; Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eb-c). The additional supplementation with L-glutamic acid or \u003cem\u003eScenedesmus\u003c/em\u003e sp. EPS further increased pepper plant height, stem diameter, leaf length, and leaf width by 14.77\u0026ndash;17.72%, 15.06\u0026ndash;17.18%, 6.43\u0026ndash;7.30%, and 11.60\u0026ndash;11.79%, respectively (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05). The single application of nitrogen-fixing bacteria increased the chlorophyll content, maximum photosynthetic efficiency, and actual photosynthetic efficiency of pepper leaves by 65.34%, 2.26% and 27.23%, respectively (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05; Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eg-i). The additional supplementation with L-glutamic acid or \u003cem\u003eScenedesmus\u003c/em\u003e sp. EPS further increased the chlorophyll content, maximum photosynthetic efficiency, and actual photosynthetic efficiency of pepper leaves by 12.63\u0026ndash;17.00%, 4.67\u0026ndash;6.20%, and 3.13\u0026ndash;4.03%, respectively (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eThis study found that the single application of \u003cem\u003eA. azotica\u003c/em\u003e increased the yield of pepper in uncultivated and cultivated soils by 36.42% and 26.91%, respectively. Conversely, the combined application of \u003cem\u003eA. azotica\u003c/em\u003e and \u003cem\u003eScenedesmus\u003c/em\u003e sp. increased the pepper yield by 107.87% and 48.05%, respectively. Thus, introducing \u003cem\u003eScenedesmus\u003c/em\u003e sp. further enhanced pepper yield. Previous study reported that \u003cem\u003eA. azotica\u003c/em\u003e promoted rice growth in barren and fertile soils and increased the yield by 38.00% and 107.00%, respectively (Li et al., \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2025\u003c/span\u003e). \u003cem\u003eScenedesmus\u003c/em\u003e sp. also enhanced the growth of tomatoes (\u003cem\u003eSolanum lycopersicum\u003c/em\u003e) and increased the yield by 32.05% (Silambarasan et al., \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). It was reported that the combined application of cyanobacteria and green algae exerted strong growth-promoting and yield-enhancing effects (Gon\u0026ccedil;alves \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2021\u003c/span\u003e), and the combined application of \u003cem\u003eA. azotica\u003c/em\u003e and \u003cem\u003eChlorella pyrenoidosa\u003c/em\u003e increased wheat yield by 21.4%, which was 7.2\u0026ndash;9.8% higher than that in treatments with single microalgal species (Do et al., 2019). Therefore, the combined application of \u003cem\u003eA. azotica\u003c/em\u003e and \u003cem\u003eScenedesmus\u003c/em\u003e sp. might be an effective approach for enhancing crop yield.\u003c/p\u003e \u003cp\u003eSoil physicochemical properties could directly affect plant growth and yield. The RDA and Mantel test results revealed that pepper yield was primarily influenced by soil AN content, urease activity, and sucrase activity. The A application increased soil AN content and sucrase activity by 7.57% and 5.36%, respectively, with no significant impact on soil urease activity. The AS application increased soil AN content, urease activity, and sucrase activity by 11.98%, 2.73%, and 9.63%, respectively. The improvement of microalgae on soil physicochemical properties might be linked to their nitrogen-fixing and phosphate-solubilizing functions (Bhardwaj et al., \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2014\u003c/span\u003e; Singh et al., \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). Nitrogen-fixing microalgae were significantly positively correlated with the soil AN content (Li et al., \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2024\u003c/span\u003e; Zhang et al., \u003cspan citationid=\"CR61\" class=\"CitationRef\"\u003e2024\u003c/span\u003e), and \u003cem\u003eA. azotica\u003c/em\u003e increased soil AN content by 16.50\u0026ndash;51.00% through nitrogen fixation (Alvarez et al., 2021; Liu et al., \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2025\u003c/span\u003e). However, studies on the function of \u003cem\u003eScenedesmus\u003c/em\u003e sp. in increasing soil AN content have not yet been reported.\u003c/p\u003e \u003cp\u003eMicroalgae could stimulate the growth and metabolic activity of soil microbial communities by releasing various extracellular metabolites and providing easily degradable organic matter. This process would considerably enhance the activity of key enzymes and promote soil nutrient cycling and transformation efficiency (Dai et al., \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; Alvarez et al., 2021). For example, green algae could increase urease activity by 2.4 times (Barone et al., \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2019\u003c/span\u003e), and inoculating \u003cem\u003eScenedesmus\u003c/em\u003e sp. into the soil significantly enhanced soil sucrase and urease activities (Zhang et al., \u003cspan citationid=\"CR61\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). In this study, both \u003cem\u003eScenedesmus\u003c/em\u003e sp. and \u003cem\u003eA. azotica\u003c/em\u003e significantly reduced the relative abundance of \u003cem\u003eCandidatus Udaeobacter\u003c/em\u003e, while introducing \u003cem\u003eScenedesmus\u003c/em\u003e sp. also significantly decreased the relative abundance of \u003cem\u003eRB41\u003c/em\u003e. \u003cem\u003eC. Udaeobacter\u003c/em\u003e is an acidophilic microorganism that exhibits a significant negative correlation with soil pH (Willms et al., \u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e2021\u003c/span\u003e), and \u003cem\u003eRB41\u003c/em\u003e expression is significantly negatively correlated with SOM and AN content (Liu et al., 2023; Shi et al., \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). Therefore, the decrease in the relative abundances of \u003cem\u003eC. Udaeobacter\u003c/em\u003e and \u003cem\u003eRB41\u003c/em\u003e were likely attributed to the capacity of \u003cem\u003eA. azotica\u003c/em\u003e and \u003cem\u003eScenedesmus\u003c/em\u003e sp. to increase SOM content and pH, with \u003cem\u003eScenedesmus\u003c/em\u003e sp. addition leading to a substantial increase in AN content. The \u003cem\u003eA. azotica\u003c/em\u003e application made eukaryotic microorganisms \u003cem\u003eHormotilopsis\u003c/em\u003e, \u003cem\u003eChlorosarcinopsis\u003c/em\u003e, and \u003cem\u003eCryothecomonas\u003c/em\u003e become the core microbial constituents. \u003cem\u003eHormotilopsis\u003c/em\u003e and \u003cem\u003eChlorosarcinopsis\u003c/em\u003e are soil green algae (Flechtner et al., \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2008\u003c/span\u003e; Novakovskaya et al., \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2023\u003c/span\u003e), and the increased abundance of these green algae might be attributed to the alterations in the soil environment conditions induced by \u003cem\u003eA. azotica\u003c/em\u003e (Suleiman et al., \u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). In addition, introducing \u003cem\u003eScenedesmus\u003c/em\u003e sp. enriched \u003cem\u003eFlavisolibacter, Sphingomonas\u003c/em\u003e, \u003cem\u003eMethylobacterium\u003c/em\u003e, \u003cem\u003eTolypothrix\u003c/em\u003e, and \u003cem\u003eMicrocoleus\u003c/em\u003e, establishing \u003cem\u003eMicrocoleus\u003c/em\u003e and \u003cem\u003eGemmatimonas\u003c/em\u003e as the core microorganisms. \u003cem\u003eFlavisolibacter\u003c/em\u003e, \u003cem\u003eSphingomonas\u003c/em\u003e, \u003cem\u003eMethylobacterium\u003c/em\u003e, and \u003cem\u003eGemmatimonas\u003c/em\u003e are plant growth-promoting bacteria with nitrogen-fixing function (Desnoues et al., \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2003\u003c/span\u003e; Jiao et al., \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2024\u003c/span\u003e; Sultana et al., \u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e2024\u003c/span\u003e) and \u003cem\u003eTolypothrix\u003c/em\u003e and \u003cem\u003eMicrocoleus\u003c/em\u003e are nitrogen-fixing cyanobacteria (Alvarez et al., 2021). Introducing \u003cem\u003eScenedesmus\u003c/em\u003e sp. also significantly upregulated the genes associated with nitrogen fixation and increased the abundances of \u003cem\u003eBradyrhizobium\u003c/em\u003e sp., \u003cem\u003eSphingomonas panaciterrae\u003c/em\u003e, \u003cem\u003eAzospirillum\u003c/em\u003e sp., and \u003cem\u003eTrichormus azotica\u003c/em\u003e by 144.20%, 712.03%, 116.76%, and 170.96%, respectively. However, the \u003cem\u003eA. azotica\u003c/em\u003e application did not affect the abundance of these nitrogen-fixing bacteria. It was reported that \u003cem\u003eBradyrhizobium\u003c/em\u003e sp. fixed nitrogen in crop roots and increase the nitrogen content in the rhizosphere soil of crops by 13.36\u0026ndash;16.82% (Terakado-Tonooka et al., \u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e2013\u003c/span\u003e)d \u003cem\u003epanaciterrae\u003c/em\u003e promoted plant growth by enhancing the AN content in plant roots by 18.65\u0026ndash;46.15% (Sultana et al., \u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). \u003cem\u003eAzospirillum\u003c/em\u003e sp. and \u003cem\u003eT. azotica\u003c/em\u003e also had nitrogen-fixing functions and increased soil AN content by 12.25% and 10.20\u0026ndash;14.30%, respectively (Gladkikh et al., \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2008\u003c/span\u003e; Cass\u0026aacute;n et al., 2016). SEM model also showed that \u003cem\u003eScenedesmus\u003c/em\u003e sp. promoted nitrogen cycling in the soil by influencing rhizosphere prokaryotic microbial communities, thereby promoting plant growth and pepper yields. Thus, \u003cem\u003eScenedesmus\u003c/em\u003e sp. increased soil AN content by enriching nitrogen-fixing bacteria, promoted the absorption and utilization of soil nitrogen by the roots, and ultimately enhanced pepper growth and yield.\u003c/p\u003e \u003cp\u003eStudies have shown that microalgae could enrich microorganisms with specific functions by producing certain substances (Thompson et al., \u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e2012\u003c/span\u003e). In this study, three nitrogen-fixing bacteria were isolated from the rhizosphere soil in the AS application group, and the enrichment effect of \u003cem\u003eScenedesmus\u003c/em\u003e sp. EPS on these bacteria was further verified. To identify the specific components of EPS responsible for this effect remain unclear, a comprehensive spectral analysis of the EPS produced by \u003cem\u003eScenedesmus\u003c/em\u003e sp. were conducted. The results revealed high levels of L-glutamic acid, 2,5-Anhydro-1-deoxy-1-[(1-hydroxy-2-butanyl)amino]-D-mannitol, and V-pyrro/NO. Previous studies found that introducing trace amounts of L-glutamic acid could reshape the plant microbiome (Kim et al., \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Wang et al., \u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). This study also found that introducing \u003cem\u003eScenedesmus\u003c/em\u003e sp. EPS and L-glutamic acid at concentrations equivalent to those of EPS significantly promoted the growth of these three nitrogen-fixing bacteria, with no significant differences between the two treatments. L-glutamic acid served as a nitrogen source for nitrogen-fixing bacteria and might act as a precursor for synthesizing essential amino acids, enzymes and other proteins, thereby promoting their growth (Rehm et al., \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e2010\u003c/span\u003e). These findings suggest that \u003cem\u003eScenedesmus\u003c/em\u003e sp. enriched nitrogen-fixing bacteria through L-glutamic acid secretion.\u003c/p\u003e \u003cp\u003eSoil elements must be absorbed and utilized by plants to manifest their effects. The \u003cem\u003eA. azotica\u003c/em\u003e application increased nitrogen content in the roots, stems, and leaves of pepper plants by 26.95%, 25.58%, and 12.54%, respectively. The combined application of \u003cem\u003eA. azotica\u003c/em\u003e and \u003cem\u003eScenedesmus\u003c/em\u003e sp. increased nitrogen content in the roots, stems, and leaves by 64.99%, 48.84%, and 29.15%, respectively. Studies have shown that microalgal biofertilizers derived from high-rate microalgal ponds dominated by green algae significantly increased the nitrogen content of maize plants, and nitrogen-fixing cyanobacteria enhanced nitrogen content in crop roots (De et al., 2021). Further analysis revealed that the \u003cem\u003eA. azotica\u003c/em\u003e application upregulated the expression of genes related to nitrate and nitrite absorption (gene-LOC107848172, gene-LOC107875603, and gene-LOC107875600) and the gene (gene-LOC107870135) responsible for synthesizing L-glutamine from ammonium nitrogen during nitrogen metabolism in pepper roots. The expression of genes (gene-LOC107841845 and gene-LOC107870135) involved in synthesizing L-glutamine from ammonium nitrogen was further upregulated after introducing \u003cem\u003eScenedesmus\u003c/em\u003e sp. Moreover, the expression of the genes responsible for converting nitrate to nitrite and nitrite to ammonium nitrogen (gene-LOC107867964, gene-LOC107853280, and gene-LOC107840295) was significantly upregulated. Nitrogen absorbed by plants from the soil is initially converted to ammonium nitrogen, which is subsequently assimilated into glutamine and glutamate via the glutamine synthetase/glutamate synthase pathway. Glutamine and glutamate are essential precursors for synthesizing various plant proteins, promoting cell division, and accelerating plant growth (Ruiz et al., \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e2000\u003c/span\u003e; Tabuchi et al., 2007). The increase in available soil nitrogen content might significantly upregulate the expression of key genes in crop roots, including transporter protein genes (e.g., \u003cem\u003eNRT1\u003c/em\u003e and \u003cem\u003eNRT2\u003c/em\u003e families) responsible for nitrate and nitrite absorption, nitrate reductase (NR), nitrite reductase (NiR) genes involved in nitrate reduction, and glutamine synthetase (GS) and glutamate synthase (GOGAT) genes that mediate ammonium assimilation pathways (Wang et al., \u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). This coordinated regulatory mechanism of gene expression enabled crops to absorb, convert and assimilate nitrogen from the soil more efficiently, thereby improving nitrogen-use efficiency and promoting plant growth and crop yields.\u003c/p\u003e"},{"header":"Conclusions","content":"\u003cp\u003eIn summary, \u003cem\u003eScenedesmus\u003c/em\u003e sp. significantly increased the abundance of nitrogen-fixing bacteria in the rhizosphere soil of pepper plants by secreting L-glutamic acid. These nitrogen-fixing bacteria further increased the soil AN content, which subsequently upregulated the expression of genes in pepper roots responsible for converting nitrate to nitrite, nitrite to ammonium nitrogen, and ammonium nitrogen to L-glutamine. Finally, the improved absorption and utilization of nitrogen by the roots boosted pepper growth and yield. This study provided important scientific evidence for the development and utilization of microalgae-based biofertilizer to improve crop yields in the sustainable agriculture.\u003c/p\u003e"},{"header":"Declarations","content":"\u003ch2\u003eCompeting interests\u003c/h2\u003e \u003cp\u003eThe authors declare no competing interests.\u003c/p\u003e\u003ch2\u003eAuthor contributions\u003c/h2\u003e \u003cp\u003eL.C. and W.H.Q.: conceived and designed the experiments, performed software analysis, formal analysis, data curation, methodology, and wrote the first version of the manuscript. X.Y.W. and S.Z.H.: performed methodology, project administration, visualization, validation, and conducted the experiments. C.J.: performed investigation and project administration. W.J.: provided resources, supervised the project, administered the study, validated the research, and contributed to writing\u0026mdash;review and editing. All authors discussed the data, contributed to the writing and reviewing of manuscript drafts. All authors reviewed and approved the submitted version.\u003c/p\u003e\u003ch2\u003eAcknowledgements\u003c/h2\u003e \u003cp\u003eWe acknowledge the financial support from the Scientific Research Project of China Metallurgical Geology Bureau (CMGBKY202501).\u003c/p\u003e\u003ch2\u003eAvailability of data and materials\u003c/h2\u003e \u003cp\u003eThe raw sequencing data of 16S rRNA and 18S rRNA in this study have been deposited in the NCBI Sequence Read Archive (SRA) under accession number PRJNA1268602. The raw metagenomic data have been deposited in the NCBI SRA under accession number PRJNA1273704, and the raw transcriptome data have been deposited in the NCBI SRA under accession number PRJNA1273509.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eBarone V et al (2019) Effect of living cells of microalgae or their extracts on soil enzyme activities. Arch Agron Soil Sci 65:712\u0026ndash;726. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1080/03650340.2018.1521513\u003c/span\u003e\u003cspan address=\"10.1080/03650340.2018.1521513\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBello AS et al (2021) Enhancement in bell pepper (\u003cem\u003eCapsicum annuum\u003c/em\u003e L.) plants with application of \u003cem\u003eRoholtiella\u003c/em\u003e sp. (\u003cem\u003eNostocales\u003c/em\u003e) under soilless cultivation. Agronomy 11:1624. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.3390/agronomy11081624\u003c/span\u003e\u003cspan address=\"10.3390/agronomy11081624\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBhardwaj D et al (2014) Biofertilizers function as key player in sustainable agriculture by improving soil fertility, plant tolerance and crop productivity. Microb Cell Fact 13:1\u0026ndash;10. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1186/1475-2859-13-66\u003c/span\u003e\u003cspan address=\"10.1186/1475-2859-13-66\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBokulich NA et al (2013) Quality-filtering vastly improves diversity estimates from Illumina amplicon sequencing. Nat Methods 10:57\u0026ndash;59. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1038/nmeth.2276\u003c/span\u003e\u003cspan address=\"10.1038/nmeth.2276\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eCass\u0026aacute;n F, Diaz-Zorita M (2016) \u003cem\u003eAzospirillum\u003c/em\u003e sp. in current agriculture: From the laboratory to the field. Soil Biol Biochem 103:117\u0026ndash;130. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.soilbio.2016.08.020\u003c/span\u003e\u003cspan address=\"10.1016/j.soilbio.2016.08.020\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eDai Z et al (2018) Long-term nitrogen fertilization decreases bacterial diversity and favors the growth of \u003cem\u003eActinobacteria\u003c/em\u003e and \u003cem\u003eProteobacteria\u003c/em\u003e in agro-ecosystems across the globe. Glob. Change Biol. 24, 3452\u0026ndash;3461. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1111/gcb.14163\u003c/span\u003e\u003cspan address=\"10.1111/gcb.14163\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eDe Paula Pereira ASA et al (2021) Organomineral fertilizers pastilles from microalgae grown in wastewater: Ammonia volatilization and plant growth. Sci Total Environ 779:146205. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.scitotenv.2021.146205\u003c/span\u003e\u003cspan address=\"10.1016/j.scitotenv.2021.146205\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eDeepika P, MubarakAli D (2020) Production and assessment of microalgal liquid fertilizer for the enhanced growth of four crop plants. Biocatal Agric Biotechnol 28:101701. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.bcab.2020.101701\u003c/span\u003e\u003cspan address=\"10.1016/j.bcab.2020.101701\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eDesnoues N et al (2003) Nitrogen fixation genetics and regulation in a \u003cem\u003ePseudomonas stutzeri\u003c/em\u003e strain associated with rice. Microbiology 149:2251\u0026ndash;2262. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1099/mic.0.26270-0\u003c/span\u003e\u003cspan address=\"10.1099/mic.0.26270-0\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eDineshkumar R et al (2019) The impact of using microalgae as biofertilizer in maize (\u003cem\u003eZea mays\u003c/em\u003e L). Waste Biomass Valoriz 10:1101\u0026ndash;1110. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1007/s12649-017-0123-7\u003c/span\u003e\u003cspan address=\"10.1007/s12649-017-0123-7\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eEdgar RC (2013) UPARSE: highly accurate OTU sequences from microbial amplicon reads. Nat Methods 10:996\u0026ndash;998. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1038/nmeth.2604\u003c/span\u003e\u003cspan address=\"10.1038/nmeth.2604\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eFeng J et al (2024) Geologically younger ecosystems are more dependent on soil biodiversity for supporting function. Nat Commun 15:4141. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1038/s41467-024-48289-y\u003c/span\u003e\u003cspan address=\"10.1038/s41467-024-48289-y\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eFlechtner VR, Johansen JR, Belnap J (2008) The biological soil crusts of the San Nicolas Island: Enigmatic algae from a geographically isolated ecosystem. West North Am Naturalist 68:405\u0026ndash;436. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.3398/1527-0904-68.4.405\u003c/span\u003e\u003cspan address=\"10.3398/1527-0904-68.4.405\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGao Y et al (2022) Metagenomics and network analysis elucidating the coordination between fermentative bacteria and microalgae in a novel bacterial-algal coupling reactor (BACR) for mariculture wastewater treatment. Water Res 215:118256. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.watres.2022.118256\u003c/span\u003e\u003cspan address=\"10.1016/j.watres.2022.118256\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGladkikh A et al (2008) Nitrogen-fixing cyanobacterium \u003cem\u003eTrichormus variabilis\u003c/em\u003e of the Lake Baikal phytoplankton. Microbiology 77:726\u0026ndash;733. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1134/S0026261708060118\u003c/span\u003e\u003cspan address=\"10.1134/S0026261708060118\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGon\u0026ccedil;alves AL (2021) The use of microalgae and cyanobacteria in the improvement of agricultural practices: a review on their biofertilising, biostimulating and biopesticide roles. Appl Sci 11:871. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.3390/app11020871\u003c/span\u003e\u003cspan address=\"10.3390/app11020871\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGuo S et al (2024) Predatory protists impact plant performance by promoting plant growth-promoting rhizobacterial consortia. ISME J 18:wrae180. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1093/ismejo/wrae180\u003c/span\u003e\u003cspan address=\"10.1093/ismejo/wrae180\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHaas BJ et al (2011) Chimeric 16S rRNA sequence formation and detection in Sanger and 454-pyrosequenced PCR amplicons. Genome Res 21:494\u0026ndash;504. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1101/gr.112730.110\u003c/span\u003e\u003cspan address=\"10.1101/gr.112730.110\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHartmann M et al (2015) Distinct soil microbial diversity under long-term organic and conventional farming. ISME J 9:1177\u0026ndash;1194. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1038/ismej.2014.210\u003c/span\u003e\u003cspan address=\"10.1038/ismej.2014.210\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eJiao L et al (2024) Dynamic microbial regulation of triiron tetrairon phosphate nanomaterials in the tomato rhizosphere. Environ Sci -Nano 11:1157\u0026ndash;1169. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1039/D3EN00797A\u003c/span\u003e\u003cspan address=\"10.1039/D3EN00797A\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKapoore RV, Wood EE, Llewellyn CA (2021) Algae biostimulants: A critical look at microalgal biostimulants for sustainable agricultural practices. Biotechnol Adv 49:107754. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.biotechadv.2021.107754\u003c/span\u003e\u003cspan address=\"10.1016/j.biotechadv.2021.107754\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKim DR et al (2021) Glutamic acid reshapes the plant microbiota to protect plants against pathogens. Microbiome 9:1\u0026ndash;18. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1186/s40168-021-01186-8\u003c/span\u003e\u003cspan address=\"10.1186/s40168-021-01186-8\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLebrun JD et al (2012) Assessing impacts of copper on soil enzyme activities in regard to their natural spatiotemporal variation under long-term different land uses. Soil Biol Biochem 49:150\u0026ndash;156. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.soilbio.2012.02.027\u003c/span\u003e\u003cspan address=\"10.1016/j.soilbio.2012.02.027\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLi C et al (2024) The influence of microalgae fertilizer on soil water conservation and soil improvement: Yield and quality of potted tomatoes. Agronomy 14:2102. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.3390/agronomy14092102\u003c/span\u003e\u003cspan address=\"10.3390/agronomy14092102\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLi S et al (2025) Enhancement of rice production and soil carbon sequestration utilizing nitrogen-fixing cyanobacteria. Appl Soil Ecol 207:105940. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.apsoil.2025.105940\u003c/span\u003e\u003cspan address=\"10.1016/j.apsoil.2025.105940\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLi Z et al (2023) Marine toxin domoic acid alters nitrogen cycling in sediments. Nat Commun 14:7873. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1038/s41467-023-43265-4\u003c/span\u003e\u003cspan address=\"10.1038/s41467-023-43265-4\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLiu G et al (2023b) Effects of thaw slump on soil bacterial communities on the Qinghai-Tibet Plateau. CATENA 232:107342. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.catena.2023.107342\u003c/span\u003e\u003cspan address=\"10.1016/j.catena.2023.107342\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLiu J et al (2025) Application of microalgae in remediation of heavy metal-contaminated soils and its stimulatory effect on wheat growth. Algal Res 88:103995. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.algal.2025.103995\u003c/span\u003e\u003cspan address=\"10.1016/j.algal.2025.103995\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLove MI, Huber W, Anders S (2014) Moderated estimation of fold change and dispersion for RNA-seq data with DESeq2. Genome Biol 15:550. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1186/s13059-014-0550-8\u003c/span\u003e\u003cspan address=\"10.1186/s13059-014-0550-8\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMartre P et al (2024) Global needs for nitrogen fertilizer to improve wheat yield under climate change. Nat Plants 10:1081\u0026ndash;1090. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1038/s41477-024-01739-3\u003c/span\u003e\u003cspan address=\"10.1038/s41477-024-01739-3\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMuller A et al (2017) Strategies for feeding the world more sustainably with organic agriculture. Nat commun 8:1\u0026ndash;13. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1038/s41467-017-01410-w\u003c/span\u003e\u003cspan address=\"10.1038/s41467-017-01410-w\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eNosengo N (2003) Fertilized to death. Nature 425:894\u0026ndash;896. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1038/425894a\u003c/span\u003e\u003cspan address=\"10.1038/425894a\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eNovakovskaya IV et al (2023) \u003cem\u003eHeterochlamydomonas uralensis\u003c/em\u003e sp. Nov.(\u003cem\u003eChlorophyta\u003c/em\u003e, \u003cem\u003eChlamydomonadaceae\u003c/em\u003e), new species described from the mountain tundra community in the Subpolar Urals (Russia). Diversity 15:673. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.3390/d15050673\u003c/span\u003e\u003cspan address=\"10.3390/d15050673\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eQuast C et al (2012) The SILVA ribosomal RNA gene database project: improved data processing and web-based tools. Nucleic Acids Res 41:D590\u0026ndash;D596. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1093/nar/gks1219\u003c/span\u003e\u003cspan address=\"10.1093/nar/gks1219\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eRodrigues S et al (2024) Effect of a zinc phosphate shell on the uptake and translocation of foliarly applied ZnO nanoparticles in pepper plants (\u003cem\u003eCapsicum annuum\u003c/em\u003e). Environ Sci Technol 58:3213\u0026ndash;3223. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1021/acs.est.3c08723\u003c/span\u003e\u003cspan address=\"10.1021/acs.est.3c08723\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eReichert JM et al (2023) Soil morphological, physical and chemical properties affecting \u003cem\u003eEucalyptus\u003c/em\u003e spp. productivity on Entisols and Ultisols[J]. Soil Tillage Res 226:105563. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.still.2022.105563\u003c/span\u003e\u003cspan address=\"10.1016/j.still.2022.105563\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eRehm N et al (2010) L-Glutamine as a nitrogen source for \u003cem\u003eCorynebacterium glutamicum\u003c/em\u003e: derepression of the AmtR regulon and implications for nitrogen sensing. Microbiology 156:3180\u0026ndash;3193. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1099/mic.0.040667-0\u003c/span\u003e\u003cspan address=\"10.1099/mic.0.040667-0\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eRuiz JM, Castilla N, Romero L (2000) Nitrogen metabolism in pepper plants applied with different bioregulators. J Agric Food Chem 48:2925\u0026ndash;2929. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1021/jf990394h\u003c/span\u003e\u003cspan address=\"10.1021/jf990394h\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eShen C et al (2024) Comparative transcriptome analysis and \u003cem\u003eArabidopsis thaliana\u003c/em\u003e overexpression reveal key genes associated with cadmium transport and distribution in root of two \u003cem\u003eCapsicum annuum\u003c/em\u003e cultivars. J Hazard Mater 465:133365. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.jhazmat.2023.133365\u003c/span\u003e\u003cspan address=\"10.1016/j.jhazmat.2023.133365\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eShi B et al (2023) Effects of the polyhalogenated carbazoles 3-bromocarbazole and 1, 3, 6, 8-tetrabromocarbazole on soil microbial communities. Environ Res 239:117379. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.envres.2023.117379\u003c/span\u003e\u003cspan address=\"10.1016/j.envres.2023.117379\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSingh JS et al (2016) Cyanobacteria: a precious bio-resource in agriculture, ecosystem, and environmental sustainability. Front Microbiol 7:529. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.3389/fmicb.2016.00529\u003c/span\u003e\u003cspan address=\"10.3389/fmicb.2016.00529\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSilambarasan S et al (2021) Removal of nutrients from domestic wastewater by microalgae coupled to lipid augmentation for biodiesel production and influence of deoiled algal biomass as biofertilizer for \u003cem\u003eSolanum lycopersicum\u003c/em\u003e cultivation. Chemosphere 268:129323. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.chemosphere.2020.129323\u003c/span\u003e\u003cspan address=\"10.1016/j.chemosphere.2020.129323\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSpohn M, Braun S, Sierra CA (2023) Continuous decrease in soil organic matter despite increased plant productivity in an 80-years-old phosphorus-addition experiment. Commun Earth Environ 4:251. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1038/s43247-023-00915-1\u003c/span\u003e\u003cspan address=\"10.1038/s43247-023-00915-1\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSolomon W et al (2023) Potential benefit of microalgae and their interaction with bacteria to sustainable crop production. Plant Growth Regul 101:53\u0026ndash;65. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1007/s10725-023-01019-8\u003c/span\u003e\u003cspan address=\"10.1007/s10725-023-01019-8\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSuleiman AKA et al (2020) From toilet to agriculture: Fertilization with microalgal biomass from wastewater impacts the soil and rhizosphere active microbiomes, greenhouse gas emissions and plant growth. Resour Conserv Recycl 161:104924. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.resconrec.2020.104924\u003c/span\u003e\u003cspan address=\"10.1016/j.resconrec.2020.104924\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSultana R et al (2024) \u003cem\u003eSphingomonas panaciterrae\u003c/em\u003e PB20 increases growth, photosynthetic pigments, antioxidants, and mineral nutrient contents in spinach (\u003cem\u003eSpinacia oleracea\u003c/em\u003e L). Heliyon 10:25596. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.heliyon.2024.e25596\u003c/span\u003e\u003cspan address=\"10.1016/j.heliyon.2024.e25596\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eTerakado-Tonooka J, Fujihara S, Ohwaki Y (2013) Possible contribution of \u003cem\u003eBradyrhizobium\u003c/em\u003e on nitrogen fixation in sweet potatoes. Plant Soil 367:639\u0026ndash;650. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1007/s11104-012-1495-x\u003c/span\u003e\u003cspan address=\"10.1007/s11104-012-1495-x\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eThompson AW et al (2012) Unicellular cyanobacterium symbiotic with a single-celled eukaryotic alga. Science 337:1546\u0026ndash;1550. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1126/science.1222700\u003c/span\u003e\u003cspan address=\"10.1126/science.1222700\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eUysal O, Uysal FO, Ekinci K (2015) Evaluation of microalgae as microbial fertilizer. Eur J Sustain Dev 4:77. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.14207/ejsd.2015.v4n2p77\u003c/span\u003e\u003cspan address=\"10.14207/ejsd.2015.v4n2p77\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eVan Dijk M et al (2021) A meta-analysis of projected global food demand and population at risk of hunger for the period 2010\u0026ndash;2050. Nat Food 2:494\u0026ndash;501. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1038/s43016-021-00322-9\u003c/span\u003e\u003cspan address=\"10.1038/s43016-021-00322-9\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eVasconcelos Fernandes T et al (2015) Closing domestic nutrient cycles using microalgae. Environ Sci Technol 49:12450\u0026ndash;12456. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1021/acs.est.5b02858\u003c/span\u003e\u003cspan address=\"10.1021/acs.est.5b02858\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWang Q et al (2007) Naive Bayesian classifier for rapid assignment of rRNA sequences into the new bacterial taxonomy. Appl Environ Microbiol 73:5261\u0026ndash;5267. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1128/AEM.00062-07\u003c/span\u003e\u003cspan address=\"10.1128/AEM.00062-07\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWang C et al (2021) Comparative transcriptome analysis reveals different low-nitrogen-responsive genes in pepper cultivars. Horticulturae 7:110. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.3390/horticulturae7050110\u003c/span\u003e\u003cspan address=\"10.3390/horticulturae7050110\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWang X et al (2019) An integrated approach to characterize intestinal metabolites of four phenylethanoid glycosides and intestinal microbe-mediated antioxidant activity evaluation in vitro using UHPLC-Q-Exactive High-Resolution Mass Spectrometry and a 1, 1-Diphenyl-2-picrylhydrazyl-based assay. Front Pharmacol 10:826. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.3389/fphar.2019.00826\u003c/span\u003e\u003cspan address=\"10.3389/fphar.2019.00826\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWang YF et al (2020) L-glutamic acid induced the colonization of high-efficiency nitrogen-fixing strain Ac63 (\u003cem\u003eAzotobacter chroococcum\u003c/em\u003e) in roots of Amaranthus tricolor. Plant Soil 451:357\u0026ndash;370. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1007/s11104-020-04531-2\u003c/span\u003e\u003cspan address=\"10.1007/s11104-020-04531-2\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWei G et al (2020) Similar drivers but different effects lead to distinct ecological patterns of soil bacterial and archaeal communities. Soil Biol Biochem 144:107759. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.soilbio.2020.107759\u003c/span\u003e\u003cspan address=\"10.1016/j.soilbio.2020.107759\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWeissberg O, Aharonovich D, Sher D (2023) Phototroph-heterotroph interactions during growth and long-term starvation across \u003cem\u003eProchlorococcus\u003c/em\u003e and \u003cem\u003eAlteromonas\u003c/em\u003e diversity. ISME J 17:227\u0026ndash;237. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1038/s41396-022-01330-8\u003c/span\u003e\u003cspan address=\"10.1038/s41396-022-01330-8\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWillms IM et al (2021) The ubiquitous soil verrucomicrobial clade \u0026lsquo;\u003cem\u003eCandidatus Udaeobacter\u003c/em\u003e\u0026rsquo;shows preferences for acidic pH. Environ Microbiol Rep 13:878\u0026ndash;883. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1111/1758-2229.13006\u003c/span\u003e\u003cspan address=\"10.1111/1758-2229.13006\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWu Y et al (2013) Development of artificially induced biological soil crusts in fields and their effects on top soil. Plant Soil 370:115\u0026ndash;124. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1007/s11104-013-1611-6\u003c/span\u003e\u003cspan address=\"10.1007/s11104-013-1611-6\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eXu F et al (2022) Coordination of root auxin with the fungus \u003cem\u003ePiriformospora indica\u003c/em\u003e and bacterium \u003cem\u003eBacillus cereus\u003c/em\u003e enhances rice rhizosheath formation under soil drying. ISME J 16:801\u0026ndash;811. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1038/s41396-021-01133-3\u003c/span\u003e\u003cspan address=\"10.1038/s41396-021-01133-3\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZhang C et al (2024) Soil health improvement by inoculation of indigenous microalgae in saline soil. Environ Geochem Health 46:23. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1007/s10653-023-01790-7\u003c/span\u003e\u003cspan address=\"10.1007/s10653-023-01790-7\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZhou Y et al (2022) Long-term fertilizer postponing promotes soil organic carbon sequestration in paddy soils by accelerating lignin degradation and increasing microbial necromass. Soil Biol Biochem 175:108839. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.soilbio.2022.108839\u003c/span\u003e\u003cspan address=\"10.1016/j.soilbio.2022.108839\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZhou Y et al (2024) Microalgal extracellular polymeric substances (EPS) and their roles in cultivation, biomass harvesting, and bioproducts extraction. Bioresour Technol 406:131054. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.biortech.2024.131054\u003c/span\u003e\u003cspan address=\"10.1016/j.biortech.2024.131054\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\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":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"plant-and-soil","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"plso","sideBox":"Learn more about [Plant and Soil](https://www.springer.com/journal/11104)","snPcode":"11104","submissionUrl":"https://submission.nature.com/new-submission/11104/3","title":"Plant and Soil","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"Scenedesmus sp., Yield improvement, nitrogen-fixing bacteria, L-glutamic acid, Pepper","lastPublishedDoi":"10.21203/rs.3.rs-9526545/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-9526545/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eBackground and aims\u003c/h2\u003e \u003cp\u003eReducing chemical fertilizer dependence while sustaining crop productivity is a key challenge for sustainable agriculture. Soil microalgae are promising biofertilizers, yet their growth-promoting mechanisms remain largely unclear. This study aimed to evaluate the effects of soil microalgae on pepper growth and rhizosphere processes, and to clarify the mechanisms underlying yield enhancement.\u003c/p\u003e\u003ch2\u003eMethods\u003c/h2\u003e \u003cp\u003eThis study isolated two soil microalgae \u003cem\u003eAnabaena azotica\u003c/em\u003e and \u003cem\u003eScenedesmus\u003c/em\u003e sp., which were applied in pot and field experiments, with three groups: chemical fertilizer alone, chemical fertilizer plus \u003cem\u003eAnabaena azotica\u003c/em\u003e (A), and chemical fertilizer plus a mixed inoculum of \u003cem\u003eA. azotica\u003c/em\u003e and \u003cem\u003eScenedesmus\u003c/em\u003e sp. (AS). Transcriptomic profiling of root and third-generation amplicon sequencing of rhizosphere soil were conducted to explore the underlying mechanism.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e \u003cp\u003eThe results showed that A and AS application increased pepper yield by 36.42% and 107.87%, respectively. Especially, AS application increased soil available nitrogen content by 11.98%. Moreover, \u003cem\u003eScenedesmus\u003c/em\u003e sp. enriched nitrogen-fixing bacteria, and thereby promoted nitrogen uptake and utilization in the roots, which consequently increased pepper yield. Furthermore, L-glutamic acid was found to be the key metabolite through which \u003cem\u003eScenedesmus\u003c/em\u003e sp. exerted its yield-enhancing effects.\u003c/p\u003e\u003ch2\u003eConclusions\u003c/h2\u003e \u003cp\u003eMicroalga-derived L-glutamic acid could recruit nitrogen-fixing bacteria, strengthened rhizosphere nitrogen cycling, increased plant nitrogen acquisition, and enhanced pepper yield. This study provided important scientific evidence for the development and utilization of microalgae-based biofertilizer to improve crop yields in the sustainable agriculture.\u003c/p\u003e","manuscriptTitle":"Soil microalga-derived L-glutamic acid enhances the growth and yield of pepper by recruiting nitrogen-fixing bacteria","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-05-11 16:16:46","doi":"10.21203/rs.3.rs-9526545/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"reviewersInvited","content":"","date":"2026-05-03T19:37:07+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"Plant and Soil","date":"2026-04-28T14:03:14+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2026-04-28T06:14:24+00:00","index":"","fulltext":""},{"type":"submitted","content":"Plant and Soil","date":"2026-04-27T04:36:44+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"plant-and-soil","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"plso","sideBox":"Learn more about [Plant and Soil](https://www.springer.com/journal/11104)","snPcode":"11104","submissionUrl":"https://submission.nature.com/new-submission/11104/3","title":"Plant and Soil","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"617d47f4-e39f-4bda-94fc-e39d5d7ec379","owner":[],"postedDate":"May 11th, 2026","published":true,"recentEditorialEvents":[{"type":"reviewersInvited","content":"","date":"2026-05-03T19:37:07+00:00","index":"","fulltext":""}],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[],"tags":[],"updatedAt":"2026-05-11T16:16:46+00:00","versionOfRecord":[],"versionCreatedAt":"2026-05-11 16:16:46","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-9526545","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-9526545","identity":"rs-9526545","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}