Enhanced lead precipitation by montmorillonite-based artificial cyanobacterial biocrusts co-validated indoors and outdoors

Enhanced lead precipitation by montmorillonite-based artificial cyanobacterial biocrusts co-validated indoors and outdoors | 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 Enhanced lead precipitation by montmorillonite-based artificial cyanobacterial biocrusts co-validated indoors and outdoors Keqiang Zhou, Yujing Bi, Cui Zhang, Zijia Zhang, Ling Xia, Shaoxian Song, and 3 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6266108/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Background and aims Biocrusts are endowed with the function of effectively fixing lead in soil, which largely depends on the rich and diverse microbial communities in them. This study aims to establish artificial cyanobacterial biocrusts through the montmorillonite intervention method, in order to shorten the formation time of biocrusts and improve the lead fixation efficiency of biocrusts. Methods We used Microcoleus steenstrupii and montmorillonite to prepare inoculum, established montmorillonite-based artificial cyanobacterial biocrusts on lead-contaminated sandy soil, observed the growth and lead fixation changes of artificial cyanobacterial biocrusts, and revealed the lead fixation mechanism. Results Montmorillonite promotes the accumulation of chlorophyll a in cyanobacteria. Indoors, Microcoleus steenstrupii drives the biomineralization process, converting lead mainly into basic lead carbonate, and the enhancement of bacterial community gene function is one of the triggering factors of this process. Outdoors, montmorillonite-based artificial cyanobacterial biocrusts present a larger proportion of fixed lead, and the proportion of Fe-Mn oxide-bound and residual forms in outdoor samples is higher than that of lead chemical forms in indoor samples. Conclusion These findings highlight that the mixture of Microcoleus steenstrupii and montmorillonite plays a key role in redistributing and stabilizing soil lead, confirming the feasibility of this technology for the remediation of naturally lead-contaminated lands. Artificial cyanobacterial biocrusts Montmorillonite Lead contamination Biomineralization Prediction of biocrust function Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Introduction Biocrusts are found worldwide, particularly in ecosystems with limited water resources (Weber et al. 2022 ). Cyanobacteria in biocrusts are equipped with the ability to entangle and cement soil particles mechanically, and they are difficult to be restricted by water and nutrient limitations, thus being essential for early soil stabilization and nutrient storage in degraded lands (Zhang and Liu 2024 ). Biocrusts are enriched with enormous and abundant microbial communities, including bacteria, fungi, cyanobacteria, etc., which closely collaborate and participate in the precipitation and transformation processes of metal cations, and serve as an essential manufacturing plant for biomineralization products (Taharia et al. 2024 ). Therefore, biocrusts have great potential for remediating degraded plots containing metal ions. Biocrusts specialize in immobilizing airborne dust, allowing them to store 30–60% of landed dust worldwide (Rodriguez-Caballero et al. 2022 ). Burning of coal and mining of metal mines lead to the emission of lead-containing dust into the air, transported by prevailing winds and falling to the ground through wet and dry deposition, triggering widespread lead contamination (Hosono et al. 2022 ). When lead enters the biocrust system, microorganisms may convert lead into chemically and morphologically stable and low bioavailable lead precipitation via biosorption and biomineralization pathways (Khaleghi and Rowshanzamir 2019 ; Tan et al. 2022 ). Compared with traditional phytoremediation, biocrust remediation not only applies to more types of land remediation and the remediation process requires no mulching and fertilization, but also circumvents the disposal of contaminated biomass that comes at the end of phytoremediation (Kuang et al. 2023 ; Shen et al. 2022 ). Therefore, biocrusts have their unique advantages for lead remediation. Artificial inoculation of cyanobacteria has been demonstrated to be an effective method for accelerating the establishment of biocrusts (Rossi et al. 2022 ; Wang et al. 2025 ; Xie et al. 2024 ). Clay promoted the development of artificial cyanobacterial biocrusts (ACBs) within a short period, as evidenced by more thriving cyanobacteria, richer extracellular polysaccharides, more intact surfaces, and enhanced photosynthesis (Wang et al. 2025 ; Zhou et al. 2023 ). Montmorillonite (MMT), as a highly reactive clay mineral, not only exhibits considerable adsorption of lead but also drives the formation of soil carbonate minerals (Georgiou et al. 2022 ; Wu et al. 2024 ; Zhang and Hou 2008 ). MMT binds sand grains to form sand aggregates, strengthening soil water retention and compression resistance (Abulimiti et al. 2023 ). MMT induces the process of lead biomineralization by microalgae via processes of facilitated metabolisms of photosynthesis and urea hydrolysis, and lead ultimately exists mainly in the form of hydrocerussite (Tan et al. 2022 ). Lead can also interact with pure MMT to be immobilized via cation exchange, surface complexation, ligand exchange, structural admixture, surface precipitation, and precipitation induced by surface redox reactions (Liu et al. 2022b ). As well, cyanobacteria, nitrifying bacteria, and polymer-producing bacteria in biocrusts respectively directed the nucleation of intracellular biominerals through the metabolic activities of oxygenic photosynthesis, ammonia oxidation, and ammonification of amino acids; thus, biominerals can be exported out of the cell or remain inside the cell (Qin et al. 2020 ). The degradation of exopolysaccharides (EPS) secreted by biocrusts enhances carbon and calcium activity, prompting metal cations such as calcium ions to form carbonate precipitates (Suosaari et al. 2022 ). Therefore, the combined application of MMT and biocrusts theoretically combines the advantages of efficient establishment and lead stabilization. This study aimed to control lead leakage behavior from lead-contaminated plots in a semi-arid region. A mixture of the filamentous cyanobacterium Microcoleus steenstrupii ( M. steenstrupii ) and MMT was used to establish and cultivate ACBs while controlling the moisture characteristics of an arid region, and then to elucidate the chemomorphological changes of lead in the ACB system. After 62 days of indoor cultivation, the results showed that MMT promoted the formation of ACBs and enhanced the lead-fixing effect of the ACBs system by strengthening the functional strength of genes in the microbial community of the ACBs system. The primary chemical forms of lead are exchangeable and carbonate-bound forms. The main mineral composition is basic lead carbonate, with the chemical formula Pb 3 (CO 3 ) 2 (OH) 2 . In contrast, the percentage of lead in Fe-Mn oxide-bound and residual forms in the 2-year-old outdoor ACBs system was significantly increased compared to indoor ACBs. This study holds great significance for the remediation and protection against pollution of non-climatic lead-contaminated land, providing strong security for the inhabitants, economy, and society of arid regions. Materials and methods Preparation of artificial biocrusts M. steenstrupii, belonging to the Microcoleus I clade, was obtained from biocrusts of the Qubqi Desert (40°21′N, 109°51′E) in the Dalat Banner area of China (de Lima et al. 2021). M. steenstrupii was cultured in BG-11 culture medium at 25 °C, with a light intensity of 40 μE m−2 s−1 and a 12:12 h light-dark cycle, in an incubator with continuous aeration, for 24 days. To obtain a high concentration of M. steenstrupii , the M. steenstrupii slurry underwent static sedimentation, and the upper turbid liquid was removed. To prepare the M. steenstrupii homogenate, the M. steenstrupii slurry was ground for 20 seconds by a crusher afterwards. The M. steenstrupii biomass in each inoculum was controlled at a constant 0.008 g, while the mass of MMT was adjusted to 0.0 g, 0.8 g, 2.4 g, 4.0 g, and 5.6 g, respectively. The biocrust inoculum was prepared by mixing M. steenstrupii , 20 mL culture medium, 30 mL distilled water, and each portion of MMT. Sand with a diameter centered in the range of 150~250 μm was loaded with Pb(NO 3 ) 2 solution to control a final lead concentration of 200 mg/kg. Furthermore, the sand was subjected to a long-term aging process to simulate the soil of lead-contaminated sites. Specifically, the lead-containing bottom sand was stirred with distilled water every three days for six months. After that, the treated sand was flattened in round Petri dishes with a diameter of 14.3 cm. The height of the dunes was about 1.0 cm, and the mass was 200 g ± 0.50 g. Use disposable droppers to evenly drop each inoculum onto the dunes' surface to form ACBs. The treatment groups were labelled as M0 , M100 , M300 , M500 , and M700 according to the mass ratio of M. steenstrupii and MMT. ACBs were cultured continuously for 62 days. During the incubation period, the samples were stored in an incubator under the same controlled conditions for temperature, light intensity, and light-dark cycles as described above for cyanobacteria cultivation. 5 mL/2 d of water was added during 14 days, and 5 mL/4 d after 14 days. No culture medium was added during the cultivation period. Measurements Round samples with a diameter of 7 mm and an area of 0.385 cm 2 were taken on days 1, 5, 9, 14, 19, 36, 44, and 62 after inoculation to determine the concentration of chlorophyll a (Chl a ), exopolysaccharides (EPS), and soluble protein (SP) in ACB per unit area. Besides, the amount of lead fixation was determined on days 14 and 62, and the microbial community was additionally analyzed on day 62. Each treatment had three replicates (Petri dishes). Images of ACBs The ACB photos were obtained using a standard optical camera. The spatial and elemental distribution of M. steenstrupii and MMT was observed using Scanning Electron Microscopy/Energy Dispersive X-ray spectroscopy (SEM/EDS, Phenom 6.0, Thermo Fisher Scientific, American). Chlorophyll a Chl a concentration, measured by the method described by Zhao et al. (2021), indirectly reflected the biomass of ACBs. The Chl a from milled ACB samples was extracted in 95% ethanol for 24 h at 4 °C in the dark. Absorbance values of each extracting solution were measured separately at wavelengths of 665 nm using an ultraviolet spectrophotometer (Orion Aquamate 8000). Chl a concentration would be calculated according to Equation (1): Exopolysaccharides EPS accounted for a more significant proportion of total cellular carbohydrate (Mager and Thomas 2011). Therefore, the carbohydrate concentration was used to calculate EPS concentration in ACBs. The traditional phenol-sulfuric acid method was used to determine and quantify EPS concentration (Lan et al. 2010). ACBs samples were ground in 2 mL distilled water, incubated with 1 mL 98% sulfuric acid, and statically cooled down for 24 h at room temperature. Then, add 6 M NaOH solution, adjust pH to 7, and bring the volume to 8 mL with distilled water. The treated samples were centrifuged at 4696 g for 10 min, and the supernatant fluids were collected for subsequent assays using an exact ultraviolet spectrophotometer. Soluble protein SP, a class of proteins equipped with various physiological functions such as protecting the photosystem or balancing osmotic pressure, were extracted and measured using the method described by Lv et al. (2020) and Slonimskiy et al. (2019). Soluble proteins were extracted in ice water using 5 mL of phosphate buffer at 50 mmol/L pH 7.8 and then centrifuged at 4696 g for 10 min. Then, the concentration of proteins was determined using the Coomassie brilliant blue colorimetric method. Chemical forms and quantity of lead Different chemical forms of lead concentration were analyzed to investigate the driving effect of ACBs on lead in the bottom sediment. A sequential extraction procedure was used to determine the lead concentration in five chemical forms (Sut-Lohmann et al. 2022). The modified Tessier extraction steps are presented in Table S1. After each step, the residue was washed three times with ultrapure water and centrifuged for 20 min at 3000 g. The extracts in step 5 were obtained using a microwave system (SINEO Master-40) at 180°C for half an hour. The elemental lead analyzer was a flame atomic absorption spectrometer (Agilent 280FS), which was calibrated with a standard lead solution, and a standard curve was plotted before use. The pipeline was cleaned with a dilute nitric acid solution after each step. The extracts from steps 2 and 3 were diluted 10:1 before testing. We concisely labeled the names of the analysis results, including intracellular (IC), adsorbed (Ads), water-soluble (WS), exchangeable form (Exc), carbonate-bound form (Car), Fe-Mn oxide-bound form (Fe), organic matter-bound form (OM), residual form (Res). FT-IR analysis of ACBs Since there was a clear distinction between the upper and lower layers of the ACBs, samples were taken from each layer of the two locations for analysis. Infrared spectra of samples collected in treatment groups of M0 0 , M500 0 , M0 UL , M0 LL , M500 UL , and M500 LL were analyzed using a Fourier transform infrared spectrometer (Nexus, Thermo Nicolet, U.S.). Among them, M0 0 : the ACB grown using pure M. steenstrupii slurry inoculated on dunes without lead contamination; M0 UL : upper layer of M0 ; M0 LL : lower layer of M0 ; M500 0 : the ACB grown on lead-free dunes using pure M. steenstrupii slurry and 500 times M. steenstrupii dry weight of MMT as inoculum; M500 UL : upper layer of M500 ; M500 LL : lower layer of M500 . A mixture of 5 mg of air-dried ACB with 150 mg of KBr was ground in an agate mortar. The ground powder was already pressed into translucent disks using a bench press under 8 tons of pressure. The spectrophotometer operated over a 4000-400 cm -1 range with a resolution of 4 cm -1 . The effects of atmospheric water and carbon dioxide were always subtracted. XRD analysis of lead-loaded M. steenstrupii and MMT To avoid sand and MMT, the main components of ACBs, from affecting the detection of biomineralized products, M . steenstrupii ( Ms ), MMT, and M . steenstrupii -MMT ( Ms -MMT) were immersed into 500 mL of 0.5 mmol/L Pb(NO 3 ) 2 solution and reacted for 6 h under stirring conditions at 300 r/min. The cyanobacterial fractions were collected and ground for detection. In this case, the biomass of M. steenstrupii was 0.0075 g, and the mass ratio of M. steenstrupii to MMT was 1 to 500. XRD spectra were generated on a Bruker D8-Focus diffraction system with a Cu Kα source (λ = 1.54056 Å). They were used to determine the mineral type and crystallinity of lead in the ACBs under conditions of a scanning speed of 0.1°/min and a scanning angle range of 10-80°. High-throughput sequencing At 62 days post inoculation, the samples were ground into a powder and then mixed homogeneously. Total genomic DNA was extracted from the samples using the MagaBio DNA kit BSC48L1E-G (Bioer Technology, China) according to the instructions provided by the manufacturer. The purity and concentration of DNA were quantified using NanoDrop One (Thermo Fisher Scientific, USA). The V4 region of bacterial 16S rRNA was amplified via forward primer 515F and reverse primer 806R. The PCR system consisted of 50 μL of reagents, including 25 μL of 2x Premix Taq, 1 μL of 10 μM Primer-F, 1 μL of 10 μM Primer-R, 50 ng of DNA, and the rest of the reaction was Nuclease-free water. The PCR reaction procedure comprised an initial denaturation at 94 degrees Celsius for 5 min, followed by 30 cycles at 94 degrees Celsius for 30 s, 52 degrees Celsius for 30 s, 72 degrees Celsius for 30 s, and another extension at 72 degrees Celsius for 10 min, with a final hold at 4 degrees Celsius (BioRad S1000, Bio-Rad Laboratory, CA). The fragment length of the PCR products was determined by agarose gel electrophoresis at 290-310 bp, and the concentration of PCR products was measured using GeneTools Analysis Software (Version 4.03.05.0, SynGene). The required volume of PCR products was calculated according to the principle of equal mass, and then three parallel sets of PCR products from each sample were mixed. The PCR mixed products were recovered using EZNA® Gel Extraction Kit (Omega, USA), and the target DNA fragments were recovered by TE buffer elution. The library construction was accomplished following the NEBNext® Ultra™ II DNA Library Prep Kit for Illumina® (New England Biolabs, USA) standard procedure. The amplicon libraries were sequenced into PE250 using the Illumina Nova 6000 platform (Guangdong Magigene Biotechnology Co., Ltd. Guangzhou, China). Outdoor experiments The outdoor experiments were conducted on the balcony of the college office building (30°30’N, 114°19’E). ACBs were set up in groups M0 and M500 , with the same lead-containing sand substrate as in indoor experiments. The thickness of the sand was 2.5 cm, the dry mass of the cyanobacterial inoculum was 0.016 g, and the concentration of Chl a was 2.05 μg/cm 2 . The treatment group remained open during the experiments, and the growth period lasted 2 years. The method for determining the lead distribution in the ACBs was adapted from Mirimanoff and Wilkinson (2000) and Li et al. (2018). The Chl a concentration, the chemical form of lead, and the SEM-EDS methods were the same as those described for indoor tests. Statistical analysis Following the acquisition of raw sequencing data, all sequence data were subjected to de-priming, mass number selection and dada2 denoising, and further compared to the Silva database using qiime2 software (Wang et al. 2024). The sequences of the microorganisms were entered in the NCBI database, and BLAST procedures were conducted to obtain the genera of the microorganisms. OTUs that shared at least 95% similarity with NCBI-assigned sequences were identified to the genus level, while those with similarity below this value were not recognized (de Lima et al. 2021). Non-metric multidimensional scaling (NMDS) analysis was used to analyze structural differences in microbial communities between treatment groups. We summarized four types of metabolisms as Environmental information processing (EIP), Cellular processes (CP), Genetic information processing (GIP), and Metabolism (Me). Redundancy analysis (RDA) and Spearman correlation analyses assessed the effects of key biochemical factors and the form of lead-containing substances on microbial community structure. In addition, data for Chl a , EPS, soluble protein, and lead concentration were analyzed using one-way ANOVA and Duncan post-hoc test at p<0.05 (n = 3). Data analyses were performed using SPSS 13.0 software (SPSS, Inc, Chicago, SPSS, Inc, Chicago, IL, USA). Results Creation of ACBs The prerequisite of the study was considered to be the successful establishment of ACBs on lead-containing dunes. According to Fig. S1 , the downward seepage of the inoculum disappeared when the mass ratio of M. steenstrupii to MMT in the prepared incubation reached 1 to 700. ACBs formed a complete surface feature on 9 days post inoculation. Thereafter, the ACB gradually exhibited edge shrinkage and surface cracks during its development; this phenomenon was consistent with previous experiments and was presumably due to the drying of the biocrust, which strengthened the cohesion of cyanobacteria and clay (Zhou et al. 2023 ). Fig. S2 showed that the diameter of cyanobacterial filaments was less than 5 µm, and they were aggregated with MMT, sand, and some sticky substances. It thus appeared that cyanobacteria had successfully colonized the dune surface. Development of ACBs According to Fig. 1 (a), on the fifth day after inoculation, the Chl a concentration of all ACBs decreased compared with the inoculation time, and increased on the ninth day. The Chl a concentration of the M0 treatment groups showed a periodic increase and decrease, while the M500 treatment group maintained a sustained increase in Chl a concentration. Figure 1 (b) showed that EPS concentration tended to decrease first and then increase. M500 and M700 possessed the advantage of EPS accumulation. MMT stimulated the SP production activity of ACBs, while M0 did not significantly increase until the 19th day. There was a negative correlation between malondialdehyde concentration and clay content. In summary, MMT was conducive to establishing ACBs on lead-containing sand, and the optimal treatment group was M500 . Lead sequestration in ACBs Based on Fig. 2 (a), on day 1 post-inoculation, the main chemical form of lead in ACBs was carbonate-bound form, followed by exchangeable form and Fe-Mn oxide-bound form. At the same time, the organic-bound and residual lead contents were close to zero. The total lead concentrations in M300 and M500 treatment groups were significantly higher than those in other treatment groups ( p < 0.05). On day 62 post-inoculation, the concentrations of lead in forms of carbonate-bound, exchangeable, and Fe-Mn oxide-bound all decreased, and the organic-bound lead and residual lead appeared. The M500 treatment group obtained the highest total lead content. There was no significant difference in the lead concentration in residual form among all treatment groups ( p > 0.05). Interestingly, the carbonate-bound lead content in all treatment groups decreased with the development of ACBs, indicating that ACBs played a role in transforming the chemical form of lead. Characterization analysis of ACBs Figure 3 (a) and Table S2 reflected the effects of lead and inoculum on the functional groups of ACBs. In contrast to ACBs on pure dunes, ACBs on lead-containing dunes showed several new absorption peaks. For example, the peak at 1035 cm − 1 was mainly attributed to C-C and C-O stretching modes in the carbohydrate fraction (Ferreira et al. 2011 ; Kochan et al. 2020 ); The peak bands at 1200 − 1000 cm − 1 represented the ionized asymmetric stretching of PO-2. The peak bands included a blueshift of the characteristic peak at 1097 cm − 1 and the appearance of new peaks at 1170 cm − 1 that could be attributed to the P-O vibrational stretching modes of the phosphate group. 873 cm − 1 and the absorption bands at 1419–1457 cm − 1 were assigned to the different vibrational modes of C-O of the carbonate group CO2-3 (Lachehab et al. 2020 ; Lee et al. 2019 ; Tan et al. 2022 ). The absorption peaks in the infrared spectra of the upper and lower layers of the ACBs were similar, where M500 presented both distinct MMT and bioproduct absorption peaks. By comparing the XRD standard cards, the three major absorption peaks of Pb 3 (CO 3 ) 2 (OH) 2 and Pb 5 (PO 4 ) 3 OH are 19.8°, 27.1°, 34.0° and 21.3°, 30°, 31.3°, respectively (Chen et al. 2019 ; Shi et al. 2020 ; Zhao et al. 2020 ). According to Fig. 3 (b), the Pb 3 (CO 3 ) 2 (OH) 2 characteristic peaks appeared in both MMT and Ms -MMT, and the Pb 3 (CO 3 ) 2 (OH) 2 and Pb 5 (PO 4 ) 3 OH characteristic peaks appeared in M. steenstrupii simultaneously. These results confirmed that M. steenstrupii and MMT, as well as a mixture of both, could form lead precipitates through the mineralization pathway. The reason that only Pb 3 (CO 3 ) 2 (OH) 2 was detected in the Ms -MMT treatment could be that the content of Pb 5 (PO 4 ) 3 OH was below the limit of detection, or the high efficiency of MMT mineralization replaced the biomineralization process of Ms . Microbial community analysis Microbial community analysis played a crucial role in elucidating the conversion of exchangeable lead in ACBs. According to Figure S4, the dominant microorganisms presented at M0 and M500 included a comparable proportion of Actinobacteriota and Proteobacteria at the phylum level. MMT intervention significantly boosted the relative abundance of Cyanobacteria and Acidobacteriota. At the genus level, the relative abundance in Fig. S5(a) revealed that MMT induced a substantial shift in the top four known advantageous microorganisms from M. steenstrupii (20.97%), uncultured Chloroflexi bacterium (12.06%), Pseudonocardia sp. (9.26%), Actinomycetospora sp. (8.42%) transformed to Chroococcidiopsis sp. (22.45), Pseudonocardia sp. (14.09%), Knufia separata (6.69%), M. steenstrupii (5.47%). The NMDS stress value in Fig. S5(b) was 0.00, less than the threshold value of 0.2, indicating that the results were reliable. The NMDS results visually represented the distances between communities, illustrating that MMT led to a notable change in the bacterial community structure. The correlations between the bacterial community of M0 and certain environmental factors, biochemical indicators, and chemical forms of lead in Fig. S5(c-d) revealed several bacteria that were significantly and positively correlated with the organic matter-bond fraction. In contrast, they were all significantly and negatively correlated with the residual fraction ( p < 0.05). Significant positive correlations for lead precipitation in the carbonate fraction occurred with Microvirga sp., Rhodocytophaga sp., and for the residual lead fraction only with Actinomycetospora sp. The sole microorganisms with significant positive correlations ( p < 0.05) with carbonate-bond fraction and residual fraction of lead in M500 were respectively an uncultured Verrucomicrobiota bacterium and an uncultured Acidobacteriota bacterium. Notable positive correlations with organic lead were observed for Candidatus Nitrosocosmicus sp., Devosia submarina , and Bdellovibrio sp. What emerged was that the inoculum appeared to have reshuffled the bacterial community of the biocrust, whose mode of driving lead precipitation also reversed the normalized perception of photosynthesis in cyanobacteria as the primary pathway for lead fixation. Analysis of KEGG (Kyoto Encyclopedia of Genes and Genomes) level 1 annotations confirmed that metabolism was the core functional category represented in the microbiota of M0 and M500 , with other functions being genetic information processing, cellular processes, and environmental information processing. KEGG level 2 annotation demonstrated that the core functions of M0 and M500 were identical, and the abundance of functional sequences in M500 was significantly greater than that in M0 , except for the synthesis and degradation of ketone bodies. According to Fig. 4 , the functions of the microbiome included biosynthesis of terpenoids and steroids, synthesis and degradation of ketone bodies, valine, leucine and isoleucine biosynthesis, fatty acid biosynthesis, lipoic acid metabolism, streptomycin biosynthesis, D-glutamine and D-glutamate metabolism, biotin metabolism, D-alanine metabolism、biosynthesis of amino acids, fatty acid metabolism, pantothenate and CoA biosynthesis, carbon fixation in photosynthetic organisms, citrate cycle (TCA cycle), peptidoglycan biosynthesis, aminoacyl-tRNA biosynthesis, protein export, mismatch repair, ribosome, sulfur relay system, cell cycle - Caulobacter, bacterial chemotaxis, and bacterial secretion system. 3.6. Outdoor experiments According to Fig. S7 (a) and (c), the cyanobacterial filaments reached a diameter of about 20 µm, and the ACBs have built agglomerated modules. Figure S7 (b) reflected that the “shell” was an organic-inorganic composite layer. Fig. S7 (c) and (d) presented rare scenarios of cyanobacterial filaments with and without EPS-clay layer encapsulation, where lead was detected both on the surface of the filaments and in the EPS-clay layer. The differential characterization of the silicon and aluminum elements suggested that the clays were not directly attached to the surface of the cyanobacteria, but rather encapsulated around the cyanobacteria in a “shell” form. Based on Fig. 5 , 2 years after outdoor inoculation, the Chl a content of M500 reached 11.49 µg/cm 2 , while M0 in the same environment was only 4.13 µg/cm 2 , a 2.78-fold increase during the same period. As shown in Figure S8, the pH range of M0 and M500 was 6.69 to 6.83. The mean values of adsorbed and intracellular lead in the M0 treatment group of ACBs were higher than those of the M500 treatment group by 26.50 µg/cm 2 and 28.38 µg/cm 2 , respectively. The levels of lead in the water-soluble state were all remarkably low. However, the M500 treatment group, grown outdoors for 2 years, fixed 94.66 µg/cm 2 more lead than the M0 treatment group. According to Fig. S9, the outdoor samples showed a significantly higher percentage of lead in the Fe-Mn oxide-bound fraction, organic matter-bound fraction, and residual fraction than the indoor-grown ACBs. Discussion Establishment of biocrusts on the exterior of lead-bearing dunes Changes in Chl a concentration generally reflected changes in biomass. The initial decrease in ACBs biomass was considered a period during which ACBs established environmental tolerance. The M500 treatment group was not impacted by the reduced water addition frequency, which might be attributed to the excellent water retention properties of MMT. When water was lacking, the ACB system released more EPS, although there might be a dynamic balance between EPS consumption and synthesis. MMT induced the production of SP by ACBs, which alleviated the stress response of the ACBs system and reduced oxidative stress (Naghisharifi et al. 2024 ). It was well recognized that M. steenstrupii could capture and preserve small particles of detritus, forming biofilms at the surface and promoting the establishment of biocrusts, thus creating conditions for the formation of native soils (Li et al. 2017 ). Optical and microscopic photographs visualized the establishment of ACBs, and the successful colonization of the cyanobacteria in the inoculum was marked by an increased concentration of Chl a , which represented the adaptation of artificial cyanobacterial biocrusts to a desiccated environment by converting the amount of water added to the average annual precipitation of 28 mm. Excluding intensive environmental stresses due to the low concentration of malondialdehyde, the color fading of the border of M700 could be caused by bacterial predators such as the specialized predatory Cyanoraptor, which contained a wide range of hydrolytic enzymes that led to the rupture and death of cyanobacterial cells (Bethany et al. 2022 ). Since Chl a concentration was more remarkable in M500 . It was mainly derived from a comparable proportion of cyanobacteria based on the phylum-level community composition, the M500 inoculum more strongly drove the accumulation of photosynthetic biomass and total biomass of the biocrust than the M0 inoculum, attributed to the fact that the abundant protective substances in the M500 reservoirs markedly mitigated the overall oxidative damage of biocrusts and shaped a favorable developmental environment for microorganisms. A plausible explanation for occasional reductions in EPS was the presence of EPS-degrading bacteria, such as Proteobacteria, Bacteroidetes, and Firmicutes, that were capable of hydrolyzing EPS and even directly lysing cyanobacterial cells (Bethany et al. 2022 ; Swenson et al. 2018 ). SP was commonly used to regulate the cellular activity of microbial fractions of ACBs due to the involvement of many enzymes, transporters, and chaperones in controlling metal uptake and delivery to specific cytoarchitectural domains (De Ricco et al. 2014 ). M. steenstrupii released vast quantities of compositionally complex carbohydrates and proteins that could be specifically isolated and utilized by symbiotic bacteria owing to the narrow and non-overlapping substrate preferences of bacteria for metabolites, which in turn promoted the diversity of bacterial communities (Baran et al. 2015 ). Previous researches suggested that phylogenetic taxa of cyanobacteria were particularly abundant in inorganic fertilizer-treated soils but not in organic fertilizer-treated soils, which might imply that M500 was endowed with a more powerful legion of microorganisms that degraded organic substrates, and thus enjoyed greater availability of inorganic nutrients delivered by benthic microorganisms (Ai et al. 2018 ). The copolymerization of cyanobacteria and clay provided natural protection to cyanobacterial biomass, as they formed band-type networks under high pH conditions, which were connected by surface-to-surface contact, thus limiting microbial movement and providing a degree of protection against external perturbations (Liu et al. 2021 ). The aggregation of cyanobacteria and clay commenced with the appearance of clay shells on the surface of the cyanobacteria, which led to surface clogging. This was followed by the cellular construction of biofilms containing cells and clay shells, thus circumventing the risk of lead invasion to a certain extent (Hao et al. 2023 ). Microalgae lacked tolerance to lead by itself, yet it was inconsistent with the trend of flourishing Chl a concentration. It suggested that the sandy surface covered with a slurry-type inoculum that was not susceptible to seepage and a desiccated condition served as a protection for M. steenstrupii , thereby accelerating the fertilization of the substrate (Naveed et al. 2019 ). Increased relative functional abundance of valine, leucine, and isoleucine was considered as precursors for the biosynthesis of some secondary metabolites (e.g., alkaloids and glycosides), which participated in biotic and abiotic stress responses and resisted efficiently to osmotic imbalance under the stress of external pollution (Zhang et al. 2023b ). Inoculum-driven biomineralization of biocrusts The key role in cyanobacterial biomineralization was assigned to EPS, where amino acids, fatty acids, and other substances could be crystal nucleation sites (Li et al. 2017 ). Furthermore, metal stress triggers changed in the EPS synthesis genes of microalgae, which in turn stimulated the secretion of EPS. The EPS components, besides polysaccharides and proteins, included nucleic acids, lipids, humic substances, uronic acids, and inorganic compounds, especially pyruvate and uronic acid acyl groups, which, due to their anionic properties, possessed an affinity for metal binding (Naveed et al. 2019 ). M500 significantly enhanced the function of D-alanine metabolism in the system, and the metabolic process of alanine converted it to pyruvate, which might reinforce the ability of M500 to fix lead. Like Microcoleus chthonoplastes , M. steenstrupii was involved in atmospheric carbon dioxide deposition (Kupriyanova et al. 2007 ). The carbon concentration mechanism (CCM) of other Microcoleus sp. provided an essential tool for acclimatization to low atmospheric CO 2 levels and maintaining adequate photosynthetic activity (Kupriyanova et al. 2016 ). Microcoleus sp. relied on the CCM to increase the CO 2 concentration around the carboxylating enzyme ribulose bisphosphate carboxylase/oxygenase (RuBisCO). Moreover, intracellular pH neutrality resulted in a significant increase in the energetic efficiency of the CCM since the inorganic carbon pool was dominated by highly cell-permeable carbonic acid under acidic conditions, which required considerable energy consumption for transport to maintain internal inorganic carbon levels (Mangan et al. 2016 ). Microcoleus decomposed inorganic carbon to CO 2 with the assistance of widespread carbonic anhydrases and concentrated CO 2 via RuBisCO, triggering the generation of carbonate precipitates when metal cations were present externally (Kupriyanova et al. 2007 ). Carbonic anhydrase contributed to the mineralization of calcium carbonate under extremely harsh conditions, permitting more calcium carbonate to be deposited in the cell envelope (Qin et al. 2020 ). Research showed that in arid and nutrient-poor areas, biocrust or topsoil accomplished hydrogen oxidation and chemosynthetic carbon fixation relying on the action of hydrogenase enzymes and the expression of RuBisCO genes, respectively, and that the carbon sequestration efficiency of this process even exceeded that of photosynthesis (Bay et al. 2021 ). These arguments implied that not only could cyanobacteria produce carbonate, but other microorganisms driven by inoculum also had great potential to produce carbonate. In addition, Microcoleus cells and the biofilm communities in which they were embedded lack key nitrogenase genes, whereas they were persistently active in pathways such as nitrate transport, urea uptake degradation, etc. (Tee et al. 2020 ). A research discovered that MMT facilitated urea hydrolysis metabolism induced lead mineralization by Chlorella to produce Pb 3 (CO 3 ) 2 (OH) 2 , and M. steenstrupii might also be involved in such a biochemical process under conditions that were highly similar to those of the minerals produced in this system (Tan et al. 2022 ). Microcoleus biofilm communities were revealed to feature a variety of phosphate acquisition mechanisms, which enabled them to scavenge and utilize multiple forms of organic phosphate, solubilize inorganic phosphate, uptake dissolved inorganic phosphate, and stockpile phosphate (Tee et al. 2020 ). For instance, alkaline phosphatase could be released from extracellular proteins of microalgae, committed to stripping phosphate groups from residual DNA and RNA in the matrix (Naveed et al. 2019 ). The experimental phenomena were probably related to these biochemical processes. Previous research demonstrated that lead was detected entrapped in polyphosphate particles inside and outside the cell, which partially corroborated the occurrence of Pb 5 (PO 4 ) 3 OH particles in the M0 system (Burnat et al. 2010 ). Inoculant-driven structural and functional transformation of bacterial communities Despite the mineralization capacity of M. steenstrupii known from mechanistic analyses, M. steenstrupii failed to show a significant positive correlation with carbonate-bond fractions and residual fractions of lead in the biocrusts, which might represent a subversive adjustment of microbial job allocation in biocrust systems, with M. steenstrupii serving as only a partial source of energy for the system. Microcoleus dominated the early stages of the biocrust as a primary producer, and there was a significant correlation between the metabolites released by it and those consumed by heterotrophic bacteria, suggesting that the metabolites of Microcoleus activate the metabolic activity of heterotrophs (Swenson et al. 2018 ). While basophilic cyanobacteria ( M. steenstrupii ) raised the overall pH of the biocrust matrix, acidophilic bacteria such as Actinomycetospora sp. were still present in the bottom sand, which might depend on the unique vertical structure of ACBs. Bacteria in the moss biocrust of one site were characterized by a markedly variable vertical distribution, including photosynthetically autotrophic bacteria (e.g., cyanobacteria) and methylotrophic bacteria (e.g., Methylobacterium, Sphingomonas) in the upper layer. The bacteria in the lower layer were primarily specialized actinomycetes (e.g., Actinomycetospora, Nocardioides, etc.) with weathering properties, and the potentially acidic conditions in the lower layer promoted the growth of acidophilic bacteria to degrade the alkaline clay (Liu et al. 2022a ). The standout cyanobacterium Chroococcidiopsis sp., initially founded in hot deserts, is a globular, unicellular cyanobacterium that reinforced its antioxidant capacity by increasing the content of cellular polysaccharides, scytonemin, phycobiliproteins, and phenolic compounds (Assunção et al. 2021 ). Similar to the community in M500 , the cyanobacterial abundance of artificial biocrusts in the Shapotou Desert showed higher abundance of Chroococcidiopsis sp. over Microcoleus sp., which was a reflection of adaptation to environmental conditions (Zhao et al. 2023 ). Focusing on microorganisms that stimulated the lead fixation process, Microvirga sp. was a heterotrophic member of the microbial community of biocrusts with an integrated TCA cycling system that metabolized carbon and nitrogen biomolecules produced by other microorganisms (Bailey et al. 2014 ). Consistent with PICRUSt predictions, the presence of acetyl CoA synthesis and TCA cycling in biocrusts indicated that acetyl CoA was ultimately broken down into water and CO 2 (Zhang et al. 2022 ). Microvirga sp. proved to exert a strong ability to remove lead, generating many insoluble lead precipitates inside the cell (Luo et al. 2014 ). Rhodocytophaga sp., a soil decomposer belonging to the family Cytophagaceae, was involved in the decomposition of complex carbon components of plant tissues, such as lignocellulose, relying on secreted carbohydrate-active enzymes (Chinta et al. 2021 ; Leadbeater et al. 2021 ). Carbohydrate-degrading genes were also detected inside the cells of uncultured Verrucomicrobiota bacterium, which might be able to utilize a wide range of carbohydrates, including chitin, cellulose, pectin, polyphenols, starch, xylan, and xyloglucan (Zhang et al. 2023a ). Without relevant reports, it seemed more plausible that their decomposition of carbohydrates drove carbon cycling processes in the microenvironment of the biocrust. Thus, this evidence might indirectly express an intrinsic mechanism for the positive correlation of Microvirga sp., Rhodocytophaga sp., and uncultured Verrucomicrobiota bacterium with lead in carbonate-bond fractions. There were two possible scenarios for the relevance of bacteria related to lead: either some metabolic or intermediate product of bacteria contributed to the formation of precipitates, or bacteria indirectly regulated the formation of precipitates as an essential influence on specific biochemical cycling processes in the microecology. Although the findings exceeded the limits of common sense, the unique functions of bacteria were being progressively developed and elucidated with the exploration of Earth’s nature, building on discoveries that already partially supported the findings of the present. Feasibility of outdoor applications The presence of silica and aluminum on the cyanobacterial surface and in the aggregation zone of the substrate in both treatment groups was attributed to the depositional effects of rainfall and wind, which supplied the clay for the ACBs (Rodriguez-Caballero et al. 2022 ). The deposition process of clay minerals encapsulating cyanobacteria was found in shallow marine environments, whereas the presence of a ‘shell’ mode in sand has not been mentioned (Liu et al. 2021 ). Outdoor samples of the M500 treatment group also showed a more substantial stimulatory effect on cyanobacterial proliferation than the M0 treatment group. pH was significantly lower than that of the indoor ACBs, which were weakly acidic. In contrast to the indoor medium, the outdoor medium contained almost no lead in the carbonate-bound state. The cause of this phenomenon was attributed to the decrease in pH resulting from rainfall, which dissolves CO 2 in the soil, leading to a reduction in pH and an increase in the solubility of carbonate minerals (He et al. 2023 ). During the cultivation period, perturbations came only from climate change, as the samples were completely enclosed in a large unoccupied terrace. The lead concentrations obtained by the Tessier extraction method were higher than those determined during the study of lead distribution, suggesting that some of the lead had been transformed into a highly stable fraction. It is worth noting that the M500 treatment group showed significantly greater lead levels in ACBs and bottom sediment than M0 treatment groups, while ensuring that samples were taken from the entire thickness of the layer and excluding the bottom layer of deposited lead. This could be due to the more advanced lead sequestration effect of the early inoculum and bottom sediment. Thus, when we ignored changes in lead during the process, montmorillonite-based ACBs retained excellent lead immobilization outdoors through retention and transformation in the biocrust layer. Prospective forecast for remediation of lead-contaminated soils with Ms -MMT inoculums Toxic metal contaminants posed a serious environmental threat, and bioremediation of ACB was a vital strategy to resolve organic pollutants. The performance-evolved Ms -MMT inoculum was exceptionally well adapted to the extreme desiccation conditions. It demonstrated outstanding biomineralization, thus containing the leakage of activated lead from the soil, while potentially providing a new method of bio-recovery of lead complexes. MMT, as a widely distributed and inexpensive clay, created the practical feasibility of mass production of Ms -MMT inoculums. The MMT-induced ACBs changed the bacterial community, while most of the physiological functions still existed with higher functional enrichment, demonstrating the environmental friendliness of the Ms -MMT inoculum, which triggered the formation of adaptive communities but did not change the ecosystem function. Therefore, Ms -MMT inoculum-constructed ACBs were considered a reasonable option for the ecological remediation of lead-contaminated areas. Conclusions The extension of arid areas provides excellent development potential for ACBs. Especially in lead-contaminated areas, the biomineralization function of ACBs is irreplaceable. MMT promotes the proliferation of M. steenstrupii and strengthens their stress resistance. M. steenstrupii drives the conversion of lead to basic lead carbonate, and M. steenstrupii -MMT further improves the efficiency of lead conversion and the total amount of lead precipitates. M. steenstrupii promotes the formation of a rich and diverse microbial community. The biomineralization processes are completed by the entire ACBs system. MMT causes significant changes in the microbial community structure, but the main functions of the system are not altered, but rather enhanced. It is equally applicable to the outdoor environment. Over time, residual lead will gradually accumulate. Therefore, the rational use of ACBs contributes to the fertilization and detoxification of heavily lead-contaminated, barren, and degraded land, which may become the mainstream business for achieving a green planet. Declarations Acknowledgements This study was supported by the National Natural Science Foundation of China (Grant No. 32061123009). We appreciated the technical assistance and research support of my colleagues and my faculty and mentor, who provided me with analytical equipment. Author contributions Conceptualization: Keqiang Zhou, Yujing Bi, Ling Xia; methodology: Keqiang Zhou, Cui Zhang, Zijia Zhang, J. Viridiana Garcia-Meza; resources: Ling Xia, Shaoxian Song; soft-ware: Keqiang Zhou, María Luciana Montes; writing-original draft preparation, Keqiang Zhou, Yujing Bi; writing-review and editing, J. Viridiana Garcia-Meza, Mostafa Benzaazoua; funding acquisition, Shaoxian Song. All authors have read and agreed to the published version of the manuscript. Funding This study was supported by the National Natural Science Foundation of China (Grant No. 32061123009). Data availability The data that support this study will be shared upon reasonable request to the corresponding author. 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J Clean Prod 260: 120980. doi: https://doi.org/10.1016/j.jclepro.2020.120980. Zhao Y, Wang N, Zhang Z, Pan Y, Jia R (2021) Accelerating the development of artificial biocrusts using covers for restoration of degraded land in dryland ecosystems. Land Degrad Dev 32: 285-295. doi: https://doi.org/10.1002/ldr.3714. Zhao Y, Zhao Y, Xu W, Wang N, Zhang Z (2023) Acquiring high‐quality and sufficient propagules/fragments for cyanobacteria crust inoculation and restoration of degraded soils in a sandy desert. Land Degrad Dev 34: 1593-1597. doi: https://doi.org/10.1002/ldr.4532. Zhou K, Zhang Z, Zhang C, Xia L, Meng D, Wu L, Song S, Rosa María Torres S, María E F (2023) Rapid artificial biocrust development by cyanobacterial inoculation and clay amendment. Land Degrad Dev 34: 3728-3743. doi: https://doi.org/10.1002/ldr.4716. 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Viridiana Garcia-Meza","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"J.","middleName":"Viridiana","lastName":"Garcia-Meza","suffix":""},{"id":470531619,"identity":"2f38d996-e238-43e5-9147-e1439cd48588","order_by":7,"name":"María Luciana Montes","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"María","middleName":"Luciana","lastName":"Montes","suffix":""},{"id":470531620,"identity":"cdf750c6-7b2b-4928-8e63-9a93c103ece1","order_by":8,"name":"Mostafa Benzaazoua","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Mostafa","middleName":"","lastName":"Benzaazoua","suffix":""}],"badges":[],"createdAt":"2025-03-20 04:56:39","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-6266108/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6266108/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":84689962,"identity":"50f73e49-ce3f-46b5-b6bc-adf50dabbfcc","added_by":"auto","created_at":"2025-06-16 09:36:47","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":166804,"visible":true,"origin":"","legend":"\u003cp\u003eConcentrations of 4 bio-indicators of ACBs in 5 treatments\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-6266108/v1/c5cf14e4b7e21efd2a19b632.png"},{"id":84689963,"identity":"edf64e0f-256a-4eaf-bb3d-e69de8fd3bc5","added_by":"auto","created_at":"2025-06-16 09:36:47","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":129206,"visible":true,"origin":"","legend":"\u003cp\u003eConcentration of lead in 5 forms in 5 treatments after 1 day and 62 days post inoculation\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-6266108/v1/f55f8f266c97b29cfdd98be0.png"},{"id":84691114,"identity":"137e070b-67f1-4dd6-aabe-d35d9bb03269","added_by":"auto","created_at":"2025-06-16 09:44:47","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":103344,"visible":true,"origin":"","legend":"\u003cp\u003eFTIR spectra (a) and XRD spectra (b) of ACBs samples on day 62\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-6266108/v1/a69d8d26e2a5dfe5a577a54f.png"},{"id":84689967,"identity":"ca4a6b31-3927-4573-9fd0-78364ce8dbfe","added_by":"auto","created_at":"2025-06-16 09:36:47","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":169328,"visible":true,"origin":"","legend":"\u003cp\u003eVariation of metabolism function profiles of bacterial community in \u003cem\u003eM0\u003c/em\u003eand \u003cem\u003eM500\u003c/em\u003e at 62 days post inoculation\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-6266108/v1/6b587b6c2d1a7472e4cb0111.png"},{"id":84689969,"identity":"d8d10b8d-a666-4542-9ea2-11aa43b30b74","added_by":"auto","created_at":"2025-06-16 09:36:48","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":201494,"visible":true,"origin":"","legend":"\u003cp\u003eChl \u003cem\u003ea\u003c/em\u003econcentration(a) lead distribution and content (b) lead chemical form and concentration (c) and total lead content (d) of ACBs and montmorillonite-based ACBs outdoors\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-6266108/v1/4fd8f5681cff3e91babe5cb9.png"},{"id":86882588,"identity":"2aa55123-2cde-4ee6-9136-0ce429adfb9f","added_by":"auto","created_at":"2025-07-16 16:52:42","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1293786,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6266108/v1/6996761a-03b3-488b-b8ca-bda2b656cae2.pdf"},{"id":84689984,"identity":"f3bdb9e9-fa9f-4980-abe4-4eaa3fc79a9c","added_by":"auto","created_at":"2025-06-16 09:36:48","extension":"docx","order_by":5,"title":"","display":"","copyAsset":false,"role":"supplement","size":54405167,"visible":true,"origin":"","legend":"","description":"","filename":"SI250531.docx","url":"https://assets-eu.researchsquare.com/files/rs-6266108/v1/b40ea42d88852724c21dcb77.docx"}],"financialInterests":"","formattedTitle":"Enhanced lead precipitation by montmorillonite-based artificial cyanobacterial biocrusts co-validated indoors and outdoors","fulltext":[{"header":"Introduction","content":"\u003cp\u003eBiocrusts are found worldwide, particularly in ecosystems with limited water resources (Weber et al. \u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). Cyanobacteria in biocrusts are equipped with the ability to entangle and cement soil particles mechanically, and they are difficult to be restricted by water and nutrient limitations, thus being essential for early soil stabilization and nutrient storage in degraded lands (Zhang and Liu \u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). Biocrusts are enriched with enormous and abundant microbial communities, including bacteria, fungi, cyanobacteria, etc., which closely collaborate and participate in the precipitation and transformation processes of metal cations, and serve as an essential manufacturing plant for biomineralization products (Taharia et al. \u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). Therefore, biocrusts have great potential for remediating degraded plots containing metal ions.\u003c/p\u003e \u003cp\u003eBiocrusts specialize in immobilizing airborne dust, allowing them to store 30\u0026ndash;60% of landed dust worldwide (Rodriguez-Caballero et al. \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). Burning of coal and mining of metal mines lead to the emission of lead-containing dust into the air, transported by prevailing winds and falling to the ground through wet and dry deposition, triggering widespread lead contamination (Hosono et al. \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). When lead enters the biocrust system, microorganisms may convert lead into chemically and morphologically stable and low bioavailable lead precipitation via biosorption and biomineralization pathways (Khaleghi and Rowshanzamir \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Tan et al. \u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). Compared with traditional phytoremediation, biocrust remediation not only applies to more types of land remediation and the remediation process requires no mulching and fertilization, but also circumvents the disposal of contaminated biomass that comes at the end of phytoremediation (Kuang et al. \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2023\u003c/span\u003e; Shen et al. \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). Therefore, biocrusts have their unique advantages for lead remediation.\u003c/p\u003e \u003cp\u003eArtificial inoculation of cyanobacteria has been demonstrated to be an effective method for accelerating the establishment of biocrusts (Rossi et al. \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Wang et al. \u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e2025\u003c/span\u003e; Xie et al. \u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). Clay promoted the development of artificial cyanobacterial biocrusts (ACBs) within a short period, as evidenced by more thriving cyanobacteria, richer extracellular polysaccharides, more intact surfaces, and enhanced photosynthesis (Wang et al. \u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e2025\u003c/span\u003e; Zhou et al. \u003cspan citationid=\"CR64\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). Montmorillonite (MMT), as a highly reactive clay mineral, not only exhibits considerable adsorption of lead but also drives the formation of soil carbonate minerals (Georgiou et al. \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Wu et al. \u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e2024\u003c/span\u003e; Zhang and Hou \u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e2008\u003c/span\u003e). MMT binds sand grains to form sand aggregates, strengthening soil water retention and compression resistance (Abulimiti et al. \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). MMT induces the process of lead biomineralization by microalgae via processes of facilitated metabolisms of photosynthesis and urea hydrolysis, and lead ultimately exists mainly in the form of hydrocerussite (Tan et al. \u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). Lead can also interact with pure MMT to be immobilized via cation exchange, surface complexation, ligand exchange, structural admixture, surface precipitation, and precipitation induced by surface redox reactions (Liu et al. \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e2022b\u003c/span\u003e). As well, cyanobacteria, nitrifying bacteria, and polymer-producing bacteria in biocrusts respectively directed the nucleation of intracellular biominerals through the metabolic activities of oxygenic photosynthesis, ammonia oxidation, and ammonification of amino acids; thus, biominerals can be exported out of the cell or remain inside the cell (Qin et al. \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). The degradation of exopolysaccharides (EPS) secreted by biocrusts enhances carbon and calcium activity, prompting metal cations such as calcium ions to form carbonate precipitates (Suosaari et al. \u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). Therefore, the combined application of MMT and biocrusts theoretically combines the advantages of efficient establishment and lead stabilization.\u003c/p\u003e \u003cp\u003eThis study aimed to control lead leakage behavior from lead-contaminated plots in a semi-arid region. A mixture of the filamentous cyanobacterium \u003cem\u003eMicrocoleus steenstrupii\u003c/em\u003e (\u003cem\u003eM. steenstrupii\u003c/em\u003e) and MMT was used to establish and cultivate ACBs while controlling the moisture characteristics of an arid region, and then to elucidate the chemomorphological changes of lead in the ACB system. After 62 days of indoor cultivation, the results showed that MMT promoted the formation of ACBs and enhanced the lead-fixing effect of the ACBs system by strengthening the functional strength of genes in the microbial community of the ACBs system. The primary chemical forms of lead are exchangeable and carbonate-bound forms. The main mineral composition is basic lead carbonate, with the chemical formula Pb\u003csub\u003e3\u003c/sub\u003e(CO\u003csub\u003e3\u003c/sub\u003e)\u003csub\u003e2\u003c/sub\u003e(OH)\u003csub\u003e2\u003c/sub\u003e. In contrast, the percentage of lead in Fe-Mn oxide-bound and residual forms in the 2-year-old outdoor ACBs system was significantly increased compared to indoor ACBs. This study holds great significance for the remediation and protection against pollution of non-climatic lead-contaminated land, providing strong security for the inhabitants, economy, and society of arid regions.\u003c/p\u003e"},{"header":"Materials and methods","content":"\u003cp\u003ePreparation of artificial biocrusts\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eM.\u003c/em\u003e\u003cem\u003e\u0026nbsp;steenstrupii,\u003c/em\u003e belonging to the \u003cem\u003eMicrocoleus\u0026nbsp;\u003c/em\u003eI clade, was obtained from biocrusts of the Qubqi Desert (40\u0026deg;21\u0026prime;N, 109\u0026deg;51\u0026prime;E) in the Dalat Banner area of China (de Lima et al. 2021). \u003cem\u003eM. steenstrupii\u003c/em\u003e was cultured in BG-11 culture medium at 25 \u0026deg;C, with a light intensity of 40 \u0026mu;E\u0026thinsp;m\u0026minus;2\u0026thinsp;s\u0026minus;1 and a 12:12 h light-dark cycle, in an incubator with continuous aeration, for 24 days. To obtain a high concentration of \u003cem\u003eM. steenstrupii\u003c/em\u003e, the \u003cem\u003eM. steenstrupii\u003c/em\u003e slurry underwent static sedimentation, and the upper turbid liquid was removed. To prepare the \u003cem\u003eM. steenstrupii\u003c/em\u003e homogenate, the \u003cem\u003eM. steenstrupii\u003c/em\u003e slurry was ground for 20 seconds by a crusher afterwards. The \u003cem\u003eM. steenstrupii\u003c/em\u003e biomass in each inoculum was controlled at a constant 0.008 g, while the mass of MMT was adjusted to 0.0 g, 0.8 g, 2.4 g, 4.0 g, and 5.6 g, respectively. The biocrust inoculum was prepared by mixing \u003cem\u003eM. steenstrupii\u003c/em\u003e, 20 mL culture medium, 30 mL distilled water, and each portion of MMT.\u003c/p\u003e\n\u003cp\u003eSand with a diameter centered in the range of 150~250 \u0026mu;m was loaded with Pb(NO\u003csub\u003e3\u003c/sub\u003e)\u003csub\u003e2\u003c/sub\u003e solution to control a final lead concentration of 200 mg/kg. Furthermore, the sand was subjected to a long-term aging process to simulate the soil of lead-contaminated sites. Specifically, the lead-containing bottom sand was stirred with distilled water every three days for six months. After that, the treated sand was flattened in round Petri dishes with a diameter of 14.3 cm. The height of the dunes was about 1.0 cm, and the mass was 200 g \u0026plusmn; 0.50 g.\u003c/p\u003e\n\u003cp\u003eUse disposable droppers to evenly drop each inoculum onto the dunes\u0026apos; surface to form ACBs. The treatment groups were labelled as \u003cem\u003eM0\u003c/em\u003e, \u003cem\u003eM100\u003c/em\u003e, \u003cem\u003eM300\u003c/em\u003e, \u003cem\u003eM500\u003c/em\u003e, and \u003cem\u003eM700\u003c/em\u003e according to the mass ratio of \u003cem\u003eM. steenstrupii\u003c/em\u003e and MMT. ACBs were cultured continuously for 62 days. During the incubation period, the samples were stored in an incubator under the same controlled conditions for temperature, light intensity, and light-dark cycles as described above for cyanobacteria cultivation. 5 mL/2 d of water was added during 14 days, and 5 mL/4 d after 14 days. No culture medium was added during the cultivation period.\u003c/p\u003e\n\u003cp\u003eMeasurements\u003c/p\u003e\n\u003cp\u003eRound samples with a diameter of 7 mm and an area of 0.385 cm\u003csup\u003e2\u003c/sup\u003e were taken on days 1, 5, 9, 14, 19, 36, 44, and 62 after inoculation to determine the concentration of chlorophyll a (Chl \u003cem\u003ea\u003c/em\u003e), exopolysaccharides (EPS), and soluble protein (SP) in ACB per unit area. Besides, the amount of lead fixation was determined on days 14 and 62, and the microbial community was additionally analyzed on day 62. Each treatment had three replicates (Petri dishes).\u003c/p\u003e\n\u003cp\u003eImages of ACBs\u003c/p\u003e\n\u003cp\u003eThe ACB photos were obtained using a standard optical camera. The spatial and elemental distribution of \u003cem\u003eM.\u003c/em\u003e \u003cem\u003esteenstrupii\u003c/em\u003e and MMT was observed using Scanning Electron Microscopy/Energy Dispersive X-ray spectroscopy (SEM/EDS, Phenom 6.0, Thermo Fisher Scientific, American).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eChlorophyll a\u003c/p\u003e\n\u003cp\u003eChl \u003cem\u003ea\u003c/em\u003e concentration, measured by the method described by Zhao et al. (2021), indirectly reflected the biomass of ACBs. The Chl \u003cem\u003ea\u003c/em\u003e from milled ACB samples was extracted in 95% ethanol for 24 h at 4 \u0026deg;C in the dark. Absorbance values of each extracting solution were measured separately at wavelengths of 665 nm using an ultraviolet spectrophotometer (Orion Aquamate 8000). Chl \u003cem\u003ea\u003c/em\u003e concentration would be calculated according to Equation (1):\u003c/p\u003e\n\u003cp\u003e\u003cimg src=\"data:image/png;base64,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\"\u003e\u003c/p\u003e\n\u003cp\u003eExopolysaccharides\u003c/p\u003e\n\u003cp\u003eEPS accounted for a more significant proportion of total cellular carbohydrate (Mager and Thomas 2011). Therefore, the carbohydrate concentration was used to calculate EPS concentration in ACBs. The traditional phenol-sulfuric acid method was used to determine and quantify EPS concentration (Lan et al. 2010). ACBs samples were ground in 2 mL distilled water, incubated with 1 mL 98% sulfuric acid, and statically cooled down for 24 h at room temperature. Then, add 6 M NaOH solution, adjust pH to 7, and bring the volume to 8 mL with distilled water. The treated samples were centrifuged at 4696 g for 10 min, and the supernatant fluids were collected for subsequent assays using an exact ultraviolet spectrophotometer.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eSoluble protein\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eSP, a class of proteins equipped with various physiological functions such as protecting the photosystem or balancing osmotic pressure, were extracted and measured using the method described by Lv et al. (2020) and Slonimskiy et al. (2019). Soluble proteins were extracted in ice water using 5 mL of phosphate buffer at 50 mmol/L pH 7.8 and then centrifuged at 4696 g for 10 min. Then, the concentration of proteins was determined using the Coomassie brilliant blue colorimetric method.\u003c/p\u003e\n\u003cp\u003eChemical forms and quantity of lead\u003c/p\u003e\n\u003cp\u003eDifferent chemical forms of lead concentration were analyzed to investigate the driving effect of ACBs on lead in the bottom sediment. A sequential extraction procedure was used to determine the lead concentration in five chemical forms (Sut-Lohmann et al. 2022). The modified Tessier extraction steps are presented in Table S1. After each step, the residue was washed three times with ultrapure water and centrifuged for 20 min at 3000 g. The extracts in step 5 were obtained using a microwave system (SINEO Master-40) at 180\u0026deg;C for half an hour. The elemental lead analyzer was a flame atomic absorption spectrometer (Agilent 280FS), which was calibrated with a standard lead solution, and a standard curve was plotted before use. The pipeline was cleaned with a dilute nitric acid solution after each step. The extracts from steps 2 and 3 were diluted 10:1 before testing. We concisely labeled the names of the analysis results, including\u0026nbsp;intracellular (IC), adsorbed (Ads), water-soluble (WS), exchangeable form (Exc), carbonate-bound form (Car), Fe-Mn oxide-bound form (Fe), organic matter-bound form (OM), residual form (Res).\u003c/p\u003e\n\u003cp\u003eFT-IR analysis of ACBs\u003c/p\u003e\n\u003cp\u003eSince there was a clear distinction between the upper and lower layers of the ACBs, samples were taken from each layer of the two locations for analysis. Infrared spectra of samples collected in treatment groups of \u003cem\u003eM0\u003c/em\u003e\u003csub\u003e0\u003c/sub\u003e, \u003cem\u003eM500\u003c/em\u003e\u003csub\u003e0\u003c/sub\u003e, \u003cem\u003eM0\u003c/em\u003e\u003csub\u003eUL\u003c/sub\u003e, \u003cem\u003eM0\u003c/em\u003e\u003csub\u003eLL\u003c/sub\u003e, \u003cem\u003eM500\u003c/em\u003e\u003csub\u003eUL\u003c/sub\u003e, and \u003cem\u003eM500\u003c/em\u003e\u003csub\u003eLL\u003c/sub\u003e were analyzed using a Fourier transform infrared spectrometer (Nexus, Thermo Nicolet, U.S.). Among them, \u003cem\u003eM0\u003c/em\u003e\u003csub\u003e0\u003c/sub\u003e: the ACB grown using pure \u003cem\u003eM. steenstrupii\u003c/em\u003e slurry inoculated on dunes without lead contamination; \u003cem\u003eM0\u003c/em\u003e\u003csub\u003eUL\u003c/sub\u003e: upper layer of \u003cem\u003eM0\u003c/em\u003e; \u003cem\u003eM0\u003c/em\u003e\u003csub\u003eLL\u003c/sub\u003e: lower layer of \u003cem\u003eM0\u003c/em\u003e; \u003cem\u003eM500\u003c/em\u003e\u003csub\u003e0\u003c/sub\u003e: the ACB grown on lead-free dunes using pure \u003cem\u003eM. steenstrupii\u003c/em\u003e slurry and 500 times \u003cem\u003eM. steenstrupii\u0026nbsp;\u003c/em\u003edry weight of MMT as inoculum; \u003cem\u003eM500\u003c/em\u003e\u003csub\u003eUL\u003c/sub\u003e: upper layer of \u003cem\u003eM500\u003c/em\u003e; \u003cem\u003eM500\u003c/em\u003e\u003csub\u003eLL\u003c/sub\u003e: lower layer of \u003cem\u003eM500\u003c/em\u003e. A mixture of 5 mg of air-dried ACB with 150 mg of KBr was ground in an agate mortar. The ground powder was already pressed into translucent disks using a bench press under 8 tons of pressure. The spectrophotometer operated over a 4000-400 cm\u003csup\u003e-1\u003c/sup\u003e range with a resolution of 4 cm\u003csup\u003e-1\u003c/sup\u003e. The effects of atmospheric water and carbon dioxide were always subtracted.\u003c/p\u003e\n\u003cp\u003eXRD analysis of lead-loaded M. steenstrupii and MMT\u003c/p\u003e\n\u003cp\u003eTo avoid sand and MMT, the main components of ACBs, from affecting the detection of biomineralized products, \u003cem\u003eM\u003c/em\u003e. \u003cem\u003esteenstrupii\u0026nbsp;\u003c/em\u003e(\u003cem\u003eMs\u003c/em\u003e), MMT, and \u003cem\u003eM\u003c/em\u003e. \u003cem\u003esteenstrupii\u003c/em\u003e-MMT (\u003cem\u003eMs\u003c/em\u003e-MMT) were immersed into 500 mL of 0.5 mmol/L Pb(NO\u003csub\u003e3\u003c/sub\u003e)\u003csub\u003e2\u003c/sub\u003e solution and reacted for 6 h under stirring conditions at 300 r/min. The cyanobacterial fractions were collected and ground for detection. In this case, the biomass of \u003cem\u003eM. steenstrupii\u0026nbsp;\u003c/em\u003ewas 0.0075 g, and the mass ratio of \u003cem\u003eM. steenstrupii\u0026nbsp;\u003c/em\u003eto MMT was 1 to 500. XRD spectra were generated on a Bruker D8-Focus diffraction system with a Cu K\u0026alpha; source (\u0026lambda; = 1.54056 \u0026Aring;). They were used to determine the mineral type and crystallinity of lead in the ACBs under conditions of a scanning speed of 0.1\u0026deg;/min and a scanning angle range of 10-80\u0026deg;.\u003c/p\u003e\n\u003cp\u003eHigh-throughput sequencing\u003c/p\u003e\n\u003cp\u003eAt 62 days post inoculation, the samples were ground into a powder and then mixed homogeneously. Total genomic DNA was extracted from the samples using the MagaBio DNA kit BSC48L1E-G (Bioer Technology, China) according to the instructions provided by the manufacturer. The purity and concentration of DNA were quantified using NanoDrop One (Thermo Fisher Scientific, USA). The V4 region of bacterial 16S rRNA was amplified via forward primer 515F and reverse primer 806R. The PCR system consisted of 50 \u0026mu;L of reagents, including 25 \u0026mu;L of 2x Premix Taq, 1 \u0026mu;L of 10 \u0026mu;M Primer-F, 1 \u0026mu;L of 10 \u0026mu;M Primer-R, 50 ng of DNA, and the rest of the reaction was Nuclease-free water. The PCR reaction procedure comprised an initial denaturation at 94 degrees Celsius for 5 min, followed by 30 cycles at 94 degrees Celsius for 30 s, 52 degrees Celsius for 30 s, 72 degrees Celsius for 30 s, and another extension at 72 degrees Celsius for 10 min, with a final hold at 4 degrees Celsius (BioRad S1000, Bio-Rad Laboratory, CA). The fragment length of the PCR products was determined by agarose gel electrophoresis at 290-310 bp, and the concentration of PCR products was measured using GeneTools Analysis Software (Version 4.03.05.0, SynGene). The required volume of PCR products was calculated according to the principle of equal mass, and then three parallel sets of PCR products from each sample were mixed. The PCR mixed products were recovered using EZNA\u0026reg; Gel Extraction Kit (Omega, USA), and the target DNA fragments were recovered by TE buffer elution. The library construction was accomplished following the NEBNext\u0026reg; Ultra\u0026trade; II DNA Library Prep Kit for Illumina\u0026reg; (New England Biolabs, USA) standard procedure. The amplicon libraries were sequenced into PE250 using the Illumina Nova 6000 platform (Guangdong Magigene Biotechnology Co., Ltd. Guangzhou, China).\u003c/p\u003e\n\u003cp\u003eOutdoor experiments\u003c/p\u003e\n\u003cp\u003eThe outdoor experiments were conducted on the balcony of the college office building (30\u0026deg;30\u0026rsquo;N, 114\u0026deg;19\u0026rsquo;E). ACBs were set up in groups \u003cem\u003eM0\u003c/em\u003e and \u003cem\u003eM500\u003c/em\u003e, with the same lead-containing sand substrate as in indoor experiments. The thickness of the sand was 2.5 cm,\u0026nbsp;the dry mass of the cyanobacterial inoculum was 0.016 g, and the concentration of Chl \u003cem\u003ea\u003c/em\u003e was 2.05 \u0026mu;g/cm\u003csup\u003e2\u003c/sup\u003e. The treatment group remained open during the experiments, and the growth period lasted 2 years. The method for determining the lead distribution in the ACBs was adapted from Mirimanoff and Wilkinson (2000) and Li et al. (2018). The Chl \u003cem\u003ea\u003c/em\u003e concentration, the chemical form of lead, and the SEM-EDS methods were the same as those described for indoor tests.\u003c/p\u003e\n\u003cp\u003eStatistical analysis\u003c/p\u003e\n\u003cp\u003eFollowing the acquisition of raw sequencing data, all sequence data were subjected to de-priming, mass number selection and dada2 denoising, and further compared to the Silva database using qiime2 software (Wang et al. 2024). The sequences of the microorganisms were entered in the NCBI database, and BLAST procedures were conducted to obtain the genera of the microorganisms. OTUs that shared at least 95% similarity with NCBI-assigned sequences were identified to the genus level, while those with similarity below this value were not recognized (de Lima et al. 2021). Non-metric multidimensional scaling (NMDS) analysis was used to analyze structural differences in microbial communities between treatment groups. We summarized four types of metabolisms as\u0026nbsp;Environmental information processing (EIP), Cellular processes (CP), Genetic information processing (GIP), and Metabolism (Me).\u0026nbsp;Redundancy analysis (RDA) and Spearman correlation analyses assessed the effects of key biochemical factors and the form of lead-containing substances on\u0026nbsp;microbial community structure.\u003c/p\u003e\n\u003cp\u003eIn addition, data for Chl \u003cem\u003ea\u003c/em\u003e, EPS, soluble protein, and lead concentration were analyzed using one-way ANOVA and Duncan post-hoc test at p\u0026lt;0.05 (n = 3). Data analyses were performed using SPSS 13.0 software (SPSS, Inc, Chicago, SPSS, Inc, Chicago, IL, USA).\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003eCreation of ACBs\u003c/p\u003e \u003cp\u003eThe prerequisite of the study was considered to be the successful establishment of ACBs on lead-containing dunes. According to Fig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e, the downward seepage of the inoculum disappeared when the mass ratio of \u003cem\u003eM. steenstrupii\u003c/em\u003e to MMT in the prepared incubation reached 1 to 700. ACBs formed a complete surface feature on 9 days post inoculation. Thereafter, the ACB gradually exhibited edge shrinkage and surface cracks during its development; this phenomenon was consistent with previous experiments and was presumably due to the drying of the biocrust, which strengthened the cohesion of cyanobacteria and clay (Zhou et al. \u003cspan citationid=\"CR64\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). Fig. S2 showed that the diameter of cyanobacterial filaments was less than 5 \u0026micro;m, and they were aggregated with MMT, sand, and some sticky substances. It thus appeared that cyanobacteria had successfully colonized the dune surface.\u003c/p\u003e \u003cp\u003eDevelopment of ACBs\u003c/p\u003e \u003cp\u003eAccording to Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e(a), on the fifth day after inoculation, the Chl \u003cem\u003ea\u003c/em\u003e concentration of all ACBs decreased compared with the inoculation time, and increased on the ninth day. The Chl \u003cem\u003ea\u003c/em\u003e concentration of the \u003cem\u003eM0\u003c/em\u003e treatment groups showed a periodic increase and decrease, while the \u003cem\u003eM500\u003c/em\u003e treatment group maintained a sustained increase in Chl \u003cem\u003ea\u003c/em\u003e concentration. Figure\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e(b) showed that EPS concentration tended to decrease first and then increase. \u003cem\u003eM500\u003c/em\u003e and \u003cem\u003eM700\u003c/em\u003e possessed the advantage of EPS accumulation. MMT stimulated the SP production activity of ACBs, while \u003cem\u003eM0\u003c/em\u003e did not significantly increase until the 19th day. There was a negative correlation between malondialdehyde concentration and clay content. In summary, MMT was conducive to establishing ACBs on lead-containing sand, and the optimal treatment group was \u003cem\u003eM500\u003c/em\u003e.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eLead sequestration in ACBs\u003c/p\u003e \u003cp\u003eBased on Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e(a), on day 1 post-inoculation, the main chemical form of lead in ACBs was carbonate-bound form, followed by exchangeable form and Fe-Mn oxide-bound form. At the same time, the organic-bound and residual lead contents were close to zero. The total lead concentrations in \u003cem\u003eM300\u003c/em\u003e and \u003cem\u003eM500\u003c/em\u003e treatment groups were significantly higher than those in other treatment groups (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05). On day 62 post-inoculation, the concentrations of lead in forms of carbonate-bound, exchangeable, and Fe-Mn oxide-bound all decreased, and the organic-bound lead and residual lead appeared. The \u003cem\u003eM500\u003c/em\u003e treatment group obtained the highest total lead content. There was no significant difference in the lead concentration in residual form among all treatment groups (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026gt;\u0026thinsp;0.05). Interestingly, the carbonate-bound lead content in all treatment groups decreased with the development of ACBs, indicating that ACBs played a role in transforming the chemical form of lead.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eCharacterization analysis of ACBs\u003c/p\u003e \u003cp\u003eFigure\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e(a) and Table S2 reflected the effects of lead and inoculum on the functional groups of ACBs. In contrast to ACBs on pure dunes, ACBs on lead-containing dunes showed several new absorption peaks. For example, the peak at 1035 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e was mainly attributed to C-C and C-O stretching modes in the carbohydrate fraction (Ferreira et al. \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2011\u003c/span\u003e; Kochan et al. \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2020\u003c/span\u003e); The peak bands at 1200\u0026thinsp;\u0026minus;\u0026thinsp;1000 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e represented the ionized asymmetric stretching of PO-2. The peak bands included a blueshift of the characteristic peak at 1097 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e and the appearance of new peaks at 1170 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e that could be attributed to the P-O vibrational stretching modes of the phosphate group. 873 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e and the absorption bands at 1419\u0026ndash;1457 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e were assigned to the different vibrational modes of C-O of the carbonate group CO2-3 (Lachehab et al. \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Lee et al. \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Tan et al. \u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). The absorption peaks in the infrared spectra of the upper and lower layers of the ACBs were similar, where \u003cem\u003eM500\u003c/em\u003e presented both distinct MMT and bioproduct absorption peaks.\u003c/p\u003e \u003cp\u003eBy comparing the XRD standard cards, the three major absorption peaks of Pb\u003csub\u003e3\u003c/sub\u003e(CO\u003csub\u003e3\u003c/sub\u003e)\u003csub\u003e2\u003c/sub\u003e(OH)\u003csub\u003e2\u003c/sub\u003e and Pb\u003csub\u003e5\u003c/sub\u003e(PO\u003csub\u003e4\u003c/sub\u003e)\u003csub\u003e3\u003c/sub\u003eOH are 19.8\u0026deg;, 27.1\u0026deg;, 34.0\u0026deg; and 21.3\u0026deg;, 30\u0026deg;, 31.3\u0026deg;, respectively (Chen et al. \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Shi et al. \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Zhao et al. \u003cspan citationid=\"CR61\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). According to Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e(b), the Pb\u003csub\u003e3\u003c/sub\u003e(CO\u003csub\u003e3\u003c/sub\u003e)\u003csub\u003e2\u003c/sub\u003e(OH)\u003csub\u003e2\u003c/sub\u003e characteristic peaks appeared in both MMT and \u003cem\u003eMs\u003c/em\u003e-MMT, and the Pb\u003csub\u003e3\u003c/sub\u003e(CO\u003csub\u003e3\u003c/sub\u003e)\u003csub\u003e2\u003c/sub\u003e(OH)\u003csub\u003e2\u003c/sub\u003e and Pb\u003csub\u003e5\u003c/sub\u003e(PO\u003csub\u003e4\u003c/sub\u003e)\u003csub\u003e3\u003c/sub\u003eOH characteristic peaks appeared in \u003cem\u003eM. steenstrupii\u003c/em\u003e simultaneously. These results confirmed that \u003cem\u003eM. steenstrupii\u003c/em\u003e and MMT, as well as a mixture of both, could form lead precipitates through the mineralization pathway. The reason that only Pb\u003csub\u003e3\u003c/sub\u003e(CO\u003csub\u003e3\u003c/sub\u003e)\u003csub\u003e2\u003c/sub\u003e(OH)\u003csub\u003e2\u003c/sub\u003e was detected in the \u003cem\u003eMs\u003c/em\u003e-MMT treatment could be that the content of Pb\u003csub\u003e5\u003c/sub\u003e(PO\u003csub\u003e4\u003c/sub\u003e)\u003csub\u003e3\u003c/sub\u003eOH was below the limit of detection, or the high efficiency of MMT mineralization replaced the biomineralization process of \u003cem\u003eMs\u003c/em\u003e.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eMicrobial community analysis\u003c/p\u003e \u003cp\u003eMicrobial community analysis played a crucial role in elucidating the conversion of exchangeable lead in ACBs. According to Figure S4, the dominant microorganisms presented at \u003cem\u003eM0\u003c/em\u003e and \u003cem\u003eM500\u003c/em\u003e included a comparable proportion of Actinobacteriota and Proteobacteria at the phylum level. MMT intervention significantly boosted the relative abundance of Cyanobacteria and Acidobacteriota. At the genus level, the relative abundance in Fig. S5(a) revealed that MMT induced a substantial shift in the top four known advantageous microorganisms from \u003cem\u003eM. steenstrupii\u003c/em\u003e (20.97%), uncultured Chloroflexi bacterium (12.06%), \u003cem\u003ePseudonocardia\u003c/em\u003e sp. (9.26%), \u003cem\u003eActinomycetospora\u003c/em\u003e sp. (8.42%) transformed to \u003cem\u003eChroococcidiopsis\u003c/em\u003e sp. (22.45), \u003cem\u003ePseudonocardia\u003c/em\u003e sp. (14.09%), \u003cem\u003eKnufia separata\u003c/em\u003e (6.69%), \u003cem\u003eM. steenstrupii\u003c/em\u003e (5.47%). The NMDS stress value in Fig. S5(b) was 0.00, less than the threshold value of 0.2, indicating that the results were reliable. The NMDS results visually represented the distances between communities, illustrating that MMT led to a notable change in the bacterial community structure. The correlations between the bacterial community of \u003cem\u003eM0\u003c/em\u003e and certain environmental factors, biochemical indicators, and chemical forms of lead in Fig. S5(c-d) revealed several bacteria that were significantly and positively correlated with the organic matter-bond fraction. In contrast, they were all significantly and negatively correlated with the residual fraction (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05). Significant positive correlations for lead precipitation in the carbonate fraction occurred with \u003cem\u003eMicrovirga\u003c/em\u003e sp., \u003cem\u003eRhodocytophaga\u003c/em\u003e sp., and for the residual lead fraction only with \u003cem\u003eActinomycetospora\u003c/em\u003e sp. The sole microorganisms with significant positive correlations (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05) with carbonate-bond fraction and residual fraction of lead in \u003cem\u003eM500\u003c/em\u003e were respectively an uncultured Verrucomicrobiota bacterium and an uncultured Acidobacteriota bacterium. Notable positive correlations with organic lead were observed for Candidatus \u003cem\u003eNitrosocosmicus\u003c/em\u003e sp., \u003cem\u003eDevosia submarina\u003c/em\u003e, and \u003cem\u003eBdellovibrio\u003c/em\u003e sp. What emerged was that the inoculum appeared to have reshuffled the bacterial community of the biocrust, whose mode of driving lead precipitation also reversed the normalized perception of photosynthesis in cyanobacteria as the primary pathway for lead fixation.\u003c/p\u003e \u003cp\u003eAnalysis of KEGG (Kyoto Encyclopedia of Genes and Genomes) level 1 annotations confirmed that metabolism was the core functional category represented in the microbiota of \u003cem\u003eM0\u003c/em\u003e and \u003cem\u003eM500\u003c/em\u003e, with other functions being genetic information processing, cellular processes, and environmental information processing. KEGG level 2 annotation demonstrated that the core functions of \u003cem\u003eM0\u003c/em\u003e and \u003cem\u003eM500\u003c/em\u003e were identical, and the abundance of functional sequences in \u003cem\u003eM500\u003c/em\u003e was significantly greater than that in \u003cem\u003eM0\u003c/em\u003e, except for the synthesis and degradation of ketone bodies. According to Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e, the functions of the microbiome included biosynthesis of terpenoids and steroids, synthesis and degradation of ketone bodies, valine, leucine and isoleucine biosynthesis, fatty acid biosynthesis, lipoic acid metabolism, streptomycin biosynthesis, D-glutamine and D-glutamate metabolism, biotin metabolism, D-alanine metabolism、biosynthesis of amino acids, fatty acid metabolism, pantothenate and CoA biosynthesis, carbon fixation in photosynthetic organisms, citrate cycle (TCA cycle), peptidoglycan biosynthesis, aminoacyl-tRNA biosynthesis, protein export, mismatch repair, ribosome, sulfur relay system, cell cycle - Caulobacter, bacterial chemotaxis, and bacterial secretion system.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e3.6. Outdoor experiments\u003c/p\u003e \u003cp\u003eAccording to Fig. S7 (a) and (c), the cyanobacterial filaments reached a diameter of about 20 \u0026micro;m, and the ACBs have built agglomerated modules. Figure S7 (b) reflected that the \u0026ldquo;shell\u0026rdquo; was an organic-inorganic composite layer. Fig. S7 (c) and (d) presented rare scenarios of cyanobacterial filaments with and without EPS-clay layer encapsulation, where lead was detected both on the surface of the filaments and in the EPS-clay layer. The differential characterization of the silicon and aluminum elements suggested that the clays were not directly attached to the surface of the cyanobacteria, but rather encapsulated around the cyanobacteria in a \u0026ldquo;shell\u0026rdquo; form.\u003c/p\u003e \u003cp\u003eBased on Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e, \u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e years after outdoor inoculation, the Chl \u003cem\u003ea\u003c/em\u003e content of \u003cem\u003eM500\u003c/em\u003e reached 11.49 \u0026micro;g/cm\u003csup\u003e2\u003c/sup\u003e, while \u003cem\u003eM0\u003c/em\u003e in the same environment was only 4.13 \u0026micro;g/cm\u003csup\u003e2\u003c/sup\u003e, a 2.78-fold increase during the same period. As shown in Figure S8, the pH range of \u003cem\u003eM0\u003c/em\u003e and \u003cem\u003eM500\u003c/em\u003e was 6.69 to 6.83. The mean values of adsorbed and intracellular lead in the \u003cem\u003eM0\u003c/em\u003e treatment group of ACBs were higher than those of the \u003cem\u003eM500\u003c/em\u003e treatment group by 26.50 \u0026micro;g/cm\u003csup\u003e2\u003c/sup\u003e and 28.38 \u0026micro;g/cm\u003csup\u003e2\u003c/sup\u003e, respectively. The levels of lead in the water-soluble state were all remarkably low. However, the \u003cem\u003eM500\u003c/em\u003e treatment group, grown outdoors for 2 years, fixed 94.66 \u0026micro;g/cm\u003csup\u003e2\u003c/sup\u003e more lead than the \u003cem\u003eM0\u003c/em\u003e treatment group. According to Fig. S9, the outdoor samples showed a significantly higher percentage of lead in the Fe-Mn oxide-bound fraction, organic matter-bound fraction, and residual fraction than the indoor-grown ACBs.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eEstablishment of biocrusts on the exterior of lead-bearing dunes\u003c/p\u003e \u003cp\u003eChanges in Chl \u003cem\u003ea\u003c/em\u003e concentration generally reflected changes in biomass. The initial decrease in ACBs biomass was considered a period during which ACBs established environmental tolerance. The \u003cem\u003eM500\u003c/em\u003e treatment group was not impacted by the reduced water addition frequency, which might be attributed to the excellent water retention properties of MMT. When water was lacking, the ACB system released more EPS, although there might be a dynamic balance between EPS consumption and synthesis. MMT induced the production of SP by ACBs, which alleviated the stress response of the ACBs system and reduced oxidative stress (Naghisharifi et al. \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e2024\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eIt was well recognized that \u003cem\u003eM. steenstrupii\u003c/em\u003e could capture and preserve small particles of detritus, forming biofilms at the surface and promoting the establishment of biocrusts, thus creating conditions for the formation of native soils (Li et al. \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). Optical and microscopic photographs visualized the establishment of ACBs, and the successful colonization of the cyanobacteria in the inoculum was marked by an increased concentration of Chl \u003cem\u003ea\u003c/em\u003e, which represented the adaptation of artificial cyanobacterial biocrusts to a desiccated environment by converting the amount of water added to the average annual precipitation of 28 mm. Excluding intensive environmental stresses due to the low concentration of malondialdehyde, the color fading of the border of \u003cem\u003eM700\u003c/em\u003e could be caused by bacterial predators such as the specialized predatory Cyanoraptor, which contained a wide range of hydrolytic enzymes that led to the rupture and death of cyanobacterial cells (Bethany et al. \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). Since Chl \u003cem\u003ea\u003c/em\u003e concentration was more remarkable in \u003cem\u003eM500\u003c/em\u003e. It was mainly derived from a comparable proportion of cyanobacteria based on the phylum-level community composition, the \u003cem\u003eM500\u003c/em\u003e inoculum more strongly drove the accumulation of photosynthetic biomass and total biomass of the biocrust than the \u003cem\u003eM0\u003c/em\u003e inoculum, attributed to the fact that the abundant protective substances in the \u003cem\u003eM500\u003c/em\u003e reservoirs markedly mitigated the overall oxidative damage of biocrusts and shaped a favorable developmental environment for microorganisms. A plausible explanation for occasional reductions in EPS was the presence of EPS-degrading bacteria, such as Proteobacteria, Bacteroidetes, and Firmicutes, that were capable of hydrolyzing EPS and even directly lysing cyanobacterial cells (Bethany et al. \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Swenson et al. \u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). SP was commonly used to regulate the cellular activity of microbial fractions of ACBs due to the involvement of many enzymes, transporters, and chaperones in controlling metal uptake and delivery to specific cytoarchitectural domains (De Ricco et al. \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2014\u003c/span\u003e). \u003cem\u003eM. steenstrupii\u003c/em\u003e released vast quantities of compositionally complex carbohydrates and proteins that could be specifically isolated and utilized by symbiotic bacteria owing to the narrow and non-overlapping substrate preferences of bacteria for metabolites, which in turn promoted the diversity of bacterial communities (Baran et al. \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). Previous researches suggested that phylogenetic taxa of cyanobacteria were particularly abundant in inorganic fertilizer-treated soils but not in organic fertilizer-treated soils, which might imply that \u003cem\u003eM500\u003c/em\u003e was endowed with a more powerful legion of microorganisms that degraded organic substrates, and thus enjoyed greater availability of inorganic nutrients delivered by benthic microorganisms (Ai et al. \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). The copolymerization of cyanobacteria and clay provided natural protection to cyanobacterial biomass, as they formed band-type networks under high pH conditions, which were connected by surface-to-surface contact, thus limiting microbial movement and providing a degree of protection against external perturbations (Liu et al. \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). The aggregation of cyanobacteria and clay commenced with the appearance of clay shells on the surface of the cyanobacteria, which led to surface clogging. This was followed by the cellular construction of biofilms containing cells and clay shells, thus circumventing the risk of lead invasion to a certain extent (Hao et al. \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). Microalgae lacked tolerance to lead by itself, yet it was inconsistent with the trend of flourishing Chl \u003cem\u003ea\u003c/em\u003e concentration. It suggested that the sandy surface covered with a slurry-type inoculum that was not susceptible to seepage and a desiccated condition served as a protection for \u003cem\u003eM. steenstrupii\u003c/em\u003e, thereby accelerating the fertilization of the substrate (Naveed et al. \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). Increased relative functional abundance of valine, leucine, and isoleucine was considered as precursors for the biosynthesis of some secondary metabolites (e.g., alkaloids and glycosides), which participated in biotic and abiotic stress responses and resisted efficiently to osmotic imbalance under the stress of external pollution (Zhang et al. \u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e2023b\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eInoculum-driven biomineralization of biocrusts\u003c/p\u003e \u003cp\u003eThe key role in cyanobacterial biomineralization was assigned to EPS, where amino acids, fatty acids, and other substances could be crystal nucleation sites (Li et al. \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). Furthermore, metal stress triggers changed in the EPS synthesis genes of microalgae, which in turn stimulated the secretion of EPS. The EPS components, besides polysaccharides and proteins, included nucleic acids, lipids, humic substances, uronic acids, and inorganic compounds, especially pyruvate and uronic acid acyl groups, which, due to their anionic properties, possessed an affinity for metal binding (Naveed et al. \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). \u003cem\u003eM500\u003c/em\u003e significantly enhanced the function of D-alanine metabolism in the system, and the metabolic process of alanine converted it to pyruvate, which might reinforce the ability of \u003cem\u003eM500\u003c/em\u003e to fix lead.\u003c/p\u003e \u003cp\u003eLike \u003cem\u003eMicrocoleus\u003c/em\u003e \u003cspan type=\"ItalicUnderline\" class=\"ItalicUnderline\" name=\"Emphasis\"\u003echthonoplastes\u003c/span\u003e, \u003cem\u003eM. steenstrupii\u003c/em\u003e was involved in atmospheric carbon dioxide deposition (Kupriyanova et al. \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2007\u003c/span\u003e). The carbon concentration mechanism (CCM) of other \u003cem\u003eMicrocoleus\u003c/em\u003e sp. provided an essential tool for acclimatization to low atmospheric CO\u003csub\u003e2\u003c/sub\u003e levels and maintaining adequate photosynthetic activity (Kupriyanova et al. \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). \u003cem\u003eMicrocoleus\u003c/em\u003e sp. relied on the CCM to increase the CO\u003csub\u003e2\u003c/sub\u003e concentration around the carboxylating enzyme ribulose bisphosphate carboxylase/oxygenase (RuBisCO). Moreover, intracellular pH neutrality resulted in a significant increase in the energetic efficiency of the CCM since the inorganic carbon pool was dominated by highly cell-permeable carbonic acid under acidic conditions, which required considerable energy consumption for transport to maintain internal inorganic carbon levels (Mangan et al. \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). \u003cem\u003eMicrocoleus\u003c/em\u003e decomposed inorganic carbon to CO\u003csub\u003e2\u003c/sub\u003e with the assistance of widespread carbonic anhydrases and concentrated CO\u003csub\u003e2\u003c/sub\u003e via RuBisCO, triggering the generation of carbonate precipitates when metal cations were present externally (Kupriyanova et al. \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2007\u003c/span\u003e). Carbonic anhydrase contributed to the mineralization of calcium carbonate under extremely harsh conditions, permitting more calcium carbonate to be deposited in the cell envelope (Qin et al. \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). Research showed that in arid and nutrient-poor areas, biocrust or topsoil accomplished hydrogen oxidation and chemosynthetic carbon fixation relying on the action of hydrogenase enzymes and the expression of RuBisCO genes, respectively, and that the carbon sequestration efficiency of this process even exceeded that of photosynthesis (Bay et al. \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). These arguments implied that not only could cyanobacteria produce carbonate, but other microorganisms driven by inoculum also had great potential to produce carbonate. In addition, \u003cem\u003eMicrocoleus\u003c/em\u003e cells and the biofilm communities in which they were embedded lack key nitrogenase genes, whereas they were persistently active in pathways such as nitrate transport, urea uptake degradation, etc. (Tee et al. \u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). A research discovered that MMT facilitated urea hydrolysis metabolism induced lead mineralization by Chlorella to produce Pb\u003csub\u003e3\u003c/sub\u003e(CO\u003csub\u003e3\u003c/sub\u003e)\u003csub\u003e2\u003c/sub\u003e(OH)\u003csub\u003e2\u003c/sub\u003e, and \u003cem\u003eM. steenstrupii\u003c/em\u003e might also be involved in such a biochemical process under conditions that were highly similar to those of the minerals produced in this system (Tan et al. \u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e2022\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003cem\u003eMicrocoleus\u003c/em\u003e biofilm communities were revealed to feature a variety of phosphate acquisition mechanisms, which enabled them to scavenge and utilize multiple forms of organic phosphate, solubilize inorganic phosphate, uptake dissolved inorganic phosphate, and stockpile phosphate (Tee et al. \u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). For instance, alkaline phosphatase could be released from extracellular proteins of microalgae, committed to stripping phosphate groups from residual DNA and RNA in the matrix (Naveed et al. \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). The experimental phenomena were probably related to these biochemical processes. Previous research demonstrated that lead was detected entrapped in polyphosphate particles inside and outside the cell, which partially corroborated the occurrence of Pb\u003csub\u003e5\u003c/sub\u003e(PO\u003csub\u003e4\u003c/sub\u003e)\u003csub\u003e3\u003c/sub\u003eOH particles in the \u003cem\u003eM0\u003c/em\u003e system (Burnat et al. \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2010\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eInoculant-driven structural and functional transformation of bacterial communities\u003c/p\u003e \u003cp\u003eDespite the mineralization capacity of \u003cem\u003eM. steenstrupii\u003c/em\u003e known from mechanistic analyses, \u003cem\u003eM. steenstrupii\u003c/em\u003e failed to show a significant positive correlation with carbonate-bond fractions and residual fractions of lead in the biocrusts, which might represent a subversive adjustment of microbial job allocation in biocrust systems, with \u003cem\u003eM. steenstrupii\u003c/em\u003e serving as only a partial source of energy for the system. \u003cem\u003eMicrocoleus\u003c/em\u003e dominated the early stages of the biocrust as a primary producer, and there was a significant correlation between the metabolites released by it and those consumed by heterotrophic bacteria, suggesting that the metabolites of \u003cem\u003eMicrocoleus\u003c/em\u003e activate the metabolic activity of heterotrophs (Swenson et al. \u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). While basophilic cyanobacteria (\u003cem\u003eM. steenstrupii\u003c/em\u003e) raised the overall pH of the biocrust matrix, acidophilic bacteria such as \u003cem\u003eActinomycetospora\u003c/em\u003e sp. were still present in the bottom sand, which might depend on the unique vertical structure of ACBs. Bacteria in the moss biocrust of one site were characterized by a markedly variable vertical distribution, including photosynthetically autotrophic bacteria (e.g., cyanobacteria) and methylotrophic bacteria (e.g., Methylobacterium, Sphingomonas) in the upper layer. The bacteria in the lower layer were primarily specialized actinomycetes (e.g., Actinomycetospora, Nocardioides, etc.) with weathering properties, and the potentially acidic conditions in the lower layer promoted the growth of acidophilic bacteria to degrade the alkaline clay (Liu et al. \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2022a\u003c/span\u003e). The standout cyanobacterium \u003cem\u003eChroococcidiopsis\u003c/em\u003e sp., initially founded in hot deserts, is a globular, unicellular cyanobacterium that reinforced its antioxidant capacity by increasing the content of cellular polysaccharides, scytonemin, phycobiliproteins, and phenolic compounds (Assun\u0026ccedil;\u0026atilde;o et al. \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Similar to the community in \u003cem\u003eM500\u003c/em\u003e, the cyanobacterial abundance of artificial biocrusts in the Shapotou Desert showed higher abundance of \u003cem\u003eChroococcidiopsis\u003c/em\u003e sp. over \u003cem\u003eMicrocoleus\u003c/em\u003e sp., which was a reflection of adaptation to environmental conditions (Zhao et al. \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). Focusing on microorganisms that stimulated the lead fixation process, \u003cem\u003eMicrovirga\u003c/em\u003e sp. was a heterotrophic member of the microbial community of biocrusts with an integrated TCA cycling system that metabolized carbon and nitrogen biomolecules produced by other microorganisms (Bailey et al. \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2014\u003c/span\u003e). Consistent with PICRUSt predictions, the presence of acetyl CoA synthesis and TCA cycling in biocrusts indicated that acetyl CoA was ultimately broken down into water and CO\u003csub\u003e2\u003c/sub\u003e (Zhang et al. \u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). \u003cem\u003eMicrovirga\u003c/em\u003e sp. proved to exert a strong ability to remove lead, generating many insoluble lead precipitates inside the cell (Luo et al. \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2014\u003c/span\u003e). \u003cem\u003eRhodocytophaga\u003c/em\u003e sp., a soil decomposer belonging to the family Cytophagaceae, was involved in the decomposition of complex carbon components of plant tissues, such as lignocellulose, relying on secreted carbohydrate-active enzymes (Chinta et al. \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Leadbeater et al. \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Carbohydrate-degrading genes were also detected inside the cells of uncultured Verrucomicrobiota bacterium, which might be able to utilize a wide range of carbohydrates, including chitin, cellulose, pectin, polyphenols, starch, xylan, and xyloglucan (Zhang et al. \u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e2023a\u003c/span\u003e). Without relevant reports, it seemed more plausible that their decomposition of carbohydrates drove carbon cycling processes in the microenvironment of the biocrust. Thus, this evidence might indirectly express an intrinsic mechanism for the positive correlation of \u003cem\u003eMicrovirga\u003c/em\u003e sp., \u003cem\u003eRhodocytophaga\u003c/em\u003e sp., and uncultured Verrucomicrobiota bacterium with lead in carbonate-bond fractions. There were two possible scenarios for the relevance of bacteria related to lead: either some metabolic or intermediate product of bacteria contributed to the formation of precipitates, or bacteria indirectly regulated the formation of precipitates as an essential influence on specific biochemical cycling processes in the microecology. Although the findings exceeded the limits of common sense, the unique functions of bacteria were being progressively developed and elucidated with the exploration of Earth\u0026rsquo;s nature, building on discoveries that already partially supported the findings of the present.\u003c/p\u003e \u003cp\u003eFeasibility of outdoor applications\u003c/p\u003e \u003cp\u003eThe presence of silica and aluminum on the cyanobacterial surface and in the aggregation zone of the substrate in both treatment groups was attributed to the depositional effects of rainfall and wind, which supplied the clay for the ACBs (Rodriguez-Caballero et al. \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). The deposition process of clay minerals encapsulating cyanobacteria was found in shallow marine environments, whereas the presence of a \u0026lsquo;shell\u0026rsquo; mode in sand has not been mentioned (Liu et al. \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Outdoor samples of the \u003cem\u003eM500\u003c/em\u003e treatment group also showed a more substantial stimulatory effect on cyanobacterial proliferation than the \u003cem\u003eM0\u003c/em\u003e treatment group. pH was significantly lower than that of the indoor ACBs, which were weakly acidic. In contrast to the indoor medium, the outdoor medium contained almost no lead in the carbonate-bound state. The cause of this phenomenon was attributed to the decrease in pH resulting from rainfall, which dissolves CO\u003csub\u003e2\u003c/sub\u003e in the soil, leading to a reduction in pH and an increase in the solubility of carbonate minerals (He et al. \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). During the cultivation period, perturbations came only from climate change, as the samples were completely enclosed in a large unoccupied terrace. The lead concentrations obtained by the Tessier extraction method were higher than those determined during the study of lead distribution, suggesting that some of the lead had been transformed into a highly stable fraction. It is worth noting that the \u003cem\u003eM500\u003c/em\u003e treatment group showed significantly greater lead levels in ACBs and bottom sediment than \u003cem\u003eM0\u003c/em\u003e treatment groups, while ensuring that samples were taken from the entire thickness of the layer and excluding the bottom layer of deposited lead. This could be due to the more advanced lead sequestration effect of the early inoculum and bottom sediment. Thus, when we ignored changes in lead during the process, montmorillonite-based ACBs retained excellent lead immobilization outdoors through retention and transformation in the biocrust layer.\u003c/p\u003e \u003cp\u003eProspective forecast for remediation of lead-contaminated soils with \u003cem\u003eMs\u003c/em\u003e-MMT inoculums\u003c/p\u003e \u003cp\u003eToxic metal contaminants posed a serious environmental threat, and bioremediation of ACB was a vital strategy to resolve organic pollutants. The performance-evolved \u003cem\u003eMs\u003c/em\u003e-MMT inoculum was exceptionally well adapted to the extreme desiccation conditions. It demonstrated outstanding biomineralization, thus containing the leakage of activated lead from the soil, while potentially providing a new method of bio-recovery of lead complexes. MMT, as a widely distributed and inexpensive clay, created the practical feasibility of mass production of \u003cem\u003eMs\u003c/em\u003e-MMT inoculums. The MMT-induced ACBs changed the bacterial community, while most of the physiological functions still existed with higher functional enrichment, demonstrating the environmental friendliness of the \u003cem\u003eMs\u003c/em\u003e-MMT inoculum, which triggered the formation of adaptive communities but did not change the ecosystem function. Therefore, \u003cem\u003eMs\u003c/em\u003e-MMT inoculum-constructed ACBs were considered a reasonable option for the ecological remediation of lead-contaminated areas.\u003c/p\u003e"},{"header":"Conclusions","content":"\u003cp\u003eThe extension of arid areas provides excellent development potential for ACBs. Especially in lead-contaminated areas, the biomineralization function of ACBs is irreplaceable. MMT promotes the proliferation of \u003cem\u003eM. steenstrupii\u003c/em\u003e and strengthens their stress resistance. \u003cem\u003eM. steenstrupii\u003c/em\u003e drives the conversion of lead to basic lead carbonate, and \u003cem\u003eM. steenstrupii\u003c/em\u003e-MMT further improves the efficiency of lead conversion and the total amount of lead precipitates. \u003cem\u003eM. steenstrupii\u003c/em\u003e promotes the formation of a rich and diverse microbial community. The biomineralization processes are completed by the entire ACBs system. MMT causes significant changes in the microbial community structure, but the main functions of the system are not altered, but rather enhanced. It is equally applicable to the outdoor environment. Over time, residual lead will gradually accumulate. Therefore, the rational use of ACBs contributes to the fertilization and detoxification of heavily lead-contaminated, barren, and degraded land, which may become the mainstream business for achieving a green planet.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study was supported by the National Natural Science Foundation of China (Grant No. 32061123009). We appreciated the technical assistance and research support of my colleagues and my faculty and mentor, who provided me with analytical equipment.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eConceptualization: Keqiang Zhou, Yujing Bi, Ling Xia; methodology: Keqiang Zhou, Cui Zhang, Zijia Zhang, J. Viridiana Garcia-Meza; resources: Ling Xia, Shaoxian Song; soft-ware: Keqiang Zhou, Mar\u0026iacute;a Luciana Montes; writing-original draft preparation, Keqiang Zhou, Yujing Bi; writing-review and editing, J. Viridiana Garcia-Meza, Mostafa Benzaazoua; funding acquisition, Shaoxian Song. All authors have read and agreed to the published version of the manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study was supported by the National Natural Science Foundation of China (Grant No. 32061123009).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData availability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe data that support this study will be shared upon reasonable request to the corresponding author.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u0026nbsp;\u003c/strong\u003e The authors have no relevant financial or non-financial interests to disclose.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n \u003cli\u003eAbulimiti M, Wang J, Li C, Zhang Y, Li S (2023) Bentonite could be an eco-friendly windbreak and sand-fixing material. 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Land Degrad Dev 34: 3728-3743. doi: https://doi.org/10.1002/ldr.4716.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"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":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Artificial cyanobacterial biocrusts, Montmorillonite, Lead contamination, Biomineralization, Prediction of biocrust function","lastPublishedDoi":"10.21203/rs.3.rs-6266108/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6266108/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eBackground and aims Biocrusts are endowed with the function of effectively fixing lead in soil, which largely depends on the rich and diverse microbial communities in them. This study aims to establish artificial cyanobacterial biocrusts through the montmorillonite intervention method, in order to shorten the formation time of biocrusts and improve the lead fixation efficiency of biocrusts.\u003c/p\u003e\n\u003cp\u003eMethods We used Microcoleus steenstrupii and montmorillonite to prepare inoculum, established montmorillonite-based artificial cyanobacterial biocrusts on lead-contaminated sandy soil, observed the growth and lead fixation changes of artificial cyanobacterial biocrusts, and revealed the lead fixation mechanism.\u003c/p\u003e\n\u003cp\u003eResults\u003cstrong\u003e \u003c/strong\u003eMontmorillonite promotes the accumulation of chlorophyll a in cyanobacteria. Indoors, Microcoleus steenstrupii drives the biomineralization process, converting lead mainly into basic lead carbonate, and the enhancement of bacterial community gene function is one of the triggering factors of this process. Outdoors, montmorillonite-based artificial cyanobacterial biocrusts present a larger proportion of fixed lead, and the proportion of Fe-Mn oxide-bound and residual forms in outdoor samples is higher than that of lead chemical forms in indoor samples.\u003c/p\u003e\n\u003cp\u003eConclusion These findings highlight that the mixture of Microcoleus steenstrupii and montmorillonite plays a key role in redistributing and stabilizing soil lead, confirming the feasibility of this technology for the remediation of naturally lead-contaminated lands.\u003c/p\u003e","manuscriptTitle":"Enhanced lead precipitation by montmorillonite-based artificial cyanobacterial biocrusts co-validated indoors and outdoors","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-06-16 09:36:43","doi":"10.21203/rs.3.rs-6266108/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"13b989c2-4cd2-40f2-8737-edf415e97ef5","owner":[],"postedDate":"June 16th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2025-07-16T16:44:35+00:00","versionOfRecord":[],"versionCreatedAt":"2025-06-16 09:36:43","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-6266108","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6266108","identity":"rs-6266108","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}