Plant-Mediated Green Silver Nanoparticles for Moss Removal on Stone Heritage Buildings

Plant-Mediated Green Silver Nanoparticles for Moss Removal on Stone Heritage Buildings | 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 Article Plant-Mediated Green Silver Nanoparticles for Moss Removal on Stone Heritage Buildings Mingzhong Long, Jinru Liu, Boyan Tang, Hong Zhao, Ziqiang Ao, and 4 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-9371654/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 9 You are reading this latest preprint version Abstract Moss colonization threatens stone heritage buildings. Silver nanoparticles (AgNPs) were green-synthesized using aqueous and ethanolic extracts of cinnamon, clove, thyme, and Agastache rugosa. The spherical AgNPs exhibited surface plasmon resonance peaks at 421–446 nm, with elemental compositions varying by extract. Their efficacy against moss was evaluated by measuring chlorophyll a and soluble protein content over 30 days at different concentrations. AgNPs significantly reduced moss growth in a dose-dependent manner, and those from aqueous extracts were generally more effective than those from ethanolic extracts. This study demonstrates that plant-mediated AgNPs offer an environmentally friendly approach for moss removal on stone heritage structures, providing a promising strategy for cultural heritage conservation. Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Introduction Mosses, despite their wide prevalence, are often overlooked in both research and conservation practice. However, they exert significant erosive effects on cultural heritage structures made of stone. Moss-induced biodeterioration can be categorized into either physical–mechanical or biochemical processes. Physically, moss rhizoids penetrate rock fissures, exerting pressure that widens cracks and potentially causing rock fragmentation[1]. Under arid conditions, the contraction and expansion of mosses can physically dislodge surface particles, accelerating rock degradation[2]. Biochemically, the morphology and cellular structure of moss leaves enable high water absorption and retention capacity[3]. By maintaining moisture on the stone surface, mosses prolong rock–water interactions and create microhabitats that facilitate further rock damage by other organisms. Climate-driven freeze–thaw cycles further induce stress within the rock matrix, leading to granular disintegration and increased porosity [4]. Additionally, moss metabolism, including CO₂ production and exudate secretion, triggers redox reactions with rock minerals. These reactions result in the dissolution of elements such as Ca, Na, and Fe, and accelerate weathering [5]. Given these processes, moss growth not only accelerates material loss but also diminishes the aesthetic value of stone monuments. Consequently, effective strategies for mitigating moss-induced biodeterioration are urgently needed. Previous approaches include thermal treatment, which can effectively kill hydrated moss at 60 °C [6]. Although effective and low-cost, this method may select for thermotolerant species capable of surviving in harsh environments[7,8]. Chemical biocides such as hydrogen peroxide and phenolics have also been applied, but this approach comes with the risk of corroding the same stone substrates to be protected or leaving toxic residues[9,10]. Silver nanoparticles (AgNPs) have emerged as promising antimicrobial agents due to their broad-spectrum activity, chemical stability, and low volatility[10]. Conventional synthesis methods, however, raise environmental concerns[11]. Plant-mediated green synthesis offers a sustainable alternative, being cost-effective, scalable, and environmentally benign[12,13]. Importantly, AgNPs have been reported to have no adverse effects on the physical or chemical properties of stone materials [14]. Despite the potential of plant-derived AgNPs, studies focusing on their application for moss removal in the context of stone heritage conservation remain limited. In our previous study, plant-mediated AgNPs were applied to algae, whereas the present study focuses on their inhibitory effects on bryophytes associated with stone heritage. This study aims to develop an environmentally friendly moss control strategy, based on the green synthesis of silver nanoparticles (AgNPs) using plant extracts, for the prevention and removal of biological colonization on stone heritage building surfaces. This study incorporates aqueous and ethanolic extracts from four plants—cinnamon, clove, thyme, and Agastache rugosa—in the synthesis of silver nanoparticles and systematically characterizes the resulting AgNPs' physicochemical properties. Subsequently, the research evaluates the inhibitory effects of the library of AgNPs on mixed moss communities found on stone surfaces in karst regions. The study: (1) determines the influence of AgNP concentration and exposure time on moss chlorophyll a content and soluble protein content; (2) elucidates the correlation between the type of plant extract and the efficacy of the AgNPs; and (3) compares the effectiveness of the AgNPs with the traditional biocide benzalkonium chloride. Ultimately, the goal is to propose an application strategy for green nanomaterials suitable for cultural heritage conservation scenarios, providing a novel approach for combating biodeterioration of stone artifacts. Methods Study Area and Sample Collection The study area is located at Yunfeng Tunpu, a nationally protected cultural heritage site situated approximately 18 km south of Anshun City, Guizhou Province, China. Yunfeng Tunpu is a historic settlement formed by the descendants of Ming Dynasty military and civilian groups who were relocated from northern China during campaigns to pacify and populate the south. Over six centuries of transformation, Yunfeng Tunpu has become a significant historical and cultural relic. The residential buildings in this area are primarily constructed of limestone, making it a typical example of a stone-built architectural complex. As shown in Fig. 1, the stone surfaces exhibit visible aesthetic alterations and material deterioration. Moss Sampling: Six exterior walls with dense moss growth were randomly selected within Yunfeng Tunpu. Moss samples were carefully collected using sterilized tweezers, placed into paper envelopes, and transported to the laboratory for subsequent analysis. Sample Identification In the laboratory, the collected moss samples were air-dried at room temperature (approximately 25 °C) in a well-ventilated and dry environment. For identification, an appropriate amount of moss sample was rehydrated in distilled water until the leaves were fully expanded. The specimens were then carefully examined under an OLYMPUS SZX16 stereomicroscope, and the leaves were gently detached. The intact leaves were subsequently mounted on glass slides and examined in detail using an OLYMPUS BX53 light microscope equipped with an E3ISPM20000KPA microscope camera. The morphological characteristics of the moss specimens were compared with the descriptions in Flora Bryophytorum Sinicorum and Moss Flora of Guizhou [15,16] to identify the species. Green Synthesis of AgNPs First, the cleaned and air-dried plant leaves (cinnamon, clove, thyme, and Agastache rugosa , all purchased from a local farmers’ market) were ground into a fine powder. For each type of leaf, 10 g of powder was added to 100 mL of deionized water or 99.7% anhydrous ethanol (Tianjin Fuyu Fine Chemical Co., Ltd., China). The mixtures were heated in a water bath at 80 °C for 30 min. During the experiment, they were maintained under reflux to prevent solvent evaporation.After heating, the solutions were centrifuged at 10,000 r/min, and the supernatants were collected. The supernatants were then filtered through Whatman Grade 1 filter paper to obtain the aqueous and ethanolic extracts for each plant. A certain volume of plant extract was then added to a silver nitrate solution (0.1 mmol/L, Shenzhen Xijing Biotechnology Co., Ltd., China) under continuous heating, and the color change of the mixture was observed. (The optimal conditions for maximum silver nanoparticle yield, as determined from preliminary experiments, are summarized in Table 1. The conditions were determined by analyzing the resulting mixtures using a UV–Vis spectrophotometer (SPECORD 200 PLUS) in the range of 250–600 nm.) The mixture was centrifuged at 10,000 r/min for 20 min, and the supernatant was discarded. The pellet was washed by adding 5 mL of deionized water and centrifuging repeatedly until the supernatant became colorless. The final pellet was dried in an oven at 60 °C for 24 h. Silver nanoparticles (AgNPs) were synthesized and physicochemically characterized as previously reported[17]. Table 1 . Experimental Parameters for the Preparation of Silver Nanoparticles Plant species Extractant Heating time (min) AgNO₃ : Plant extract Temperature (°C) Cinnamon Distilled water 150 8:2 90 Ethanol 120 9:1 90 Clove Distilled water 30 9:1 80 Ethanol 180 8:2 90 Thyme Distilled water 120 9:1 90 Ethanol 30 8:2 90 Patchouli Distilled water 30 9:1 90 Ethanol 150 9:1 90 Finally, the dried AgNPs were redispersed to prepare stock solutions with concentrations of 1 μg/mL, 3 μg/mL, and 5 μg/mL for subsequent use. (The AgNPs are abbreviated as follows: the first letter indicates the plant species (C: cinnamon, S: clove, T: thyme, P: Agastache rugosa), the second letter indicates the extractant (W: aqueous, A: ethanolic), and ‘Ag’ denotes silver nanoparticles. Accordingly, the samples used are: CWAg – AgNPs synthesized from aqueous extract of cinnamon leaves, CAAg – from ethanolic extract of cinnamon leaves, SWAg – from aqueous extract of clove leaves, SAAg – from ethanolic extract of clove leaves, TWAg – from aqueous extract of thyme leaves, TAAg – from ethanolic extract of thyme leaves, PWAg – from aqueous extract of Agastache rugosa , PAAg – from ethanolic extract of Agastache rugosa , BC – benzalkonium chloride). Removal of Moss from the Surface of Stone Heritage Buildings After washing, the moss samples were evenly distributed into Petri dishes. Antimicrobial agents were added at different concentrations (AgNPs: 1 μg/mL, 3 μg/mL, 5 μg/mL; benzalkonium chloride: 0.1%, 0.3%, 0.5%), with 10 mL added to each dish containing moss. Chlorophyll a content and soluble protein content were measured on days 1, 15, and 30. Determination of Chlorophyll a Content: Moss treated with antimicrobial agents was washed and blotted dry. A 0.2 g sample was placed into a mortar, to which silica, calcium carbonate, and 2–3 mL of 95% ethanol were added. The sample was ground into a homogeneous slurry, followed by the addition of 10 mL of 95% ethanol. Grinding was continued until the tissue became colorless. The homogenate was allowed to stand for 3–5 min and then filtered into a 25 mL brown volumetric flask. The residue and filter paper were rinsed with a small amount of ethanol, and the filtrate was made up to the mark. The absorbance of the solution in a 1 cm cuvette was measured at 665 nm and 649 nm, and chlorophyll a content was calculated according to the following equation: Determination of Soluble Protein Content: A 0.3 g sample of moss was ground with silica, calcium carbonate, and 5 mL of distilled water. The homogenate was centrifuged at 8,000 r/min for 10 min. A 1 mL aliquot of the supernatant was mixed with 3 mL of Coomassie Brilliant Blue G-250 solution in a test tube, thoroughly vortexed, and incubated at room temperature for 3–5 min. Absorbance was measured at 595 nm, and each measurement was repeated three times to obtain the mean value. A standard curve was prepared using protein standards ranging from 0 to 100 μg/mL, and soluble protein content was expressed as μg per gram of fresh moss (μg/g). Results Characterization of AgNPs Mixing the plant extracts with silver nitrate solution induced a visible color change in most samples from colorless to brown (Fig. 2 ), indicating the formation of AgNPs due to the excitation of surface plasmon resonance (SPR). UV–Vis spectroscopy further confirmed nanoparticle synthesis, as all eight samples exhibited characteristic SPR peaks in the range of 421–446 nm. The peaks were observed at 422 nm, 421 nm, 445 nm, 435 nm, 442 nm, 446 nm, 437 nm, and 426 nm, with each sample displaying distinct spectral profiles (Fig. 3 ). Morphological characterization using scanning electron microscopy (SEM) revealed that the synthesized nanoparticles were predominantly spherical, with a relatively uniform distribution in size. Elemental composition was assessed by energy-dispersive X-ray spectroscopy (EDX), which verified the presence of Ag as the major constituent across all samples. CWAg, CAAg, and TAAg were composed mainly of C, O, and Ag, whereas TWAg contained only C and Ag. SWAg displayed signals for C, O, Cl, and Ag, while SAAg and PWAg were characterized by C, Cl, and Ag. PAAg exhibited a more complex composition including C, O, Al, Cl, and Ag (Fig. 4 ). These results collectively confirm the successful synthesis of AgNPs with distinct chemical signatures depending on the plant extract used, suggesting that phytochemicals present in different plant matrices influence nanoparticle composition and potentially their bioactivity while maintaining the spherical morphology. Evaluation of Biocide Performance of AgNPs Moss Identification Results Examination of numerous samples revealed that the dominant bryophyte species on Yunfeng Tunpu encompassed a variety of mosses, including Brachythecium procumbens , Erythrodontium julaceum , and Entodon prorepens , among others (Table 2 ). Table 2 Species composition of mosses on Yunfeng Tunpu. Phylum Family Genus Species Bryophyta Entodontaceae Erythrodontium Julaceous Erythrodontium Moss Bryophyta Entodontaceae Entodon Prostrate Entodon Moss Bryophyta Brachytheciaceae Brachythecium Procumbent Brachythecium Moss Bryophyta Anomodontaceae Anomodon Lesser Anomodon Moss Bryophyta Pottiaceae Barbula Unguiculate Barbula Moss Bryophyta Sphagnaceae Sphagnum Blunt-leaved Bog Moss / Prairie Sphagnum Anthocerotophyta Anthocerotaceae Anthoceros Spotted Hornwort Bryophyta Andreaeaceae Andreaea Rock Moss (Black Moss) Chlorophyll a Content in Mosses Observation of moss samples treated with biocides revealed a reduction in green pigments within the cells, resulting in an overall yellow coloration (Fig. 5 ). Figure 6 illustrates the relationship between chlorophyll a content and both biocide concentration and exposure time. On Day 1, at a biocide concentration of 0.1%, CWAg (cinnamon aqueous extract AgNPs) and BC (benzalkonium chloride) exhibited the greatest reduction of chlorophyll a content. At a concentration of 0.3%, the impact of all biocides on chlorophyll a reduction was enhanced, with CWAg and CAAg demonstrating the most outstanding efficacy, followed by BC and PAAg. At a concentration of 0.5%, CWAg and CAAg continued to show the best performance, while the effectiveness of BC began to fall below that of PAAg. By Day 15, the effects of the biocides on the reduction of chlorophyll a content became more distinct across different concentrations. At the 0.5% concentration, the chlorophyll a content in the BC-treated group dropped to 0.09 mg/L, whereas the TWAg-treated group showed a content of 2.65 mg/L, indicating relatively poor efficacy. On Day 30, even at a concentration of 0.1%, BC treatment reduced the chlorophyll a content to below 1 mg/L. At the 0.5% concentration, CAAg achieved the best results, with a chlorophyll a content of merely 0.06 mg/L, followed by BC at 0.08 mg/L. Although SAAg resulted in the lowest efficacy, it still reduced the chlorophyll a content to 2.2 mg/L. The gradual decline in chlorophyll a content following biocide treatment is consistent with the observed coloration of moss shown in Fig. 5 . These results indicate that biocide treatment significantly affects moss chlorophyll a content, and the effectiveness is more pronounced with increasing biocide concentration and prolonged treatment duration. Soluble Protein Content in Mosses As observed in Fig. 7 , the soluble protein content in normally growing mosses ranged from 324 to 339 µg/g. Experimental results indicated that on Day 1, all biocide treatments led to varying degrees of reduction of the soluble protein content. Among them, CAAg, CWAg, BC, and PAAg exhibited particularly pronounced effects. With increasing biocide concentration, the effectiveness of SWAg, SAAg, TWAg, TAAg, and PWAg also became increasingly evident. As time progressed, the performance of the nine biocides became more distinct. By Day 30, all biocide treatments had reduced the soluble protein content in the mosses to below 100 µg/g. Among all tested biocides, BC demonstrated the most outstanding efficacy, followed by AgNPs synthesized from cinnamon extract, while those synthesized from thyme extract showed relatively inferior performance. Overall, the declining trend in soluble protein content was generally consistent with the changes in chlorophyll a content (with the exception of the BC group), indicating that biocide treatment not only affected photosynthesis but also influenced the overall protein metabolism in the mosses. Discussion UV-Vis spectroscopy is a commonly used method for detecting the presence of colloidal AgNPs, which exhibit a strong absorption band due to SPR, resulting in intense light scattering between 400–450 nm [ 18 ]. In this experiment, the mixtures of eight plant extracts with AgNO₃ showed absorption bands at 420–450 nm, corresponding to the characteristic light scattering from AgNPs. Additionally, all reaction solutions turned brown, which is another indicator of the formation of AgNPs. The morphology and elemental composition of AgNPs have been described in our previous study [ 17 ] and are not repeated here. This morphology has been associated with enhanced antimicrobial activity. De Melo reported that spherical AgNPs exhibit greater antibacterial activity than their rod-shaped analogs, suggesting that the spherical nanoparticles synthesized in this study should possess high antimicrobial potential[ 19 ]. Although spheres have the smallest surface area-to-volume ratio among three-dimensional shapes, their enhanced activity may be attributed to other physicochemical properties. EDX analysis indicated variations in the elemental composition of AgNPs synthesized from different extracts of the same plant. For instance, PAAg (ethanolic extract of Agastache rugosa) contained oxygen (O) and aluminum (Al) elements not detected in PWAg (aqueous extract of Agastache rugosa), possibly because the compounds containing these elements were more efficiently extracted by ethanol and subsequently adsorbed onto the nanoparticle surfaces. The presence of elements such as O and Cl on the nanoparticle surface may influence their physicochemical properties and contribute to their overall bioactivity. The green synthesis of AgNPs employed in this study involves natural products present in the plant extracts. For example, eugenol in clove leaves contains abundant hydroxyl groups (–OH) that can act as proton sources. The electron-donating groups in the ortho - and para -positions enhance electrochemical reducing ability through inductive effects, facilitating electron transfer from eugenol and promoting the reduction of Ag⁺ to Ag⁰[ 20 ]. Additionally, compounds such as α-pinene, camphene, and carvacrol in thyme exhibit mild reducing properties, enabling the reduction of silver ions to metallic AgNPs [ 19 ]. This suggests that other plant extracts may also contain similar natural reducing agents, highlighting a benefit of the use of plant-derived components for AgNP synthesis. Given that this study aimed to evaluate the effectiveness of biocides against mixed moss communities commonly found on stone cultural heritage structures—primarily consisting of Brachythecium procumbens , Erythrodontium julaceum , and Entodon prorepens —the physiological responses of mosses to AgNP treatment were assessed based on chlorophyll a content and soluble protein content. Chlorophyll a content is a key indicator of photosynthetic capacity and directly reflects the growth status of mosses [ 21 ]. Chloroplasts, as critical organelles in plant cells, are known to be highly sensitive to biocides. For instance, mosses in environments contaminated with heavy metals such as cadmium, lead, and nickel often exhibit structural damage to chloroplasts and a consequent decrease in chlorophyll a content [ 22 ]. In this experiment, chlorophyll a content gradually decreased with increasing biocide concentration and exposure time. Particularly at the highest tested concentration of 5 µg/mL, treatments with CWAg, CAAg, and PAAg resulted in a sharp decline in chlorophyll a content within one day. Consistent with this observation, Liang et al. [ 23 ] reported that AgNPs at 2 µg/mL significantly affected the protonemata of Physcomitrella patens within 10 days.Over the 30-day observation period, even the least effective biocide treatments (TWAg and TAAg) led to a notable decrease in chlorophyll a content. However, current research on the effects of AgNPs on mosses has mainly focused on juvenile gametophytes, with limited studies on mature mosses. Thus, the precise mechanisms of chloroplast sensitivity to AgNP require further investigation. Liang et al. [ 23 ] did not detect AgNPs inside chloroplasts, suggesting that AgNPs may be unable to penetrate the chloroplast membrane. Instead, the observed reduction in thylakoid number may indirectly affect photosynthesis and chlorophyll content. Therefore, it is hypothesized that AgNPs mainly influence chlorophyll levels through extracellular or indirect mechanisms, such as generating reactive oxygen species (ROS) that damage other organelles, interacting with or disrupting the cell membrane, or causing DNA damage. In summary, AgNPs may affect chlorophyll a content through one or more mechanisms, with efficacy depending on the type of plant extract, nanoparticle size, and moss species. Soluble proteins play essential roles in nutrient regulation and cellular metabolic activity, serving as key biomarkers in plant stress biology [ 24 ]. Studies have shown that soluble protein content in plants adjusts in response to external environmental stressors to enable the plant to adapt to complex conditions[ 25 ]. Low concentrations of metal ions in the environment may promote an increase in soluble protein content, while high metal concentrations can disrupt protein integrity, leading to a decrease in content and even eventual moss death[ 26 ]. In this study, low concentrations of AgNPs significantly reduced soluble protein content as early as Day 1, with CAAg treatment reducing content threefold. Benzalkonium chloride (BC) treatment also reduced soluble protein to approximately 130 µg/g. As hypothesized, with increasing biocide concentration and exposure time, soluble protein content did not recover. This result is likely due to ROS accumulation beyond the scavenging capacity of the moss antioxidant system [ 27 ]. Concurrently, AgNP entry may enhance proteolytic enzyme activity, accelerating protein hydrolysis and further reducing content. These results indicate that low concentrations of AgNPs and BC as biocides impose significant stress on mosses, hindering their adaptation to external pressures. Combined with the diminished chlorophyll a content, it is inferred that AgNPs at 3 µg/mL and 5 µg/mL can cause moss death within one month. Compared with non-plant-mediated AgNPs, such as those synthesized by chemical reduction methods, the plant-mediated AgNPs in this study exhibited similar or enhanced biocidal activity against mosses at lower concentrations [ 10 , 14 ]. This may be attributed to the synergistic effect of phytochemicals adsorbed on the nanoparticle surface, which can enhance membrane disruption and oxidative stress [ 12 ]. In conclusion, This study focused on bryophytes as the primary research subject and investigated green-synthesized AgNPs as a potential moss removal agent. The results demonstrate that AgNPs synthesized from plant extracts effectively inhibit the growth of mosses isolated from Yunfeng Tunpu that are associated with the deterioration of ancient stone walls. The efficacy of inhibition was closely related to the type of plant extract; for example, CWAg (from cinnamon extracts) consistently outperformed TWAg (from thyme extracts). Unlike other heritage conservation studies, this research used both aqueous and ethanolic extracts from four plants to synthesize AgNPs and maintained AgNP concentrations within ranges considered acceptable for human and environmental safety. To our knowledge, this is the first exploratory study investigating the potential ability of AgNPs to control moss growth on stone heritage buildings, and it demonstrated promising results. However, the long-term efficacy of AgNPs against moss colonies on stone surfaces requires further evaluation. Effective treatment should inactivate organisms within an acceptable timeframe and prevent regrowth. Moreover, the application of AgNPs should be context-specific, as resistance levels of organisms may vary significantly across regions. Continuous improvement in AgNP synthesis technology and concentration adjustment is recommended for effective biological control. These findings are consistent with our previous study on algae [ 17 ] and further demonstrate that plant-mediated AgNPs may be effective against different photosynthetic colonizers, including bryophytes, on stone surfaces. Declarations Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. Author Contribution M.L. and J.L. wrote the main manuscript and performed experiments. B.T. and H.Z. conducted material characterization. Z.A. and Y.G. carried out field sampling and biocide treatments. L.G. and Y.C. analyzed the data and prepared the figures. M.L. and X.L. supervised the work and secured funding. All authors reviewed the manuscript. Acknowledgement This work was supported by the National Natural Science Foundation of China (32460314, 32360309, 42561014); the Science and Technology Programs of Guizhou Province (Qiankehe Jichu - ZK [2023] Yiban 147, 427). Data Availability All data generated or analysed during this study are included in this published article. The datasets are available from the corresponding author upon reasonable request. References Elgohary YM, Mansour MMA, Salem MZM. Assessment of the potential effects of plants with their secreted biochemicals on the biodeterioration of archaeological stones. Biomass Convers Biorefin. 2022. https://doi.org/10.1007/s13399-022-03300-8 . Lian B, Chen Y, Zhu LJ, et al. Degradation of carbonate rocks by microbes. Earth Sci Front. 2008;15:90–6. 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Ecotoxicol Environ Saf. 2015;113:499–505. https://doi.org/10.1016/j.ecoenv.2014.12.035 . Additional Declarations No competing interests reported. Cite Share Download PDF Status: Under Review Version 1 posted Reviews received at journal 13 May, 2026 Reviewers agreed at journal 13 May, 2026 Reviewers agreed at journal 11 May, 2026 Reviewers agreed at journal 11 May, 2026 Reviewers agreed at journal 01 May, 2026 Reviewers invited by journal 30 Apr, 2026 Editor assigned by journal 30 Apr, 2026 Submission checks completed at journal 30 Apr, 2026 First submitted to journal 30 Apr, 2026 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. 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Long","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAy0lEQVRIiWNgGAWjYBACefbm459/VNTw2B9vIFKLYc+xNGaGM8fkGM4cINaaGzlqzIwtzMYMNxKI1ME4I4ftcWEDW2LjzMcbbzDU2EQT1MLO8/a48cwdMonN0mnFFgzH0nIbCNrSnpcgwXuGLbFNOsdMgrHhMGEtDAdyDCR425gTeyTPEKvlRI6ZNFCLsYQED5FagIGcbDgDGMgGPEC/JBDjF2BUHnzwARiVBuyHN974UGNDhMOQgIFEAinKIVpI1TEKRsEoGAUjAwAAVtRB3gvdOz4AAAAASUVORK5CYII=","orcid":"","institution":"Guizhou Minzu University","correspondingAuthor":true,"prefix":"","firstName":"Mingzhong","middleName":"","lastName":"Long","suffix":""},{"id":636105089,"identity":"fb1389dc-79dc-4b4c-b8c4-87a774461bc6","order_by":1,"name":"Jinru Liu","email":"","orcid":"","institution":"Guizhou Minzu University","correspondingAuthor":false,"prefix":"","firstName":"Jinru","middleName":"","lastName":"Liu","suffix":""},{"id":636105090,"identity":"fde30de3-e49d-41e5-9321-3b206ee9f6e5","order_by":2,"name":"Boyan Tang","email":"","orcid":"","institution":"Guizhou Minzu University","correspondingAuthor":false,"prefix":"","firstName":"Boyan","middleName":"","lastName":"Tang","suffix":""},{"id":636105092,"identity":"e3c798d6-4b94-4ebf-acb0-902998b393a1","order_by":3,"name":"Hong Zhao","email":"","orcid":"","institution":"Guizhou Minzu University","correspondingAuthor":false,"prefix":"","firstName":"Hong","middleName":"","lastName":"Zhao","suffix":""},{"id":636105093,"identity":"8e69211e-9eac-436b-8fa9-46687f32f47f","order_by":4,"name":"Ziqiang Ao","email":"","orcid":"","institution":"Guizhou Minzu University","correspondingAuthor":false,"prefix":"","firstName":"Ziqiang","middleName":"","lastName":"Ao","suffix":""},{"id":636105096,"identity":"f216881f-60fc-4327-9a18-edd1fafa7552","order_by":5,"name":"Yun Guo","email":"","orcid":"","institution":"Guizhou Minzu University","correspondingAuthor":false,"prefix":"","firstName":"Yun","middleName":"","lastName":"Guo","suffix":""},{"id":636105098,"identity":"5cd442be-a2cc-4563-8d18-3851cc4e1e7d","order_by":6,"name":"Li Gan","email":"","orcid":"","institution":"Guizhou Minzu University","correspondingAuthor":false,"prefix":"","firstName":"Li","middleName":"","lastName":"Gan","suffix":""},{"id":636105102,"identity":"078636d6-3e3a-4add-a1fc-13f4aaa6fd85","order_by":7,"name":"Yingqiu Chen","email":"","orcid":"","institution":"Guizhou Minzu University","correspondingAuthor":false,"prefix":"","firstName":"Yingqiu","middleName":"","lastName":"Chen","suffix":""},{"id":636105103,"identity":"11a2c957-94b9-4b18-ac96-a56b6504f182","order_by":8,"name":"Xiaona Li","email":"","orcid":"","institution":"Guizhou Normal University","correspondingAuthor":false,"prefix":"","firstName":"Xiaona","middleName":"","lastName":"Li","suffix":""}],"badges":[],"createdAt":"2026-04-09 18:09:44","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-9371654/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-9371654/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":108961302,"identity":"d15b1420-34f8-43d2-99d6-78175a97dc34","added_by":"auto","created_at":"2026-05-11 08:46:34","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":2080395,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eThe location of Yunfeng Tunpu in the study area, the moss coverage, and the microscopic dissolution of rock surfaces induced by moss.\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"floatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-9371654/v1/178038877b2306ef26c4a4c4.png"},{"id":108961303,"identity":"b6a2de54-2229-4808-80a4-12371067ec17","added_by":"auto","created_at":"2026-05-11 08:46:34","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":114990,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003e(a) Plant extract and (b) Silver nanoparticles\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"floatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-9371654/v1/e87cf959cb90eaa07b53df2d.png"},{"id":108977829,"identity":"6ce44519-7a42-4103-a696-c98b6ad06d1f","added_by":"auto","created_at":"2026-05-11 11:33:06","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":616075,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eUV–Vis spectra of eight silver nanoparticle solutions\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"floatimage3.png","url":"https://assets-eu.researchsquare.com/files/rs-9371654/v1/3d20a3dac4376f68c510a11a.png"},{"id":108961299,"identity":"3f963de3-d7ee-462b-a89c-225e7aa8389d","added_by":"auto","created_at":"2026-05-11 08:46:34","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":301207,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eRepresentative SEM micrographs and EDX spectra of AgNPs. Data adapted from our previous study (Long et al., 2014).Abbreviations: CWAg (a), CAAg (b), SWAg (c), SAAg (d), TWAg (e), TAAg (f), PWAg (g), PAAg (h).\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"floatimage4.png","url":"https://assets-eu.researchsquare.com/files/rs-9371654/v1/2686e3c7cce5227b9d9d1f04.png"},{"id":108961304,"identity":"cbb8ab38-fe94-4119-896a-9470b50afb0f","added_by":"auto","created_at":"2026-05-11 08:46:35","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":476685,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eMicroscopic images of moss cells before (a) and after (b) treatment with silver nanoparticles\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"floatimage5.png","url":"https://assets-eu.researchsquare.com/files/rs-9371654/v1/e1f193812de469ad93c63ee2.png"},{"id":108961300,"identity":"3ffe68ba-1a9a-4f16-8753-93fe8a00c674","added_by":"auto","created_at":"2026-05-11 08:46:34","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":428833,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eChlorophyll a concentration of moss after treatment with various fungicides at different concentrations.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eNote: The color gradient (red → yellow → blue) indicates a decrease in chlorophyll a. The left y-axis shows fungicide types, the right y-axis shows chlorophyll a concentration (mg/L), and the x-axis shows fungicide concentrations. Data are shown for day 1, day 15, and day 30 after treatment.\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"floatimage6.png","url":"https://assets-eu.researchsquare.com/files/rs-9371654/v1/f70b43dc882b2e18574cbf9f.png"},{"id":108961301,"identity":"f1a87a94-7623-48db-bbab-419bf7986d15","added_by":"auto","created_at":"2026-05-11 08:46:34","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":1144465,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSoluble protein content of moss after treatment with various fungicides at different concentrations.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eNote: The x-axis represents fungicide types, and the y-axis represents soluble protein content. Panels a–c show day 1, d–f day 15, and g–i day 30 under three fungicide concentrations.\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"floatimage7.png","url":"https://assets-eu.researchsquare.com/files/rs-9371654/v1/22ea1700d7e5eb742471824b.png"},{"id":108979836,"identity":"f6ae5f3d-1fb9-4652-858f-fe747218873f","added_by":"auto","created_at":"2026-05-11 12:01:49","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":5432319,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-9371654/v1/1f75102c-4ccf-497d-82e4-f5fdec4cbd4d.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Plant-Mediated Green Silver Nanoparticles for Moss Removal on Stone Heritage Buildings","fulltext":[{"header":"Introduction","content":"\u003cp\u003eMosses, despite their wide prevalence, are often overlooked in both research and conservation practice. However, they exert significant erosive effects on cultural heritage structures made of stone. Moss-induced biodeterioration can be categorized into either physical\u0026ndash;mechanical or biochemical processes. Physically, moss rhizoids penetrate rock fissures, exerting pressure that widens cracks and potentially causing rock fragmentation[1]. Under arid conditions, the contraction and expansion of mosses can physically dislodge surface particles, accelerating rock degradation[2].\u0026nbsp;Biochemically, the morphology and cellular structure of moss leaves enable high water absorption and retention capacity[3]. By maintaining moisture on the stone surface, mosses prolong rock\u0026ndash;water interactions and create microhabitats that facilitate further rock damage by other organisms. Climate-driven freeze\u0026ndash;thaw cycles further induce stress within the rock matrix, leading to granular disintegration and increased porosity [4]. Additionally, moss metabolism, including CO₂ production and exudate secretion, triggers redox reactions with rock minerals. These reactions result in the dissolution of elements such as Ca, Na, and Fe, and accelerate weathering [5].\u003c/p\u003e\n\u003cp\u003eGiven these processes, moss growth not only accelerates material loss but also diminishes the aesthetic value of stone monuments. Consequently, effective strategies for mitigating moss-induced biodeterioration are urgently needed. Previous approaches include thermal treatment, which can effectively kill hydrated moss at 60 \u0026deg;C [6]. Although effective and low-cost, this method may select for thermotolerant species capable of surviving in harsh environments[7,8]. Chemical biocides such as hydrogen peroxide and phenolics have also been applied, but this approach comes with the risk of corroding the same stone substrates to be protected or leaving toxic residues[9,10]. Silver nanoparticles (AgNPs) have emerged as promising antimicrobial agents due to their broad-spectrum activity, chemical stability, and low volatility[10]. Conventional synthesis methods, however, raise environmental concerns[11]. Plant-mediated green synthesis offers a sustainable alternative, being cost-effective, scalable, and environmentally benign[12,13]. Importantly, AgNPs have been reported to have no adverse effects on the physical or chemical properties of stone materials [14]. Despite the potential of plant-derived AgNPs, studies focusing on their application for moss removal in the context of stone heritage conservation remain limited. In our previous study, plant-mediated AgNPs were applied to algae, whereas the present study focuses on their inhibitory effects on bryophytes associated with stone heritage. This study aims to develop an environmentally friendly moss control strategy, based on the green synthesis of silver nanoparticles (AgNPs) using plant extracts, for the prevention and removal of biological colonization on stone heritage building surfaces. This study incorporates aqueous and ethanolic extracts from four plants\u0026mdash;cinnamon, clove, thyme, and Agastache rugosa\u0026mdash;in the synthesis of silver nanoparticles and systematically characterizes the resulting AgNPs\u0026apos; physicochemical properties. Subsequently, the research evaluates the inhibitory effects of the library of AgNPs on mixed moss communities found on stone surfaces in karst regions. The study: (1) determines the influence of AgNP concentration and exposure time on moss chlorophyll a content and soluble protein content; (2) elucidates the correlation between the type of plant extract and the efficacy of the AgNPs; and (3) compares the effectiveness of the AgNPs with the traditional biocide benzalkonium chloride. Ultimately, the goal is to propose an application strategy for green nanomaterials suitable for cultural heritage conservation scenarios, providing a novel approach for combating biodeterioration of stone artifacts.\u003c/p\u003e"},{"header":"Methods","content":"\u003cp\u003e\u003cstrong\u003eStudy Area and Sample Collection\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe study area is located at Yunfeng Tunpu, a nationally protected cultural heritage site situated approximately 18 km south of Anshun City, Guizhou Province, China. Yunfeng Tunpu is a historic settlement formed by the descendants of Ming Dynasty military and civilian groups who were relocated from northern China during campaigns to pacify and populate the south. Over six centuries of transformation, Yunfeng Tunpu has become a significant historical and cultural relic. The residential buildings in this area are primarily constructed of limestone, making it a typical example of a stone-built architectural complex. As shown in Fig. 1, the stone surfaces exhibit visible aesthetic alterations and material deterioration.\u003c/p\u003e\n\u003cp\u003eMoss Sampling: Six exterior walls with dense moss growth were randomly selected within Yunfeng Tunpu. Moss samples were carefully collected using sterilized tweezers, placed into paper envelopes, and transported to the laboratory for subsequent analysis.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSample Identification\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eIn the laboratory, the collected moss samples were air-dried at room temperature (approximately 25 \u0026deg;C) in a well-ventilated and dry environment. For identification, an appropriate amount of moss sample was rehydrated in distilled water until the leaves were fully expanded. The specimens were then carefully examined under an OLYMPUS SZX16 stereomicroscope, and the leaves were gently detached. The intact leaves were subsequently mounted on glass slides and examined in detail using an OLYMPUS BX53 light microscope equipped with an E3ISPM20000KPA microscope camera. The morphological characteristics of the moss specimens were compared with the descriptions in \u003cem\u003eFlora Bryophytorum\u003c/em\u003e \u003cem\u003eSinicorum\u003c/em\u003e and \u003cem\u003eMoss Flora of Guizhou\u003c/em\u003e [15,16] to identify the species.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eGreen Synthesis of AgNPs\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFirst, the cleaned and air-dried plant leaves (cinnamon, clove, thyme, and \u003cem\u003eAgastache rugosa\u003c/em\u003e, all purchased from a local farmers\u0026rsquo; market) were ground into a fine powder. For each type of leaf, 10 g of powder was added to 100 mL of deionized water or 99.7% anhydrous ethanol (Tianjin Fuyu Fine Chemical Co., Ltd., China). The mixtures were heated in a water bath at 80 \u0026deg;C for 30 min. During the experiment, they were maintained under reflux to prevent solvent evaporation.After heating, the solutions were centrifuged at 10,000 r/min, and the supernatants were collected. The supernatants were then filtered through Whatman Grade 1 filter paper to obtain the aqueous and ethanolic extracts for each plant.\u003c/p\u003e\n\u003cp\u003eA certain volume of plant extract was then added to a silver nitrate solution (0.1 mmol/L, Shenzhen Xijing Biotechnology Co., Ltd., China) under continuous heating, and the color change of the mixture was observed. (The optimal conditions for maximum silver nanoparticle yield, as determined from preliminary experiments, are summarized in Table 1. The conditions were determined by analyzing the resulting mixtures using a UV\u0026ndash;Vis spectrophotometer (SPECORD 200 PLUS) in the range of 250\u0026ndash;600 nm.) The mixture was centrifuged at 10,000 r/min for 20 min, and the supernatant was discarded. The pellet was washed by adding 5 mL of deionized water and centrifuging repeatedly until the supernatant became colorless. The final pellet was dried in an oven at 60 \u0026deg;C for 24 h. Silver nanoparticles (AgNPs) were synthesized and physicochemically characterized as previously reported[17].\u003c/p\u003e\u003cp\u003e\u003cstrong\u003eTable\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003e1\u003c/strong\u003e\u003cstrong\u003e.\u003c/strong\u003e \u003cstrong\u003eExperimental Parameters for the Preparation of Silver Nanoparticles\u003c/strong\u003e\u003c/p\u003e\n \u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\" width=\"85%\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 84px;\"\u003e\n \u003cp\u003e\u003cstrong\u003ePlant species\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 97px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eExtractant\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 84px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eHeating time (min)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 145px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eAgNO₃ : Plant extract\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 62px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eTemperature (\u0026deg;C)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd rowspan=\"2\" valign=\"top\" style=\"width: 84px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eCinnamon\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 97px;\"\u003e\n \u003cp\u003eDistilled water\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 84px;\"\u003e\n \u003cp\u003e150\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 145px;\"\u003e\n \u003cp\u003e8:2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 62px;\"\u003e\n \u003cp\u003e90\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 97px;\"\u003e\n \u003cp\u003eEthanol\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 84px;\"\u003e\n \u003cp\u003e120\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 145px;\"\u003e\n \u003cp\u003e9:1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 62px;\"\u003e\n \u003cp\u003e90\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd rowspan=\"2\" valign=\"top\" style=\"width: 84px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eClove\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 97px;\"\u003e\n \u003cp\u003eDistilled water\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 84px;\"\u003e\n \u003cp\u003e30\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 145px;\"\u003e\n \u003cp\u003e9:1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 62px;\"\u003e\n \u003cp\u003e80\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 97px;\"\u003e\n \u003cp\u003eEthanol\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 84px;\"\u003e\n \u003cp\u003e180\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 145px;\"\u003e\n \u003cp\u003e8:2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 62px;\"\u003e\n \u003cp\u003e90\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd rowspan=\"2\" valign=\"top\" style=\"width: 84px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eThyme\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 97px;\"\u003e\n \u003cp\u003eDistilled water\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 84px;\"\u003e\n \u003cp\u003e120\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 145px;\"\u003e\n \u003cp\u003e9:1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 62px;\"\u003e\n \u003cp\u003e90\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 97px;\"\u003e\n \u003cp\u003eEthanol\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 84px;\"\u003e\n \u003cp\u003e30\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 145px;\"\u003e\n \u003cp\u003e8:2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 62px;\"\u003e\n \u003cp\u003e90\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd rowspan=\"2\" valign=\"top\" style=\"width: 84px;\"\u003e\n \u003cp\u003e\u003cstrong\u003ePatchouli\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 97px;\"\u003e\n \u003cp\u003eDistilled water\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 84px;\"\u003e\n \u003cp\u003e30\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 145px;\"\u003e\n \u003cp\u003e9:1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 62px;\"\u003e\n \u003cp\u003e90\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 97px;\"\u003e\n \u003cp\u003eEthanol\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 84px;\"\u003e\n \u003cp\u003e150\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 145px;\"\u003e\n \u003cp\u003e9:1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 62px;\"\u003e\n \u003cp\u003e90\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n\u003c/div\u003e\n\u003cp\u003eFinally, the dried AgNPs were redispersed to prepare stock solutions with concentrations of 1 \u0026mu;g/mL, 3 \u0026mu;g/mL, and 5 \u0026mu;g/mL for subsequent use. (The AgNPs are abbreviated as follows: the first letter indicates the plant species (C: cinnamon, S: clove, T: thyme, P: Agastache rugosa), the second letter indicates the extractant (W: aqueous, A: ethanolic), and \u0026lsquo;Ag\u0026rsquo; denotes silver nanoparticles. Accordingly, the samples used are: CWAg \u0026ndash; AgNPs synthesized from aqueous extract of cinnamon leaves, CAAg \u0026ndash; from ethanolic extract of cinnamon leaves, SWAg \u0026ndash; from aqueous extract of clove leaves, SAAg \u0026ndash; from ethanolic extract of clove leaves, TWAg \u0026ndash; from aqueous extract of thyme leaves, TAAg \u0026ndash; from ethanolic extract of thyme leaves, PWAg \u0026ndash; from aqueous extract of \u003cem\u003eAgastache rugosa\u003c/em\u003e, PAAg \u0026ndash; from ethanolic extract of \u003cem\u003eAgastache rugosa\u003c/em\u003e, BC \u0026ndash; benzalkonium chloride).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eRemoval of Moss from the Surface of Stone Heritage Buildings\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAfter washing, the moss samples were evenly distributed into Petri dishes. Antimicrobial agents were added at different concentrations (AgNPs: 1 \u0026mu;g/mL, 3 \u0026mu;g/mL, 5 \u0026mu;g/mL; benzalkonium chloride: 0.1%, 0.3%, 0.5%), with 10 mL added to each dish containing moss. Chlorophyll a content and soluble protein content were measured on days 1, 15, and 30.\u003c/p\u003e\n\u003cp\u003eDetermination of Chlorophyll a Content: Moss treated with antimicrobial agents was washed and blotted dry. A 0.2 g sample was placed into a mortar, to which silica, calcium carbonate, and 2\u0026ndash;3 mL of 95% ethanol were added. The sample was ground into a homogeneous slurry, followed by the addition of 10 mL of 95% ethanol. Grinding was continued until the tissue became colorless. The homogenate was allowed to stand for 3\u0026ndash;5 min and then filtered into a 25 mL brown volumetric flask. The residue and filter paper were rinsed with a small amount of ethanol, and the filtrate was made up to the mark. The absorbance of the solution in a 1 cm cuvette was measured at 665 nm and 649 nm, and chlorophyll a content was calculated according to the following equation:\u003c/p\u003e\n\u003cp\u003e\u003cimg width=\"265\" height=\"17\" src=\"data:image/png;base64,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\" v:shapes=\"_x0000_i1025\" alt=\"image\"\u003e\u003c/p\u003e\n\u003cp\u003eDetermination of Soluble Protein Content: A 0.3 g sample of moss was ground with silica, calcium carbonate, and 5 mL of distilled water. The homogenate was centrifuged at 8,000 r/min for 10 min. A 1 mL aliquot of the supernatant was mixed with 3 mL of Coomassie Brilliant Blue G-250 solution in a test tube, thoroughly vortexed, and incubated at room temperature for 3\u0026ndash;5 min. Absorbance was measured at 595 nm, and each measurement was repeated three times to obtain the mean value. A standard curve was prepared using protein standards ranging from 0 to 100 \u0026mu;g/mL, and soluble protein content was expressed as \u0026mu;g per gram of fresh moss (\u0026mu;g/g).\u003c/p\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003eCharacterization of AgNPs\u003c/h2\u003e \u003cp\u003eMixing the plant extracts with silver nitrate solution induced a visible color change in most samples from colorless to brown (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e), indicating the formation of AgNPs due to the excitation of surface plasmon resonance (SPR). UV\u0026ndash;Vis spectroscopy further confirmed nanoparticle synthesis, as all eight samples exhibited characteristic SPR peaks in the range of 421\u0026ndash;446 nm. The peaks were observed at 422 nm, 421 nm, 445 nm, 435 nm, 442 nm, 446 nm, 437 nm, and 426 nm, with each sample displaying distinct spectral profiles (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eMorphological characterization using scanning electron microscopy (SEM) revealed that the synthesized nanoparticles were predominantly spherical, with a relatively uniform distribution in size. Elemental composition was assessed by energy-dispersive X-ray spectroscopy (EDX), which verified the presence of Ag as the major constituent across all samples. CWAg, CAAg, and TAAg were composed mainly of C, O, and Ag, whereas TWAg contained only C and Ag. SWAg displayed signals for C, O, Cl, and Ag, while SAAg and PWAg were characterized by C, Cl, and Ag. PAAg exhibited a more complex composition including C, O, Al, Cl, and Ag (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e). These results collectively confirm the successful synthesis of AgNPs with distinct chemical signatures depending on the plant extract used, suggesting that phytochemicals present in different plant matrices influence nanoparticle composition and potentially their bioactivity while maintaining the spherical morphology.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eEvaluation of Biocide Performance of AgNPs\u003c/h3\u003e\n\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eMoss Identification Results\u003c/h2\u003e \u003cp\u003eExamination of numerous samples revealed that the dominant bryophyte species on Yunfeng Tunpu encompassed a variety of mosses, including \u003cem\u003eBrachythecium procumbens\u003c/em\u003e, \u003cem\u003eErythrodontium julaceum\u003c/em\u003e, and \u003cem\u003eEntodon prorepens\u003c/em\u003e, among others (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eSpecies composition of mosses on Yunfeng Tunpu.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"4\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePhylum\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eFamily\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eGenus\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eSpecies\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eBryophyta\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eEntodontaceae\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eErythrodontium\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eJulaceous Erythrodontium Moss\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eBryophyta\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eEntodontaceae\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eEntodon\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eProstrate Entodon Moss\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eBryophyta\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eBrachytheciaceae\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eBrachythecium\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eProcumbent Brachythecium Moss\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eBryophyta\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eAnomodontaceae\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eAnomodon\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eLesser Anomodon Moss\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eBryophyta\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003ePottiaceae\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eBarbula\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eUnguiculate Barbula Moss\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eBryophyta\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eSphagnaceae\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eSphagnum\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eBlunt-leaved Bog Moss / Prairie Sphagnum\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAnthocerotophyta\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eAnthocerotaceae\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eAnthoceros\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eSpotted Hornwort\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eBryophyta\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eAndreaeaceae\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eAndreaea\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eRock Moss (Black Moss)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eChlorophyll a Content in Mosses\u003c/h3\u003e\n\u003cp\u003eObservation of moss samples treated with biocides revealed a reduction in green pigments within the cells, resulting in an overall yellow coloration (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e). Figure\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e illustrates the relationship between chlorophyll a content and both biocide concentration and exposure time. On Day 1, at a biocide concentration of 0.1%, CWAg (cinnamon aqueous extract AgNPs) and BC (benzalkonium chloride) exhibited the greatest reduction of chlorophyll a content. At a concentration of 0.3%, the impact of all biocides on chlorophyll a reduction was enhanced, with CWAg and CAAg demonstrating the most outstanding efficacy, followed by BC and PAAg. At a concentration of 0.5%, CWAg and CAAg continued to show the best performance, while the effectiveness of BC began to fall below that of PAAg.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eBy Day 15, the effects of the biocides on the reduction of chlorophyll a content became more distinct across different concentrations. At the 0.5% concentration, the chlorophyll a content in the BC-treated group dropped to 0.09 mg/L, whereas the TWAg-treated group showed a content of 2.65 mg/L, indicating relatively poor efficacy.\u003c/p\u003e \u003cp\u003eOn Day 30, even at a concentration of 0.1%, BC treatment reduced the chlorophyll a content to below 1 mg/L. At the 0.5% concentration, CAAg achieved the best results, with a chlorophyll a content of merely 0.06 mg/L, followed by BC at 0.08 mg/L. Although SAAg resulted in the lowest efficacy, it still reduced the chlorophyll a content to 2.2 mg/L. The gradual decline in chlorophyll a content following biocide treatment is consistent with the observed coloration of moss shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e. These results indicate that biocide treatment significantly affects moss chlorophyll a content, and the effectiveness is more pronounced with increasing biocide concentration and prolonged treatment duration.\u003c/p\u003e\n\u003ch3\u003eSoluble Protein Content in Mosses\u003c/h3\u003e\n\u003cp\u003eAs observed in Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e, the soluble protein content in normally growing mosses ranged from 324 to 339 \u0026micro;g/g. Experimental results indicated that on Day 1, all biocide treatments led to varying degrees of reduction of the soluble protein content. Among them, CAAg, CWAg, BC, and PAAg exhibited particularly pronounced effects. With increasing biocide concentration, the effectiveness of SWAg, SAAg, TWAg, TAAg, and PWAg also became increasingly evident. As time progressed, the performance of the nine biocides became more distinct. By Day 30, all biocide treatments had reduced the soluble protein content in the mosses to below 100 \u0026micro;g/g. Among all tested biocides, BC demonstrated the most outstanding efficacy, followed by AgNPs synthesized from cinnamon extract, while those synthesized from thyme extract showed relatively inferior performance.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eOverall, the declining trend in soluble protein content was generally consistent with the changes in chlorophyll a content (with the exception of the BC group), indicating that biocide treatment not only affected photosynthesis but also influenced the overall protein metabolism in the mosses.\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eUV-Vis spectroscopy is a commonly used method for detecting the presence of colloidal AgNPs, which exhibit a strong absorption band due to SPR, resulting in intense light scattering between 400\u0026ndash;450 nm [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. In this experiment, the mixtures of eight plant extracts with AgNO₃ showed absorption bands at 420\u0026ndash;450 nm, corresponding to the characteristic light scattering from AgNPs. Additionally, all reaction solutions turned brown, which is another indicator of the formation of AgNPs.\u003c/p\u003e \u003cp\u003eThe morphology and elemental composition of AgNPs have been described in our previous study [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e] and are not repeated here. This morphology has been associated with enhanced antimicrobial activity. De Melo reported that spherical AgNPs exhibit greater antibacterial activity than their rod-shaped analogs, suggesting that the spherical nanoparticles synthesized in this study should possess high antimicrobial potential[\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. Although spheres have the smallest surface area-to-volume ratio among three-dimensional shapes, their enhanced activity may be attributed to other physicochemical properties. EDX analysis indicated variations in the elemental composition of AgNPs synthesized from different extracts of the same plant. For instance, PAAg (ethanolic extract of Agastache rugosa) contained oxygen (O) and aluminum (Al) elements not detected in PWAg (aqueous extract of Agastache rugosa), possibly because the compounds containing these elements were more efficiently extracted by ethanol and subsequently adsorbed onto the nanoparticle surfaces. The presence of elements such as O and Cl on the nanoparticle surface may influence their physicochemical properties and contribute to their overall bioactivity.\u003c/p\u003e \u003cp\u003eThe green synthesis of AgNPs employed in this study involves natural products present in the plant extracts. For example, eugenol in clove leaves contains abundant hydroxyl groups (\u0026ndash;OH) that can act as proton sources. The electron-donating groups in the \u003cem\u003eortho\u003c/em\u003e- and \u003cem\u003epara\u003c/em\u003e-positions enhance electrochemical reducing ability through inductive effects, facilitating electron transfer from eugenol and promoting the reduction of Ag⁺ to Ag⁰[\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. Additionally, compounds such as α-pinene, camphene, and carvacrol in thyme exhibit mild reducing properties, enabling the reduction of silver ions to metallic AgNPs [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. This suggests that other plant extracts may also contain similar natural reducing agents, highlighting a benefit of the use of plant-derived components for AgNP synthesis.\u003c/p\u003e \u003cp\u003eGiven that this study aimed to evaluate the effectiveness of biocides against mixed moss communities commonly found on stone cultural heritage structures\u0026mdash;primarily consisting of \u003cem\u003eBrachythecium procumbens\u003c/em\u003e, \u003cem\u003eErythrodontium julaceum\u003c/em\u003e, and \u003cem\u003eEntodon prorepens\u003c/em\u003e\u0026mdash;the physiological responses of mosses to AgNP treatment were assessed based on chlorophyll a content and soluble protein content.\u003c/p\u003e \u003cp\u003eChlorophyll a content is a key indicator of photosynthetic capacity and directly reflects the growth status of mosses [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. Chloroplasts, as critical organelles in plant cells, are known to be highly sensitive to biocides. For instance, mosses in environments contaminated with heavy metals such as cadmium, lead, and nickel often exhibit structural damage to chloroplasts and a consequent decrease in chlorophyll a content [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eIn this experiment, chlorophyll a content gradually decreased with increasing biocide concentration and exposure time. Particularly at the highest tested concentration of 5 \u0026micro;g/mL, treatments with CWAg, CAAg, and PAAg resulted in a sharp decline in chlorophyll a content within one day. Consistent with this observation, Liang et al. [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e] reported that AgNPs at 2 \u0026micro;g/mL significantly affected the protonemata of Physcomitrella patens within 10 days.Over the 30-day observation period, even the least effective biocide treatments (TWAg and TAAg) led to a notable decrease in chlorophyll a content. However, current research on the effects of AgNPs on mosses has mainly focused on juvenile gametophytes, with limited studies on mature mosses. Thus, the precise mechanisms of chloroplast sensitivity to AgNP require further investigation. Liang et al. [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e] did not detect AgNPs inside chloroplasts, suggesting that AgNPs may be unable to penetrate the chloroplast membrane. Instead, the observed reduction in thylakoid number may indirectly affect photosynthesis and chlorophyll content. Therefore, it is hypothesized that AgNPs mainly influence chlorophyll levels through extracellular or indirect mechanisms, such as generating reactive oxygen species (ROS) that damage other organelles, interacting with or disrupting the cell membrane, or causing DNA damage. In summary, AgNPs may affect chlorophyll a content through one or more mechanisms, with efficacy depending on the type of plant extract, nanoparticle size, and moss species.\u003c/p\u003e \u003cp\u003eSoluble proteins play essential roles in nutrient regulation and cellular metabolic activity, serving as key biomarkers in plant stress biology [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. Studies have shown that soluble protein content in plants adjusts in response to external environmental stressors to enable the plant to adapt to complex conditions[\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. Low concentrations of metal ions in the environment may promote an increase in soluble protein content, while high metal concentrations can disrupt protein integrity, leading to a decrease in content and even eventual moss death[\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. In this study, low concentrations of AgNPs significantly reduced soluble protein content as early as Day 1, with CAAg treatment reducing content threefold. Benzalkonium chloride (BC) treatment also reduced soluble protein to approximately 130 \u0026micro;g/g. As hypothesized, with increasing biocide concentration and exposure time, soluble protein content did not recover. This result is likely due to ROS accumulation beyond the scavenging capacity of the moss antioxidant system [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. Concurrently, AgNP entry may enhance proteolytic enzyme activity, accelerating protein hydrolysis and further reducing content.\u003c/p\u003e \u003cp\u003eThese results indicate that low concentrations of AgNPs and BC as biocides impose significant stress on mosses, hindering their adaptation to external pressures. Combined with the diminished chlorophyll a content, it is inferred that AgNPs at 3 \u0026micro;g/mL and 5 \u0026micro;g/mL can cause moss death within one month. Compared with non-plant-mediated AgNPs, such as those synthesized by chemical reduction methods, the plant-mediated AgNPs in this study exhibited similar or enhanced biocidal activity against mosses at lower concentrations [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e, \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. This may be attributed to the synergistic effect of phytochemicals adsorbed on the nanoparticle surface, which can enhance membrane disruption and oxidative stress [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. In conclusion, This study focused on bryophytes as the primary research subject and investigated green-synthesized AgNPs as a potential moss removal agent. The results demonstrate that AgNPs synthesized from plant extracts effectively inhibit the growth of mosses isolated from Yunfeng Tunpu that are associated with the deterioration of ancient stone walls. The efficacy of inhibition was closely related to the type of plant extract; for example, CWAg (from cinnamon extracts) consistently outperformed TWAg (from thyme extracts). Unlike other heritage conservation studies, this research used both aqueous and ethanolic extracts from four plants to synthesize AgNPs and maintained AgNP concentrations within ranges considered acceptable for human and environmental safety. To our knowledge, this is the first exploratory study investigating the potential ability of AgNPs to control moss growth on stone heritage buildings, and it demonstrated promising results. However, the long-term efficacy of AgNPs against moss colonies on stone surfaces requires further evaluation. Effective treatment should inactivate organisms within an acceptable timeframe and prevent regrowth. Moreover, the application of AgNPs should be context-specific, as resistance levels of organisms may vary significantly across regions. Continuous improvement in AgNP synthesis technology and concentration adjustment is recommended for effective biological control. These findings are consistent with our previous study on algae [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e] and further demonstrate that plant-mediated AgNPs may be effective against different photosynthetic colonizers, including bryophytes, on stone surfaces.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e \u003ch2\u003eDeclaration of competing interest\u003c/h2\u003e \u003cp\u003eThe authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.\u003c/p\u003e \u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eM.L. and J.L. wrote the main manuscript and performed experiments. B.T. and H.Z. conducted material characterization. Z.A. and Y.G. carried out field sampling and biocide treatments. L.G. and Y.C. analyzed the data and prepared the figures. M.L. and X.L. supervised the work and secured funding. All authors reviewed the manuscript.\u003c/p\u003e\u003ch2\u003eAcknowledgement\u003c/h2\u003e\u003cp\u003eThis work was supported by the National Natural Science Foundation of China (32460314, 32360309, 42561014); the Science and Technology Programs of Guizhou Province (Qiankehe Jichu - ZK [2023] Yiban 147, 427).\u003c/p\u003e\u003ch2\u003eData Availability\u003c/h2\u003e\u003cp\u003eAll data generated or analysed during this study are included in this published article. The datasets are available from the corresponding author upon reasonable request.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eElgohary YM, Mansour MMA, Salem MZM. Assessment of the potential effects of plants with their secreted biochemicals on the biodeterioration of archaeological stones. 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Ecotoxicol Environ Saf. 2015;113:499\u0026ndash;505. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.ecoenv.2014.12.035\u003c/span\u003e\u003cspan address=\"10.1016/j.ecoenv.2014.12.035\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"npj-heritage-science","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"hsci","sideBox":"Learn more about [Heritage Science](http://heritagesciencejournal.springeropen.com)","snPcode":"40494","submissionUrl":"https://submission.nature.com/new-submission/40494/3","title":"npj Heritage Science","twitterHandle":"@SpringerOpen","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"","lastPublishedDoi":"10.21203/rs.3.rs-9371654/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-9371654/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eMoss colonization threatens stone heritage buildings. Silver nanoparticles (AgNPs) were green-synthesized using aqueous and ethanolic extracts of cinnamon, clove, thyme, and Agastache rugosa. The spherical AgNPs exhibited surface plasmon resonance peaks at 421–446 nm, with elemental compositions varying by extract. Their efficacy against moss was evaluated by measuring chlorophyll a and soluble protein content over 30 days at different concentrations. AgNPs significantly reduced moss growth in a dose-dependent manner, and those from aqueous extracts were generally more effective than those from ethanolic extracts. This study demonstrates that plant-mediated AgNPs offer an environmentally friendly approach for moss removal on stone heritage structures, providing a promising strategy for cultural heritage conservation.\u003c/p\u003e","manuscriptTitle":"Plant-Mediated Green Silver Nanoparticles for Moss Removal on Stone Heritage Buildings","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-05-11 08:45:53","doi":"10.21203/rs.3.rs-9371654/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"editorInvitedReview","content":"","date":"2026-05-14T03:01:07+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"45141900200635455262027460293551626702","date":"2026-05-14T01:26:28+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"65907629245738275307558358065076172120","date":"2026-05-12T02:31:13+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"39881842224659940139233595897622471323","date":"2026-05-12T02:25:24+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"154114811280781174327742781942685572442","date":"2026-05-01T04:10:54+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2026-05-01T02:59:19+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2026-04-30T20:09:37+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2026-04-30T17:50:00+00:00","index":"","fulltext":""},{"type":"submitted","content":"npj Heritage Science","date":"2026-04-30T16:07:40+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"npj-heritage-science","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"hsci","sideBox":"Learn more about [Heritage Science](http://heritagesciencejournal.springeropen.com)","snPcode":"40494","submissionUrl":"https://submission.nature.com/new-submission/40494/3","title":"npj Heritage Science","twitterHandle":"@SpringerOpen","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"46cd3aa1-9be6-4394-95cb-eb2309d986a7","owner":[],"postedDate":"May 11th, 2026","published":true,"recentEditorialEvents":[{"type":"editorInvitedReview","content":"","date":"2026-05-14T03:01:07+00:00","index":38,"fulltext":""},{"type":"reviewerAgreed","content":"45141900200635455262027460293551626702","date":"2026-05-14T01:26:28+00:00","index":37,"fulltext":""},{"type":"reviewerAgreed","content":"65907629245738275307558358065076172120","date":"2026-05-12T02:31:13+00:00","index":36,"fulltext":""},{"type":"reviewerAgreed","content":"39881842224659940139233595897622471323","date":"2026-05-12T02:25:24+00:00","index":35,"fulltext":""},{"type":"reviewerAgreed","content":"154114811280781174327742781942685572442","date":"2026-05-01T04:10:54+00:00","index":24,"fulltext":""},{"type":"reviewersInvited","content":"20","date":"2026-05-01T02:59:19+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2026-04-30T20:09:37+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2026-04-30T17:50:00+00:00","index":"","fulltext":""},{"type":"submitted","content":"npj Heritage Science","date":"2026-04-30T16:07:40+00:00","index":"","fulltext":""}],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[],"tags":[],"updatedAt":"2026-05-11T08:45:54+00:00","versionOfRecord":[],"versionCreatedAt":"2026-05-11 08:45:53","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-9371654","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-9371654","identity":"rs-9371654","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}