Immobilization mechanisms of Sr(II), Ni(II), and Cd(II) on glomalin-related soil protein in mangrove sediments at the microscopic scale.

Glomalin-related soil protein (GRSP) is a significant component in the sequestration of heavy metal in soils, but its mechanisms for metal adsorption are poorly known. This study combined spectroscopic data with molecular docking simulations to reveal metal adsorption onto GRSP's surface functional groups at the molecular level. The EXAFS combined with FTIR and XPS analyses indicated that the adsorption of Cd(II), Sr(II), and Ni(II) by GRSP occurred mainly through the coordination of -OH and -COOH groups with the metal. The -COOH and -OH groups bound to the metal as electron donors and the electron density of the oxygen atom decreased, suggesting that electrostatic attraction might be involved in the adsorption process. Two-dimensional correlation spectroscopy revealed that preferential adsorption occurred on GRSP for the metal in sequential order of -COOH groups followed by -OH groups. The presence of the Ni-C shell in the Ni EXAFS spectrum suggested that Ni formed organometallic complexes with the GRSP surface. However, Sr-C and Cd-C were absent in the second shell of the Sr and Cd spectra, which was attributed to the adsorption of Sr and Cd ions with large hydration ion radius by GRSP to form outer-sphere complexes. Through molecular docking simulations, negatively charged residues such as ASP151 and ASP472 in GRSP were found to provide electrostatic attraction and ligand combination for the metal adsorption, which was consistent with the spectroscopic analyses. Overall, these findings provided new insights into the interaction mechanisms between GRSP and metals, which will help deepen our understanding of the ecological functions of GRSP in metal sequestration.

Uncovering the disposable face masks as vectors of metal ions (Pb(â ¡), Cd(â ¡), Sr(â ¡)) during the COVID-19 pandemic.

The demand for disposable face masks (DFMs) increased sharply in response to the COVID-19 pandemic. However, information regarding the underlying roles of the largely discarded DFMs in the environment is extremely lacking. This study focused on the pristine and UV-aged DFMs as vectors of metal ions (Pb(Ⅱ), Cd(Ⅱ), and Sr(Ⅱ)). Further, the aging mechanism of DFMs with UV radiation as well as the interaction mechanisms between DFMs and metal ions were investigated. Results revealed that the aging process would help to promote more metal ions adsorbed onto DFMs, which was mainly attributed to the presence of oxygen-containing groups on the aged DFMs. The adsorption affinity of pristine and aged DFMs for the metal ions followed Pb(Ⅱ) > Cd(Ⅱ) > Sr(Ⅱ), which was positively corrected with the electronegativity of the metals. Interestingly, we found that even if DFMs were not disrupted, DFMs had similar or even higher adsorption affinity for metals compared with other existing microplastics. Besides, regarding environmental factors, including salinity and solution pH played a crucial role in the adsorption processes, with greater adsorption capacities for pristine and aged DFMs at higher pH values and low salinity. Fourier transform infrared spectroscopy, X-ray photoelectron spectroscopy, and density functional theory further confirmed that the pristine DFMs interacted with the metals mainly through electrostatic interaction, while electrostatic interaction and surface complexation jointly regulated the adsorption of the metals onto aged DFMs. Overall, these findings would help to evaluate environmental behaviors and risks of DFMs associated with metals.

Immobilization of lead(â ¡) and zinc(â ¡) onto glomalin-related soil protein (GRSP): Adsorption properties and interaction mechanisms.

Glomalin-related soil protein (GRSP), a microbial product that can be used as a bioflocculant, is critical to metal sequestration in the ecosystem. However, the relationship between GRSP and heavy metal has not been well explored. In this study, the adsorption behaviors and mechanisms of Pb(II) and Zn(II) ions on GRSP were investigated. Results reveal that the Pb(II) and Zn(II) adsorption closely conform to the pseudo second-order model, which indicates that the chemisorption of GRSP occurred after intra-particle diffusion. The adsorption process is influenced by the degree of pollution, pH value, GRSP content in the environment. In addition, scanning electron microscopy coupled with microanalysis (SEM-EDX) reveals that the surface structure of GRSP is irregularly blocky or flaky and metal ions are uniformly distributed on the surface of GRSP. Fourier transform infrared (FT-IR), X-ray photoelectron spectroscopy (XPS), and X-ray diffraction (XRD) analysis show that the carboxyl and nitro groups on GRSP act as ligands to form complexes with two divalent metal ions. The interaction between GRSP and the metals is mainly surface complexation. This research further reveals the dynamic response of its structural components when GRSP sequestrates heavy metals in mangrove sediment and aqueous ecosystems, demonstrating a new perspective for the transport and transformation of heavy metals onto GRSP.

Glomalin-related soil protein: The particle aggregation mechanism and its insight into coastal environment improvement.

Glomalin-related soil protein (GRSP), a ubiquitous microbial product, plays a crucial role in particle aggregation and metal adsorption, but the underlying mechanisms remain unknown. Here, GRSP fraction was extracted from estuarine ecosystems and systematically characterized to elucidate the aggregation mechanisms and its impact on coastal environment improvement. We found that GRSP fraction (gravimetric mass of extracted GRSP, 5.1-24.3 mg g-1) was a globally relevant novel bioflocculant and that protein (linked to Bradford protein assay, 1.64-4.37 mg g-1) was the active flocculant constituent. The zeta potential, FTIR, XPS, and 13C NMR analyses identified its key constituents and structure, and revealed that the charge neutralization and bridging were GRSP fraction aggregation mechanisms. Thermogravimetric-infrared spectrometry analysis showed that GRSP fraction was highly thermostable, and the main volatile pyrolysis products included H2O, CO2, CO, and CH4. The SEM-EDX and XPS Fe valence spectroscopy suggested that GRSP fraction contained rich Fe (11.91 ± 0.48%) and could form Fe-rich flocs with particles. We also found that GRSP fraction has a high adsorption capacity (76-95%) for Cu, Zn, Pb and Cd, and its flocculation properties provide new insights into metal adsorption. The analysis of particle aggregation mechanism and its metal adsorption capacity is of great significance to elucidate the role of GRSP fraction in coastal environment improvement.

Dissolved organic matter byproducts from combined low-level alkaline and mechanical treatment of sludge: species, and their bioavailability.

Low-level alkalinity (pH 9-10) coupled with ultrasonic or mechanical cutting with different energy input for obtaining carbon sources were tested for sludge pretreatment process before anaerobic sludge digestion. The differences between the primary sludge (PS) and waste activated sludge (WAS)-derived dissolved organic matter (DOM) species were evaluated for their bioavailability and affinity (in the form of amino acids) to the bio-nutrient removal (BNR) biomass. Soluble microbial by-product-like substances as the predominant DOM components in the raw PS and WAS increased by 23 and 22%, respectively, after low-level alkaline treatment (pH 9-10) and ultrasonication. In addition, the protein components were degraded further as free amino acids (FAAs). The sludge-derived aspartate, glutamate, followed by arginine were the most commonly used FAAs by the BNR biomass. The pattern of recovering this special sludge-derived carbon source to enhance P removal and recovery in the BNR process is depicted.

Aluminum-Tailored Energy Level and Morphology of Co3- x Alx O4 Porous Nanosheets toward Highly Efficient Electrocatalysts for Water Oxidation.

Tuning energy levels plays a crucial role in developing cost-effective, earth-abundant, and highly active oxygen evolution catalysts. However, to date, little attention has been paid to the effect of using heteroatom-occupied lattice sites on the energy level to engineer electrocatalytic activity. In order to explore heteroatom-engineered energy levels of spinel Co3 O4 for highly-effective oxygen electrocatalysts, herein Al atoms are directly introduced into the crystal lattice by occupying the Co2+ ions in the tetrahedral sites and Co3+ ions in the octahedral sites (denoted as Co2+ Td and Co3+ Oh , respectively). Experimental and theoretical simulations demonstrate that Al3+ ions substituting Co2+ Td and Co3+ Oh active sites, especially Al3+ ions occupying the Co2+ Td sites, optimizes the adsorption, activation, and desorption features of intermediate species during oxygen evolution reaction (OER) processes. As a result, the optimized Co1.75 Al1.25 O4 nanosheet exhibit unprecedented OER activity with an ultralow overpotential of 248 mV to deliver a current of 10 mA cm-2 , among the best Co-based OER electrocatalysts. This work should not only provide fundamental understanding of the effect of Al-occupied different Co sites in Co3-x Alx O4 composites on OER performance, but also inspire the design of low-cost, earth-abundant, and high-active electrocatalysts toward water oxidation.

Phenolic metabolism and related heavy metal tolerance mechanism in Kandelia Obovata under Cd and Zn stress.

In the present study, a set of pot culture experiments was conducted to reveal how the metabolism process of phenolic compounds was affected by cadmium (Cd) and zinc (Zn) and to further uncover heavy metal tolerance mechanisms in Kandelia obovata. After 60d of treatment, the biomass and chlorophyll a content in the leaves were suppressed, but total phenolic compounds in roots and leaves were improved by the increasing gradient of Cd or Zn concentrations; Total phenolic compounds significantly increased by 3.6-44.6% in the roots, and by 0.4-126.6% in the leaves. At the meantime, the activity of Shikimate dehydrogenase (SKDH), cinnamyl alcohol dehydrogenase (CAD), and polyphenol oxidase (PPO) in the roots increased by 11.2-307.6%, 12.4-175.4% and - 2.7-392.8%, and the results were 3.4-69.5%, 1.7-40.0%, 16.0-99.7% in the leaves. Higher toxicity of Cd than Zn, as well as slight alleviating effect of 100 mg kg-1 Zn on 2.5 mg kg-1 Cd were found. Additionally, a significantly positive correlation coefficients for relationship between phenolic metabolism related enzyme activity and Cd/Zn contamination levels was found, and leaf SKDH, leaf CAD, and leaf PPO activities were moderately correlated with leaf Cd (r = 0.39, r = 0.43, and r = 0.57, respectively) and leaf Zn (r = 0.44, r = 0.41, r = 0.19, respectively) content, which indicate that Cd and Zn play a previously unrecognized but major role in phenolic compounds synthesis, transport, and metabolism in K. obovata. The results also provided evidence that the application of high levels of Cd and Zn was accompanied by three phenolic metabolism pathways participating in heavy metal tolerance process.

Single-Nanostructured Electrochemical Detection for Intrinsic Mechanism of Energy Storage: Progress and Prospect.

Energy storage appliances are active by means of accompanying components for renewable energy resources that play a significant role in the advanced world. To further improve the electrochemical properties of the lithium-ion batteries (LIBs), sodium-ion batteries (SIBs), and lithium-sulfur (Li-S) batteries, the electrochemical detection of the intrinsic mechanisms and dynamics of electrodes in batteries is required to guide the rational design of electrodes. Thus, several researches have conducted in situ investigations and real-time observations of electrode evolution, ion diffusion pathways, and side reactions during battery operation at the nanoscale, which are proven to be extremely insightful. However, the in situ cells are required to be compatible for electrochemical tests and are therefore often challenging to operate. In the past few years, tremendous progresses have been made with novel and more advanced in situ electrochemical detection methods for mechanism studies, especially single-nanostructured electrodes. Herein, a comprehensive review of in situ techniques based on single-nanostructured electrodes for studying electrodes changes in LIBs, SIBs, and Li-S batteries, including structure evolution, phase transition, interface formation, and the ion diffusion pathway is provided, which is instructive and meaningful for the optimization of battery systems.

Response of phenolic metabolism to cadmium and phenanthrene and its influence on pollutant translocations in the mangrove plant Aegiceras corniculatum (L.) Blanco (Ac).

Polyphenolic compounds are abundant in mangrove plants, playing a pivotal role in the detoxification of pollutants extruded from surrounding environments into plant tissues. The present study aimed to examine the variations of phenolic compounds, namely total polyphenolics, soluble tannins, condensed tannins and lignin, in the mangrove plant Aegiceras corniculatum (L.) due to the presence of exogenous cadmium and phenanthrene and to explore the influence of phenolic metabolism on biological translocation of these pollutants from roots to leaves. After a 6-week exposure to cadmium and phenanthrene, significant accumulations of both pollutants were observed. All determined phenolic compounds in both leaves and roots at high dosage levels were enhanced compared to the uncontaminated plant. Elevations of polyphenols in both treatments are possibly a result of stimulation in the activity of phenylalanine ammonia-lyase (PAL) and the enrichment of soluble sugar. Additionally, a significantly positive dosage relationship between polyphenolic metabolism intensity and phenanthrene contamination levels was found, while the trend observed in cadmium treatment was weak since cadmium at high levels inhibited phenolic production. The enrichment of polyphenols led to a decline in the biological translocation of these pollutants from roots to leaves. The immobilization of pollutants in the plant roots is possibly linked to the adsorption potential of polyphenols. These results will improve the understanding of the tolerance of mangrove plants to exogenous pollutants and will guide the selection of plants in phytoremediation because of the variability of polyphenol concentrations among species.

Phosphorus mediation of cadmium stress in two mangrove seedlings Avicennia marina and Kandelia obovata differing in cadmium accumulation.

Mangrove ecosystems are vulnerable to environmental threats. In order to elucidate the effect of phosphorus (P) on cadmium (Cd) tolerance and physiological responses in mangroves under Cd stress, a mangrove specie with salt exclusion Kandelia obovata and a specie with salt secretion Avicennia marina were compared in a hydroponic experiment. The results showed that most Cd was accumulated in mangrove roots and that P addition induced Cd immobilisation in them. Cd stress significantly increased malonaldehyde content, whereas P significantly decreased malonaldehyde in mangroves. Phosphorus positively regulated the photosynthetic pigment, proline content and synthesis of non-protein thiols, glutathione and phytochelatins in the leaves under Cd stress conditions. The results suggest different adaptive strategies adopted by two mangroves in a complex environment and A. marina showed a stronger Cd tolerance than K. obovata. The study provides a theoretical basis for P mediated detoxification of Cd in mangrove plants.

Toxicological evaluation of silver nanoparticles and silver nitrate in rats following 28 days of repeated oral exposure.

The increasing application of silver nanoparticles (AgNPs) has been raising concerns about their potential adverse effects to human and the environment. However, the knowledge on the systemic toxicity of AgNPs in mammalian systems is still limited. The present study investigated the toxicity of PVP-coated AgNPs in rats treated with repeated oral administration, and compared that with equivalent dose of AgNO3 . Specifically, one hundred male and female rats were orally administrated with particulate or ionic forms of silver (Ag) separately at doses of 0.5 and 1 mg kg-1 body weight daily for 28 days. The results reveal no significant toxic effects of AgNPs and AgNO3 up to 1 mg kg-1 body weight, with respect to the body weight, organ weight, food intake, and histopathological examination. Ag distribution pattern in organs of rats treated with AgNPs was similar to that of AgNO3 treated rats, showing liver and kidneys are the main target organs followed by testis and spleen. The total Ag contents in organs were significantly lower in the AgNPs treated rats than those in the AgNO3 treated rats. However, the comparisons between AgNPs and AgNO3 treatments further indicated more potent of AgNPs in biochemical and hematological parameters in rats, including red blood cell count (RBC), platelet count (PLT), white blood cell count (WBC) and aspartate transaminase (AST). Results of this study suggested that particulate Ag at least partially contributed to the observed toxicity of AgNPs, and both ionic and particulate Ag should be taken into consideration in toxicological evaluation of AgNPs. © 2016 Wiley Periodicals, Inc. Environ Toxicol 32: 609-618, 2017.

The influence of flavonoid amendment on the absorption of cadmium in Avicennia marina roots.

Flavonoid is a key factor for the tolerance to cadmium in plants. Concentration-dependent kinetics experiment was conducted to investigate the influence of flavonoid amendment on the Cd(2+) uptake in Avicennia marina (Forsk) Vierh. roots. We found that compared with the control, saturation concentration and maximal absorption rate of Cd was higher under flavonoid amendment (p<0.05). When roots were exposed to ion transport inhibitor (LaCl3), flavonoid amendment also facilitated Cd transport in roots. Flavonoids had no influence on Cd(2+) uptake in root cell walls. In conclusion, flavonoids enhance the tolerance to Cd and have a significant stimulative effect on symplasm transport of Cd in A. marina roots. Ca(2+)-channel was not the unique means of symplasm transport for Cd(2+) absorption.

Impact of Predator Cues on Responses to Silver Nanoparticles in Daphnia carinata.

The past decades have witnessed a boom in nanotechnology that has led to increasing production and application of silver nanoparticles (AgNPs) in the textile industry due to their antimicrobial properties. Increase in the manufacture and use of NPs inevitably has resulted in their increased release into aquatic environments resulting in the exposure of organisms living in these environments. Recently, the risk of exposure to NPs and the potential interaction with biological systems has received increasing attention. The present study investigated the potential effects of predator cues on the toxicity of environmentally relevant concentrations of AgNPs in Daphnia carinata at organismal and biochemical levels. The results of this study show that exposure to environmentally relevant concentrations of AgNPs can result in adverse effects on daphnids with 24- and 48-h LC50 values of 3.56 and 1.75 µg/L, respectively. Furthermore, significant inhibition of reproduction was observed at concentrations as low as 0.5 µg/L. Exposure to predator cues alone resulted in an increase in reproduction and inhibition of superoxide dismutase activity in daphnids. However, coexposure to predator cues interacted in an antagonistic manner with AgNPs with a 24-h LC50 value of 10.81 µg/L compared with 3.56 µg/L for AgNPs alone. In summary, AgNPs could pose risks to aquatic invertebrates at environmentally relevant concentrations. Interestingly, the presence of other factors, such as predator cues, moderated the effects of exposure to AgNPs. Therefore, there is a need to further investigate the potential interactions between NPs and biological factors that can modulate toxicity of NPs for application to the risk assessment of aquatic invertebrates.

Alleviated toxicity of cadmium by the rhizosphere of Kandelia obovata (S., L.) Yong.

A pot experiment was conducted to investigate the Cadmium (Cd) toxicity alleviated by the rhizosphere of Kandelia obovata (S., L.) Yong (K. obovata), using a rhizobox with Cd concentrations of 0, 12.5, 25, 50 and 100 mg kg(-1) soil. The rhizobox used to plant K. obovata was divided into five sections by nylon cloth (S2-S5). Our results showed that pH was lower in rhizosphere than in non-rhizosphere soil. Microbial biomass C (Cmic) and N (Nmic) was stimulated in rhizosphere zone, but inhibited by Cd additions. Soil enzyme activities were significantly higher in rhizosphere (S2) than in non-rhizosphere soil (S5) (p < 0.05). In addition, DTPA-extractable Cd in different rhizosphere zone soil (S2-S5) was influenced directly under different rates of Cd supply. Moreover, Cd treatments could induce the enhancement of DTPA-extractable Cd. This study suggests that Cd toxicity can be alleviated in the rhizosphere even under high Cd supply.

Making Healthcare Accessible: A Rapid Clean-Room-Free Fabrication Strategy for Microfluidics-Driven Biosensors Based on Coupling Stereolithography and Hot Embossing.

Microfluidics offers transformative potential in healthcare by enabling miniaturized, user-friendly, and cost-effective devices for disease diagnostics among other biomedical applications; however, their meaningful adoption is severely hindered, especially in developing countries and resource-limited settings, by the cost, time, and complexity of their fabrication. To overcome this barrier of access, this work develops a novel approach for highly efficient (<4 h), cost-effective, and clean-room-free fabrication of functional polydimethylsiloxane (PDMS)-based microfluidic devices based on coupling stereolithography three-dimensional (3D) printing with hot embossing. The strategy exhibits high fidelity between the digital design and final device, remarkable transfer accuracy between the 3D print and poly(methyl methacrylate) (PMMA) mold, in addition to highly smooth surfaces (R a < 1 µm). To establish the versatility of the approach and performance quality of the fabricated devices, three advanced microfluidics-driven biosensing platforms are developed: a microsphere droplet generator, a stop-flow lithography-based hydrogel microparticle synthesizer, and a hydrogel postembedded microfluidic device for multiplexed biomarker detection. As a proof-of-concept, the latter platform was applied to the multiplexed detection of microRNA, a highly promising class of liquid biopsy biomarkers for many diseases including cancer. Notably, the ability to demonstrate multiplexed sensing of disease biomarkers within devices made through a facile, rapid, and clean-room-free strategy demonstrates the immense potential of this fabrication approach to accelerate the adoption and advancement of biomedical microfluidic devices in practice and in resource-limited settings.

Enhanced Cr(VI) stabilization by terrestrial-derived soil protein: Photoelectrochemical properties and reduction mechanisms.

Glomalin-related soil protein (GRSP) is a stable iron-organic carbon mixture that can enhance heavy metal sequestration in soils. However, the roles of GRSP in the transformation and fate of Cr(VI) have been rarely reported. Herein, we investigated the electrochemical and photocatalytic properties of GRSP and its mechanisms in Cr(VI) adsorption and reduction. Results showed that GRSP had a stronger ability for Cr(VI) adsorption and reduction than other biomaterials, with the highest adsorption amount of up to 0.126 mmol/g. The removal efficiency of Cr(VI) by GRSP was enhanced (4-7%) by ultraviolet irradiation due to the hydrated electrons produced by GRSP. Fe(II) ions, persistent free radicals, and oxygen-containing functional groups on the GRSP surface as electron donors participated in the reduction of Cr(VI) under dark condition. Moreover, Cr(III) was mainly adsorbed on the -COOH groups of GRSP via electrostatic interactions. Based on 2D correlation spectroscopy, the preferential adsorption occurred on the GRSP surface for Cr(VI) in the sequential order of CO → COO- → O-H → C-O. This work provides new insights into the Cr(VI) adsorption and reduction mechanism by GRSP. Overall, GRSP can serve as a natural iron-organic carbon for the photo-reduction of Cr(VI) pollution in environments.

Microplastics emerge as a hotspot for dibutyl phthalate sources in rivers and oceans: Leaching behavior and potential risks.

Dibutyl phthalate (DBP) as a plasticizer has been widely used in the processing of plastic products. Nevertheless, these DBP additives have the potential to be released into the environment throughout the entire life cycle of plastic products. Herein, the leaching behavior of DBP from PVC microplastics (MPs) in freshwater and seawater and its potential risks were investigated. The results show that the plasticizer content, UV irradiation, and hydrochemical conditions have a great influence on the leaching of DBP from the MPs. The release of DBP into the environment increases proportionally with higher concentrations of additive DBP in MPs, particularly when it exceeds 15 %. The surface of MPs undergoes accelerated oxidation and increased hydrophilicity under UV radiation, thereby facilitating the leaching of DBP. Through 30 continuous leaching experiments, the leaching of DBP from MPs in freshwater and seawater can reach up to 12.28 and 5.42 mg g-1, respectively, indicating that MPs are a continuous source of DBP pollution in the aquatic environment. Moreover, phthalate pollution index (PPI) indicates that MPs can significantly increase DBP pollution in marine environment through land and sea transport processes. Therefore, we advocate that the management of MPs waste containing DBP be prioritized in coastal sustainable development.

Arsenic(III) sequestration by terrestrial-derived soil protein: Roles of redox-active moieties and Fe(III).

Glomalin-related soil protein (GRSP) has demonstrated significant potential for water purification and remediation of heavy metals in soils; however, its redox reactivity for As(III) sequestration and the corresponding redox-active component are still poorly understood. This study investigated the photochemical properties of GRSP and its mechanism of oxidation/adsorption of As(III). The results showed that UV irradiation triggered electron transfer and the production of reactive oxygen species (ROS) in GRSP, thereby facilitating As(III) oxidation with promotion rates ranging from 43.34 % to 111.1 %. The oxidation of As(III) occurred both on the GRSP photoforming holes and in the ROS reaction from the oxygen reduction products of the photoforming electrons. OH• and H2O2 played an important role in the oxidation of As(III) by GRSP, especially under alkaline conditions. Moreover, the presence of Fe(III) in GRSP facilitated the formation of OH• and its the oxidation capacity towards As(III). The binding of As(III) to the -COOH, -OH, and -FeO groups on the GRSP surface occurred through surface complexation. Overall, these findings provided new insights into the roles of the redox-active moieties and Fe(III) on GRSP in the promoted oxidation of As(III), which would help to deepen our understanding of the migration and transformation of As(III) in soils.

Comparative analysis of size-fractional eukaryotic microbes in subtropical riverine systems inferred from 18S rRNA gene V4 and V9 regions.

Eukaryotic microbes play key ecological roles in riverine ecosystems. Amplicon sequencing has greatly facilitated the identification and characterization of eukaryotic microbial communities. Currently, 18S rRNA gene V4 and V9 hypervariable regions are widely used for sequencing eukaryotic microbes. Identifying optimal regions for the profiling of size-fractional eukaryotic microbial communities is critical for microbial ecological studies. In this study, we spanned three rivers with typical natural-human influenced transition gradients to evaluate the performance of the 18S rRNA gene V4 and V9 hypervariable regions for sequencing size-fractional eukaryotic microbes (>180 µm, 20-180 µm, 5-20 µm, 3-5 µm, 0.8-3 µm). Our comparative analysis revealed that amplicon results depend on the specific species and microbial size. The V9 region was most effective for detecting a broad taxonomic range of species. The V4 region was superior to the V9 region for the identification of microbes in the minor 3 µm and at the family and genus levels, especially for specific microbial groups, such as Labyrinthulomycetes. However, the V9 region was more effective for studies of diverse eukaryotic groups, including Archamoebae, Heterolobosea, and Microsporidia, and various algae, such as Haptophyta, Florideophycidae, and Bangiales. Our results highlight the importance of accounting for potential misclassifications when employing both V4 and V9 regions for the identification of microbial sequences. The use of optimal regions for amplification could enhance the utility of amplicon sequencing in environmental studies. The insights gained from this work will aid future studies that employ amplicon-based identification approaches for the characterization of eukaryotic microbial communities and contribute to our understanding of microbial ecology within aquatic systems.