What Is the Molecular Weight of "High" Molecular Weight Dissolved Organic Matter?

The use of ultrafiltration to isolate high molecular weight dissolved organic matter (HMWDOM) from seawater is a fundamental tool in the environmental organic chemist's toolbox. Yet, important characteristics of HMWDOM relevant to its origin and cycling, such as its molecular weight distribution, remain poorly defined. We used diffusion-ordered NMR spectroscopy coupled with mixed-mode chromatography to separate and characterize two major components of marine HMWDOM: acylpolysaccharides (APS) and high molecular weight humic substances (HS). The molecular weights (MWs) of APS and HS both fell within distinct, narrow envelopes; 2.0-16 kDa for APS and 0.9-6.5 kDa for HS. In water samples from the North Pacific Ocean the average MW of both components decreased with depth through the mesopelagic. However, the minimum MW of APS was >2 kDa, well above the molecular weight cutoff of the ultrafilter, suggesting APS removal processes below 2 kDa are highly efficient. The MW distribution of APS shows only small variations with depth, while the MW distribution of HS narrowed due to removal of HMW components. Despite the narrowing of the MW distribution, the concentration of HS did not decrease with depth between 15 and 915 m. This suggests that HMW HS produced in surface waters was either degraded into lower MW compounds without significant remineralization, or that HMW HS was remineralized but replaced by an additional source of HS in the mesopelagic ocean. Based on these results, we propose potential pathways for the production and removal of these major components of HMWDOM.

Dietary fulvic acid improves immune, digestive and antioxidant parameters in juvenile white-leg shrimp (Litopenaeus vannamei) in a super-intensive system.

In the current study, the effects of dietary fulvic acid supplementation at levels of 0.5, 1 and 2% were examined in white-leg shrimp, Litopenaeus vannamei. A significant increase in the weight of the shrimp was observed in the group treated with 2% fulvic acid in comparison to the control group. This may have been associated with an increased digestive efficiency, with the food conversion ratio reducing from 2.4 to 1.9, and increased hepatopancreatic amylase, protease, and lipase enzyme activities. Enhanced activity of hemolymph superoxide dismutase was suggestive of an enhanced immune capacity, while hemolymph cell count increased by 16.4 and 13.6% in shrimp receiving diets supplemented with 1 and 2% fulvic acid, respectively. Additionally, the number of large granular cells increased by 37.3% and 40.8% relative to the control in these two groups. Furthermore, the lysozyme activity increased in shrimp receiving dietary supplementation of 1% and 2% fulvic acid by 16.7% and 24.7%, respectively. Phenol oxidase activity, which activates phagocytosis and encapsulation of invading pathogens, increased in all groups supplemented with fulvic acid, with the highest activity in the 1% fulvic acid group. Overall the present results suggest that fulvic acid is a promising feed additive for white-leg shrimp super-intensive culture.

Effect of Modified Forms of Sodium Humate PowHumus on the Balance of Arginine in Peritoneal Macrophages of Intact Mice.

Substances of silver nanoparticles dialyzed through a 13 kDa membrane, synthesized in a medium of humic ligands modified with hydroquinone and 2-hydroxynaphthoquinone from PowHumus brown coal, specifically enhance the M2 properties of peritoneal macrophages due to inhibition of NO synthase and significant activation of arginase, thus enhancing anti-inflammatory properties of cells. In small, but effective concentrations, they do not have cytotoxic properties and do not contain pyrogenic impurities. The studied humates are able to influence the mechanisms of immune response formation and are an effective means for correcting inflammation and regeneration.

Microbial community assemblage altered by coprecipitation of artificial humic substances and ferrihydrite: Implications for carbon fixation pathway transformation.

The suppression of soil carbon mineralization has been demonstrated to be effectively facilitated by carbon­iron interactions, yet the specific mechanisms by which artificial humic substances (A-HS) coupled with ferrihydrite influence this process remain insufficiently explored. This study is to investigate how the A-HS, specifically artificial fulvic acid (A-FA) and artificial humic acid (A-HA), coupled with ferrihydrite, affect carbon mineralization under anaerobic system that simulates paddy flooding conditions. The object is to investigate trends in carbon emissions and to delineate microbial community structure and functional pathways. The findings indicate that A-HA and A-FA substantially reduce CO2 and CH4 emissions, with A-FA having a particularly pronounced effect on carbon fixation, halving CO2 concentrations. The low concentration of Fe(II) observed suggest that A-FA and A-HA impede the dissimilatory iron reduction (DIR) process. Detailed 16S rDNA sequencing and gene prediction analyses reveal changes in microbial community structures and functions, highlighting Methanobacterium as the dominant hydrogenotrophic methanogens. The reductive citric acid cycle, predominantly utilized by Clostridium carboxidivorans, was identified as the principal carbon fixation pathway. This work provides a novel insight into the microbial mechanisms of carbon sequestration and highlights the potential of A-HS in improving soil fertility and contributing to climate change mitigation through enhancing soil carbon storage.

Improving Tree Seedling Quality Using Humates Combined with Bacteria to Address Decarbonization Challenges through Forest Restoration.

Improving the quality of tree planting material for carbon sequestration through reforestation can help solve environmental problems, including the need to reduce the concentration of carbon dioxide in the atmosphere. The purpose of this study was to investigate the possibility of using humic substances in combination with rhizosphere microorganisms Pseudomonas protegens DA1.2 and Pseudomonas sp. 4CH as a means to stimulate the growth of seedlings of pine, poplar, large-leaved linden, red oak, horse chestnut, and rowan. Humic substances stimulated the growth of shoots and roots of pine, large-leaved linden, and horse chestnut seedlings. The effects of bacteria depended on both plant and bacteria species: Pseudomonas protegens DA1.2 showed a higher stimulatory effect than Pseudomonas sp. 4CH on pine and linden, and Pseudomonas sp. 4CH was more effective in the case of chestnut. An additive effect of humates and Pseudomonas protegens DA1.2 on the growth rate of pine and linden saplings was discovered. Poplar, red oak, and rowan seedlings were unresponsive to the treatments. The growth-stimulating effects of the treatments are discussed in connection with the changes in carbon, chlorophyll, and nitrogen contents in plants. The results show the need for further research in bacterial species capable of stimulating the growth of plant species that were unresponsive in the present experiments.

Seasonal studies of aquatic humic substances from Amazon rivers: characterization and interaction with Cu (II), Fe (II), and Al (III) using EEM-PARAFAC and 2D FTIR correlation analyses.

Aquatic humic substances (AHS) are defined as an important components of organic matter, being composed as small molecules in a supramolecular structure and can interact with metallic ions, thereby altering the bioavailability of these species. To better understand this behavior, AHS were extracted and characterized from Negro River, located near Manaus city and Carú River, that is situated in Itacoatiara city, an area experiencing increasing anthropogenic actions; both were characterized as blackwater rivers. The AHS were characterized by 13C nuclear magnetic ressonance and thermochemolysis GC-MS to obtain structural characteristics. Interaction studies with Cu (II), Al (III), and Fe (III) were investigated using fluorescence spectroscopy applied to parallel factor analysis (PARAFAC) and two-dimensional correlation spectroscopy with Fourier transform infrared spectroscopy (2D-COS FTIR). The AHS from dry season had more aromatic fractions not derived from lignin and had higher content of alkyls moities from microbial sources and vegetal tissues of autochthonous origin, while AHS isolated in the rainy season showed more metals in its molecular architecture, lignin units, and polysacharide structures. The study showed that AHS composition from rainy season were able to interact with Al (III), Fe (III), and Cu (II). Two fluorescent components were identified as responsible for interaction: C1 (blue-shifted) and C2 (red-shifted). C1 showed higher complexation capacities but with lower complexation stability constants (KML ranged from 0.3 to 7.9 × 105) than C2 (KML ranged from 3.1 to 10.0 × 105). 2D-COS FTIR showed that the COO- and C-O in phenolic were the most important functional groups for interaction with studied metallic ions.

Assessing sorption of fluoroquinolone antibiotics in soils from a Kd compilation based on pure organic and mineral components.

The presence of fluoroquinolone (FQ) antibiotics in soils may cause a threat to human health due to overexposure and the generation of antibiotic resistance genes. Understanding their sorption behavior in soils is important to predict subsequent FQ (bio) availability. Here, FQ sorption in pure soil organic (i.e., humic substances) and mineral (i.e., metal oxides; phyllosilicates) components is evaluated through a solid-liquid distribution coefficient (Kd (FQ)) dataset consisting of 243 entries originated from 80 different studies, to elucidate their respective contribution to the overall Kd (FQ) in bulk soils. First, different factors affecting FQ sorption and desorption in each of these soil phases are critically discussed. The strong role of pH in Kd (FQ), due to the simultaneous effect on both FQ speciation and surface charge changes, encouraged the derivation of normalized sorption coefficients for the cationic, zwitterionic and anionic FQ species in humic substances and in different phyllosilicates. Kd (FQ) in metal oxides revealed a key role of metal nature and material specific surface area due to complexation sorption mechanisms at neutral pH. Cumulative distribution functions (CDF) were applied to each dataset to establish a sorption affinity range for each phase and to derive best estimate Kd (FQ) values for those materials where normalized sorption coefficients to FQ species were unavailable. The data analysis conducted in the different soil phases set the basis for a Kd (FQ) prediction model, which combined the respective sorption affinity of each phase for FQ and phase abundance in soil to estimate Kd (FQ) in bulk soils. The model was subsequently validated with sorption data in well characterized soils compiled from the literature.

Anoxic nitrification with carbon-based materials as terminal electron acceptors.

This study investigates the potential of humic substances (HS) and graphene oxide (GO), as extracellular electron acceptors (EEA) for nitrification, aiming to explore alternatives to sustain this process in wastewater treatment systems. Experimental results demonstrate the conversion of ammonium to nitrate (up to 87 % of conversion) coupled to the reduction of either HS or GO by anaerobic consortia. Electron balance confirmed the contribution of HS and GO to ammonium oxidation. Tracer analysis in incubations performed with 15NH4+ demonstrated 15NO3- as the main product with a minor fraction ending as 29N2. Phylogenetic analysis identified Firmicutes, Euryarchaeota, and Chloroflexi as the microbial lineages potentially involved in anoxic nitrification linked to HS reduction. This study introduces a new avenue for research in which carbon-based materials with electron-accepting capacity may support the anoxic oxidation of ammonium, for instance in bioelectrochemical systems in which carbon-based anodes could support this novel process.

Copper dynamics in vineyard topsoils as affected by the supply of aerated compost tea: Insights from a batch experiment.

Aerated compost teas (ACTs) are rich in soluble humic substances (SHS) that have high affinity for metals, notably Cu. Using a batch experiment, we measured the extent to which two ACTs altered Cu dynamics in vineyard topsoils one day and 21 days after their addition. Soils were extracted with 0.01 M KCl, and total Cu concentration, free Cu ion fraction and size distribution of Cu ligands were measured in the extraction solution to assess the impact of ACT on the mobility of Cu. Diffusive gradient in thin film (DGT) measurements were carried out to assess the effect of ACT on Cu bioavailability, and the dissociation rate of Cu-SHS complexes was measured. The results revealed that ACT increased the mobility of Cu from a factor 1.2 to 5.8 depending on the soil, the ACT and the incubation time. Cu mobilization was associated with an increase in absorbance at 254 nm and a decrease in the free Cu ion fraction in the KCl extract. Associated with the strong agreement between the size distribution of SHS and that of Cu ligands in the KCl extract of soils treated with ACT, these results showed that Cu was mobilized through complexation by the SHS present in ACTs. A fraction of the SHS supplied with ACTs sorbed onto the soil constituents, notably in calcareous soils where this fraction reached 86% for ACT B. Between 15% and 50% of the SHS remaining in solution degraded between day one and day 21 under the presumed action of microflora. This explains why the Cu mobilization efficiency of ACTs was on average lower in calcareous soils than in non-calcareous soils, and decreased with time. Lastly, ACT increased the bioavailability of Cu in soils from a factor 1.3 to 4.2, due to the relatively high dissociation rate of Cu-SHS complexes.

Humic Acid Derived from Agricultural Biomass Mitigates Alveolar Bone Loss and Modulates Systemic Inflammatory Cytokines in Rats with Periodontitis.

BACKGROUND: Humic acid (HA) is a bioproduct that can be extracted from different sources and has anti-inflammatory properties that have been little explored in the treatment and prevention of Periodontal Disease (PD). Thus, we aimed to investigate the effects of oral administration of HA on the progression of PD in rats. METHODS: Twenty-four male Wistar rats were distributed into three experimental groups (Control/ Sham, PD, and PD + HA). HA was administered by gavage (80 mg/kg/day) for 28 days, and PD was induced 14 days after the beginning of treatment. Bone loss, bone topography, and surface elemental composition were analyzed. Circulating IL1-beta, TNF-alpha, and IL-10 levels were evaluated through Enzyme-Linked Immunosorbent Assay (ELISA). RESULTS: The animals treated with HA showed lower bone loss (p < 0.05). Calcium and phosphorus levels on the alveolar bone surface were lower in the PD group (p < 0.05) compared to the control group, whereas the animals treated with HA exhibited attenuation in this loss (p < 0.05). The animals treated with HA showed reduced TNF-alpha, IL1-beta, IL-10, and the TNF-alpha/IL-10 ratio compared to those with PD (p < 0.05). CONCLUSION: Treatment with HA attenuated the parameters of alveolar bone loss and modulated systemic inflammatory parameters in rats with ligature-induced PD.

Differential responses of the electron transfer capacities of soil humic acid and fulvic acid to long-term wastewater irrigation.

Wastewater irrigation is used to supplement agricultural irrigation because of its benefits and freshwater resource scarcity. However, whether wastewater irrigation for many years affects the electron transfer capacity (ETC) of natural organic matter in soil remains unclear, and organic matter could influence the decomposition and mineralization of substances with redox characteristics in soil through electron transfer, ultimately affecting the soil environment. The composition of soil humic substances (HS) is highly complex, and the effects of soil humic acid (HA) and fulvic acid (FA) on ETC is poorly understood. In this study, we separately evaluated the responses of the electron-accepting capacity (EAC) and electron-donating capacity (EDC) of soil HA and FA in agricultural fields to various durations of wastewater irrigation. Results showed that the EAC of HA and FA increased significantly with increasing the duration of wastewater irrigation. When wastewater irrigation lasted for 56 years, the EAC of HA showed a higher increment (590 %) than that of FA (223 %). The EDC of soil HA and FA, conversely, decreased compared to the control, with the highest reduction of 35.6 % for HA and 65.9 % for FA. Specifically, the EDC of HA gradually decreased starting from 29 years of wastewater irrigation, whereas the decrease in the EDC of FA exhibited no clear pattern in relation to the duration of wastewater irrigation. Increased soil organic matter and total nitrogen content under long-term wastewater irrigation led to an increase in sucrase and phosphatase activities, along with an increase in EAC and a decrease in EDC of HS. This suggests that soil enzyme activities may ultimately lead to changes in ETC. The results of this research provide practical insights into the redox system in soil and its driving role in soil organic matter transformation and nutrient cycling under wastewater irrigation.

Synergistic effects of coal waste derived humic substances and inorganic fertilizer as soil amendments for barley in sandy soil.

Increasing pressures on land resources requires increased land use efficiency. Over 900 million ha of sandy soils throughout the world are extensively used for agricultural crop production, most requiring nutrient inputs. Although use of humic substances together with inorganic fertilizer as soil amendments has been introduced, their synergistic effects on plant growth in sandy soils are not well addressed. We assessed the efficacy of a lignite waste derived humic substance on barley (Hordeum vulgare L.) growth, with and without inorganic fertilizer. Ten treatments were applied to sandy soils, comprising sole application of the humic product at four rates (NH1, NH2, NH3, NH4), sole application of fertilizer (F), and their combinations (F + NH1, F + NH2, F + NH3, F + NH4). Synergistic effects of nano humus and fertilizer were more notable than the corresponding sole application, particularly on plant biomass and seed production. Combined application with inorganic fertilizer increased root biomass by 92 % (0.1 g per plant), shoot biomass by 80 % (0.5 g per plant), root length by 24 % (3.6 cm), and seed production by 38 % (5 seeds per head) averagely relative to the untreated control, suggesting a strong synergistic effect. The increased seed production was particularly important from an agricultural perspective. Four application rates of nano humus all showed beneficial effects on barley growth with no significant differences. The most distinct positive effect of the humic product as a sole application was on root growth. Our study confirmed that a lignite waste derived humic product, nano humus, together with fertilizer may be an effective soil amendment to enhance agricultural plant growth in sandy soil regions.

Geospatial Variability of Fluorescent Dissolved Organic Matter in Urban Watersheds: Relationships with Land Cover and Wastewater Infrastructure.

We investigated the fluorescent dissolved organic matter (FDOM) composition in two watersheds with variable land cover and wastewater infrastructure, including sanitary sewers and septic systems. A four-component parallel factor analysis model was constructed from 295 excitation-emission matrices recorded for stream samples to examine relationships between FDOM and geospatial parameters. The contributions of humic acid- and fulvic acid-like fluorescence components (e.g., C1, C2, C3) were fairly consistent across a 12 month period for the 27 sampling sites. In contrast, the protein-like fluorescence component (C4) and a related ratiometric wastewater indicator (C4/C3) exhibited high variability in urban tributaries, suggesting that some sites were impacted by leaking sewer infrastructure. Principal component analysis indicated that urban areas clustered with impervious surfaces and sanitary sewer density, and cross-covariance analysis identified strong positive correlations between C4, impervious surfaces, and sanitary sewer density at short lag distances. The presence of wastewater was confirmed by detection of sucralose (up to 1,660 ng L-1) and caffeine (up to 1,740 ng L-1). Our findings not only highlight the potential for C4 to serve as an indicator of nearby, compromised sanitary sewer infrastructure, but also suggest that geospatial data can be used to predict areas vulnerable to wastewater contamination.

The impact of microbial inoculants on large-scale composting of straw and manure under natural low-temperature conditions.

Understanding large-scale composting under natural conditions is essential for improving waste management and promoting sustainable agriculture. In this study, corn straw (400 tons) and pig manure (200 tons) were composted with microbial inoculants. The thermophilic phase of composting lasted for fourteen weeks, resulting in an alkaline final product. Microbial systems with low-temperature initiation and high-temperature fermentation played a crucial role in enhancing lignocellulose degradation and humic substances (HS) formation. Adding microbes, including Rhodanobacter, Pseudomonas, and Planococcus, showed a positive correlation with degradation rates of cellulose, hemicellulose, and lignin. Bacillus, Planococcus, and Acinetobacter were positively correlated with HS formation. Microorganisms facilitated efficient hydrolysis of lignocelluloses, providing humic precursors to accelerate composting humification through phenolic protein and Maillard pathways. This study provides significant insights into large-scale composting under natural conditions, contributing to the advancement of waste management strategies and the promotion of sustainable agriculture.

Coal-Derived Humic Substances: Insight into Chemical Structure Parameters and Biomedical Properties.

An investigation was carried out on humic substances (HSs) isolated from the coal of the Kansk-Achinsk basin (Krasnoyarsk Territory, Russia). The coal HSs demonstrate the main parameters of molecular structure inherent to this class of natural compounds. An assessment was performed for the chemical, microbiological, and pharmacological safety parameters, as well as the biological efficacy. The HS sample meets the safety requirements in microbiological purity, toxic metals content (lead, cadmium, mercury, arsenic), and radionuclides. The presence of 11 essential elements was determined. The absence of general, systemic toxicity, cytotoxicity, and allergenic properties was demonstrated. The coal HS sample was classified as a Class V hazard (low danger substances). High antioxidant and antiradical activities and immunotropic and cytoprotective properties were identified. The ability of the HS to inhibit hydroxyl radicals and superoxide anion radicals was revealed. Pronounced actoprotective and nootropic activities were also demonstrated in vivo. Intragastric administration of the HS sample resulted in the improvement of physical parameters in mice as assessed by the "swim exhaustion" test. Furthermore, intragastric administration in mice with cholinergic dysfunction led to a higher ability of animals with scopolamine-induced amnesia to form conditioned reflexes. These findings suggest that the studied HS sample is a safe and effective natural substance, making it suitable for use as a dietary bioactive supplement.

Humic acid improves wheat growth by modulating auxin and cytokinin biosynthesis pathways.

Humic acids have been widely used for centuries to enhance plant growth and productivity. The beneficial effects of humic acids have been attributed to different functional groups and phytohormone-like compounds enclosed in macrostructure. However, the mechanisms underlying the plant growth-promoting effects of humic acids are only partially understood. We hypothesize that the bio-stimulatory effect of humic acids is mainly due to the modulation of innate pathways of auxin and cytokinin biosynthesis in treated plants. A physiological investigation along with molecular characterization was carried out to understand the mechanism of bio-stimulatory effects of humic acid. A gene expression analysis was performed for the genes involved in auxin and cytokinin biosynthesis pathways in wheat seedlings. Furthermore, Arabidopsis thaliana transgenic lines generated by fusing the auxin-responsive DR5 and cytokinin-responsive ARR5 promoter to ß-glucuronidase (GUS) reporter were used to study the GUS expression analysis in humic acid treated seedlings. This study demonstrates that humic acid treatment improved the shoot and root growth of wheat seedlings. The expression of several genes involved in auxin (Tryptophan Aminotransferase of Arabidopsis and Gretchen Hagen 3.2) and cytokinin (Lonely Guy3) biosynthesis pathways were up-regulated in humic acid-treated seedlings compared to the control. Furthermore, GUS expression analysis showed that bioactive compounds of humic acid stimulate endogenous auxin and cytokinin-like activities. This study is the first report in which using ARR5:GUS lines we demonstrate the biostimulants activity of humic acid.

Role of the molecular structure of humified organic matter in rice plant response to environmental lead pollution.

Humified organic matter has been shown to decrease Pb toxicity in plants. However, there are still gaps in our understanding of the mechanism by which this phenomenon occurs. In this study, we aimed to assess the ability of humic substances (HSs), humic acids (HAs), and fulvic acids (FAs) to enhance defense mechanisms in rice plants under lead (Pb)-stressed conditions. HS fractions were isolated from vermicompost using the chemical fractionation methodology established by the International Humic Substances Society. These fractions were characterized by solid-state NMR and FTIR. Chemometric analysis was used to compare humic structures and correlate them with bioactivity. Three treatments were tested to evaluate the protective effect of humic fractions on rice plants. The first experiment involved the application of humic fractions along with Pb. The second comprised pretreatment with humic fractions followed by subsequent exposure to Pb stress. The third experiment involved Pb stress and subsequent treatment with humic fractions. The root morphology and components of the antioxidative defense system were evaluated and quantified. The results showed that HS + Pb, HA + Pb, and FA + Pb treatment preserved root growth and reduced the levels of O2- and malondialdehyde (MDA) in the roots by up to 5% and 2%, respectively. Pretreatment of the plants with humic fractions promoted the maintenance of root growth and reduced the contents of O2-, H2O2, and MDA by up to 48%, 22%, and 20%, respectively. Combined application of humic fractions and Pb reduced the Pb content in plant tissues by up to 60%, while pretreatment reduced it by up to 80%. The protective capacity of humic fractions is related to the presence of peptides, lignin, and carbohydrate fragments in their molecular structures. These results suggest that products could be developed that can mitigate the adverse effects of heavy metals on agricultural crops.

The Study of Humic Substances' Impact on Anion Exchangers.

Humic substances (HSs) present in water and wastewater cause fouling of anion exchange resins (AERs), which mainly results in reducing the ion exchange capacity (IEC). In this paper, an attempt was made to investigate fouling of two polystyrene and one polyacrylic AER using water from the Oder River, treated wastewater after the ultrafiltration process (UFTW) and digester reject water from sludge dewatering at the Janówek Wastewater Treatment Plant (WWTP) in Wroclaw. HSs contained in digester reject water were characterised by the lowest aromaticity and molecular weights (MWs), the highest proportion of hydrophilic fraction and the highest amount of oxygenated functional groups. The Fourier-transform infrared (FTIR) analyses made it possible to identify chemical bonds characteristic of HSs and determine the mechanism of their retention on the surface of AER beads. The conducted experiments brought unexpected results, as the IEC increased with the amount of organic matter in the feed. Presumably, the humic substances accumulated on the beads and in the porosity of the anion exchangers themselves participated in the ion exchange process.

The effects of different electrode materials on the electric field-assisted co-composting system for the soil remediation of heavy metal pollution.

The electric field-assisted composting system (EACS) is an emerging technology that can enhance composting efficiency, but little attention has been given to electrode materials. Herein, an EACS was established to investigate the effects of electrode materials on humic substance formation and heavy metal speciation. Excitation-emission matrix analysis showed that carbon-felt and stainless-steel electrodes increased humic acid (HA) by 48.57 % and 47.53 %, respectively. In the EACS with the carbon-felt electrode, the bioavailability factors (BF) of Cu and Cr decreased by 18.00 % and 7.61 %, respectively. Despite that the stainless-steel electrodes decreased the BF of As by 11.26 %, the leaching of Cr, Ni, Cu, and Fe from the electrode itself is an inevitable concern. Microbial community analyses indicated that the electric field increased the abundance of Actinobacteria and stimulated the multiplication of heavy metal-tolerant bacteria. Redundancy analysis indicates that OM, pH, and current significantly affect the evolution of heavy metal speciation in the EACS. This study first evaluated the metal leaching risk of stainless-steel electrode, and confirmed that carbon-felt electrode is environment-friendly material with high performance and low risk in future research with EACS.