Use of plant growth-promoting bacteria to enhance chromium phytoremediation by Salvinia biloba

Use of plant growth-promoting bacteria to enhance chromium phytoremediation by Salvinia biloba | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Use of plant growth-promoting bacteria to enhance chromium phytoremediation by Salvinia biloba María de los Ángeles Martínez Saucedo, Pamela Romina Bernabeu This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7178802/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 14 Nov, 2025 Read the published version in Environmental Science and Pollution Research → Version 1 posted 6 You are reading this latest preprint version Abstract Heavy metals, such as chromium (Cr), are toxic even at low concentrations and are a significant environmental concern. Phytoremediation is an emerging biotechnological method to clean contaminated environments, using plant biomass to absorb and accumulate contaminants. To enhance the phytoremediation capacity, plant growth-promoting bacteria (PGPB) have been proposed as a strategy. This study aimed to evaluate the effect of PGPB isolated from sediment samples of the Paraná River Delta (Argentina) on the phytoremediation of Cr by Salvinia biloba , a floating macrophyte native to the region. The plants were exposed to 1, 3, and 5 ppm of Cr, exhibiting phytotoxicity characterized by turgor loss, which resulted in an increased vegetation cover; however, the photosynthetic pigment content remained unaffected at these concentrations. The total Cr concentration decreased by 32%, 26%, and 21% in the 1, 3, and 5 ppm treatments, respectively, while hexavalent chromium (Cr(VI)) was reduced by 82%, 45%, and 33%. Three microorganisms exhibiting the highest plant growth-promoting activity were selected, as they positively impacted plant growth and tolerated different Cr(VI) concentrations. To assess the effect of PGPB on the phytoremediation process, inoculated S. biloba plants were exposed to 5 ppm of Cr(VI). Notably, inoculation with microorganism ER-Y, identified as Serratia nematodiphila , a previously reported PGPB, enhanced plant growth in contaminated systems. Furthermore, inoculation significantly increased vegetation cover compared to the uninoculated control; however, unlike the increase observed in uninoculated plants, this expansion was not due to turgor loss but rather to actual plant growth stimulated by the PGPB under Cr(VI) exposure. Additionally, ER-Y contributed to an almost 9% greater Cr(VI) reduction compared to the uninoculated group, highlighting its potential role in improving both plant resilience and remediation efficiency. Biotechnology Chromium Phytoremediation Plant growth-promoting bacteria Salvinia biloba Serratia nematodiphila Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 1. Introduction Heavy metals are a group of metals and metalloids characterized by high atomic weight (greater than 40.04 g/mol), high relative density (greater than 5 g/cm 3 ), and toxicity, even at low concentrations in some cases (Haldar and Ghosh 2020 ; Yadav et al. 2019 ). These metals naturally occur in the environment; however, the primary source of heavy metal pollution comes from human activities, such as mining, industrial processes, industrial and domestic wastewater discharge, and excessive use of pesticides and fertilizers in agriculture (Ali et al. 2020 ). Consequently, heavy metal concentrations in the environment have risen to dangerous levels (Vareda et al. 2019 ). Common examples of heavy metal pollutants include arsenic (As), lead (Pb), mercury (Hg), chromium (Cr), zinc (Zn), cadmium (Cd), copper (Cu), and nickel (Ni) (Pratush et al. 2018 ). While some of these heavy metals are essential for life, acting as cofactors in metabolic and enzymatic pathways, they can become toxic when present in high concentrations (Briffa et al. 2020 ; Mishra et al. 2019 ). The toxicity of heavy metals depends on their chemical nature, concentration, oxidation state, and bioavailability (Haldar and Ghosh 2020 ). In the case of Cr, toxicity is primarily determined by its oxidation state and solubility (Tchounwou et al. 2012 ). Cr can exist in various oxidation states in the environment, with trivalent chromium (Cr(III)) and hexavalent chromium (Cr(VI)) being the most prevalent forms. Cr(VI) predominantly exists as chromate (CrO 4 2− ), dichromate (Cr 2 O 7 2− ), and chromium trioxide (CrO 3 ) (Pratush et al. 2018 ). These two oxidation states exhibit distinct physicochemical properties, chemical behavior, mobility, bioavailability, and toxicity (Malaviya et al. 2020 ). Cr(VI) is more toxic than Cr(III) due to its higher redox potential, greater solubility, and ability to easily diffuse across cell membranes. Once inside the cell, Cr(VI) is reduced to Cr(III), generating free radicals and reactive oxygen species (ROS) (Ahemad 2015 ; Briffa et al. 2020 ; Prado et al. 2016 ). High oxygen levels in the environment can reconvert Cr(III) into Cr(VI), making Cr a significant water pollutant (Malaviya et al. 2020 ). Water pollution by heavy metals is considered one of the most severe environmental problems, affecting not only aquatic organisms but also plants, since metals are transported to sediments and soils where they accumulate (Aziz et al. 2023 ). Once heavy metals enter aquatic environments, they tend to bind with iron, manganese oxides, sulfides, organic compounds, and clay minerals in sediments, where they are quickly deposited. Depending on environmental conditions, these metals can be released back into the water (Zhao et al. 2021 ). Industrial wastewater discharges are one of the main sources of heavy metals in the environment, and thus many rivers located near industries that use heavy metals are heavily polluted (Briffa et al. 2020 ). To comply with regulations, both water intended for human consumption and wastewater must undergo treatment (Vareda et al. 2019 ). However, many water treatment techniques used in developing countries are ineffective at removing heavy metals (Joseph et al. 2019 ). Various physical and chemical methods exist for removing heavy metals from liquid media, such as membrane filtration, ion exchange, adsorption, and chemical precipitation (Fei and Hu 2023 ). However, these methods are not always efficient, sustainable, or cost-effective (Ali et al. 2020 ; Emiliani et al. 2020 ). For this reason, alternative methods that are more environmentally friendly and economical for remediating contaminated sites have been investigated, such as phytoremediation. This is a biotechnological technique that uses plant biomass to absorb and accumulate contaminants from soil, air, and water through different mechanisms (Kafle et al. 2022 ; Tiwari et al. 2019 ). A wide range of organic and inorganic contaminants, such as insecticides, chlorinated solvents, hydrocarbons, surfactants, and heavy metals, can be remediated through phytoremediation (Ali et al. 2020 ; Tufail et al. 2022 ). To be effectively used in phytoremediation, a plant must be native to the contaminated site, have rapid growth, and be capable of absorbing and tolerating contaminants (Ali et al. 2020 ). The main objective of most phytoremediation studies is to extract contaminants through plant roots, translocate them to photosynthetically active biomass (leaves), and concentrate them for subsequent processing (Malaviya et al. 2020 ). Aquatic plants, which often use rhizofiltration as a mechanism for removing heavy metals, are considered for treating contaminated waters due to their rapid growth rate, high biomass production, and potential for contaminant removal, as well as their ability to develop an extensive root system (Ali et al. 2020 ; Bora and Sarma 2020 ). They play an important role in aquatic ecosystems as they act as natural filters for contaminants transported by water and serve as bioindicators of various stressors in ecosystems (Eid et al. 2020 ; Emiliani et al. 2021 ). Furthermore, many of the aquatic plants studied for their phytoremediation ability are typically invasive species that are resistant to nutrient deficiencies and environmental variations (Newete and Byrne 2016 ). Several species of aquatic plants, such as Eichhornia spp., Salvinia spp., Pistia stratiotes , Lemna minor , Azolla spp., among others, have been shown to be capable of removing heavy metals from wastewater (Rezania et al. 2016 ). The Salviniaceae family is one of the most studied in terms of its phytoremediation capabilities (Delgado-González et al. 2021 ). Salviniaceae is a family of floating aquatic ferns consisting of the genera Azolla and Salvinia , with around 20 species distributed in tropical to temperate regions (Xu and Deng 2017 ). The genus Salvinia includes 10 species that inhabit slow-moving freshwater bodies such as lakes, ponds, and lagoons (Contreras and Robledo 2021 ). These species lack true roots and have a stem that grows horizontally across the water surface, where three leaves develop at each node: two upper floating leaves folded over each other along the central vein, and one submerged lower leaf, highly divided and structurally similar to roots (hereafter referred to as “roots” for simplicity), where sporocarps develop (Nagalingum et al. 2006 ). In Argentina, the most abundant Salvinia species are S. auriculata , S. minima , S. natans , and S . biloba (Emiliani et al. 2020 ). Particularly, S.biloba , native to the Paraná River region, exhibits a high growth rate, can survive adverse conditions, and is capable of absorbing heavy metals such as Cd, Pb, Cu, Zn, Ni and Cr (Emiliani et al. 2021 ). Generally, Salvinia populations are sterile and expand through vegetative multiplication due to the ease with which they fragment, with each node capable of generating new individuals (Miranda and Schwartsburd 2019 ). In stagnant or slow-flowing waters, they can form large communities that cover the entire water surface where they grow, and in some cases, they can become invasive weeds (Motitsoe et al., 2020 ). Although phytoremediation is considered an economic and ecological technique for the in-situ remediation of contaminated environments compared to conventional methods (Newete and Byrne 2016 ), the main disadvantage is the required time. The use of plants does not allow for complete cleanup, as the absorption rate decreases as the concentration of the contaminant diminishes (Phieler et al. 2014 ). Efficient phytoremediation within a reasonable timeframe can be achieved by increasing the plant’s yield and the accumulation of contaminants (Rezania et al. 2016 ). To achieve this, the use of plant growth-promoting bacteria (PGPB) has been proposed due to their ability to improve plant growth and stress tolerance (Mesa-Marín et al. 2020 ). PGPB are microorganisms associated with plant tissues or free-living that have a beneficial effect on plant growth and protect plants from pathogens and abiotic stress. For PGPB to have a beneficial effect on plants, they must be able to colonize the rhizosphere or root surface for an extended period in a competitive environment with other present microorganisms (Zvinavashe et al. 2021 ). Colonization is related to exudation, as microorganisms exhibit chemotaxis toward the exudates, attracting them to the plant. Some exudates can have negative effects by acting as antimicrobial agents against certain microorganisms while also serving as stimuli for establishing beneficial interactions with other microorganisms (Compant et al. 2010 ; Souza et al. 2015 ). Others may activate genes in PGPB involved in root adhesion and colonization, such as genes that encode secretion systems, fimbriae, flagella, lytic enzymes (cellulases, xylanases, pectinases, endoglucanases, proteases, lipases), and quorum-sensing systems (Gómez-Godínez et al. 2023 ). On the other hand, due to the high competitiveness present in the rhizosphere, the production of siderophores, lytic enzymes, and antibiotics by PGPB contributes to the colonization process by reducing the growth of phytopathogens (Compant et al. 2010 ). PGPB can promote plant growth through direct and indirect mechanisms. Direct mechanisms result in plant growth and development, such as increasing available nutrients and/or regulating phytohormone levels. Indirect mechanisms, on the other hand, act by inhibiting plant pathogens like fungi and bacteria (Kong and Glick 2017 ; Olanrewaju et al. 2017 ). The main mechanisms involved in nutrient provision are phosphate solubilization, biological nitrogen fixation, and iron chelation through siderophore production. As for the regulation of phytohormone levels, there is the production of auxins, cytokinins, gibberellins and the modulation of ethylene levels through ACC deaminase activity (Gamalero and Glick 2011 ). These phytohormones can stimulate plant growth and developmental stages, such as cell elongation, cell division, root development, shoot initiation, and tissue differentiation (Ajijah et al. 2023 ). The production of antibiotics, lytic enzymes, degradation of toxins produced by pathogens, and competition for nutrients and space are some of the mechanisms involved in disease inhibition (Chepsergon and Moleleki 2023 ). Different PGPB may use one or more mechanisms to promote plant growth, and the mechanism employed can vary depending on the environmental situation (Olanrewaju et al. 2017 ; Stegelmeier et al. 2022 ). PGPB have been used to promote plant growth while simultaneously mitigating the level of toxicity or damage caused by exposure to various contaminants, such as heavy metals, in a process known as microorganism-assisted phytoremediation (Ahemad 2015 ). The efficiency of heavy metal phytoremediation is limited by metal availability in the soil, root system development, production of photosynthetically active biomass, and the plant’s tolerance to each specific metal (Gamalero et al. 2009 ). For this reason, PGPB play a crucial role in heavy metal phytoremediation by enhancing the process’s efficiency through promoting plant growth, increasing plant tolerance to metal toxicity, increasing metal solubility and mobility, and/or biotransforming metals into less toxic compounds. Most studies on the application of PGPB have focused on terrestrial plants; however, research has recently started investigating their application in hydroponic crops and aquatic plants (Ishizawa et al. 2017 , 2019 ; Jewell et al. 2023 ; Makino et al. 2022 ; Stegelmeier et al. 2022 ; Toyama et al. 2022 ). The application of PGPB in hydroponic crops presents some differences compared to terrestrial plants. Since an aquatic system is different from the soil surrounding the rhizosphere, the same plant may have different microbial communities depending on whether it grows in soil or water. Many microorganisms that promote plant growth in soil do not survive the transition to a hydroponic medium (Stegelmeier et al. 2022 ). On the other hand, the inoculation of PGPB in aquatic systems does not always produce the desired effects due to failure in colonization or competition with the native microorganisms in the inoculated plant (Ishizawa et al. 2017 ). Considering these findings and the inherent capacity of PGPB to stimulate plant growth, their integration into phytoremediation processes has emerged as a promising strategy to enhance both remediation efficiency and plant health under stress conditions, a concept known as assisted phytoremediation. 2. Materials and methods 2.1. Procurement and maintenance of Salvinia biloba plants S. biloba specimens used in the experiments were collected from Reserva Ecológica Costanera Sur, Ciudad Autónoma de Buenos Aires, Argentina (34°36′14″S 58°21′09″O), which belongs to the Paraná Delta and Islands Ecoregion. To remove potential contaminants adhered to the plants, they were thoroughly rinsed several times with running water. After washing, the plants were transferred to containers filled with a nutrient solution (CaCl 2 0.588 mg/l, MgSO 4 0.246 mg/l, NaHCO 3 0.126 mg/l, KCl 0.055 mg/l; pH 7.0) (Mendes et al. 2021 ). The containers were then placed in a controlled culture chamber under regulated temperature (26°C) and light conditions (photoperiod of 14 h of light and 10 h of darkness) (Prado et al. 2010 ) until the plants were used in the assays. 2.2. Tolerance of Salvinia biloba to different Cr(VI) concentrations 2.2.1. Evaluation of morphological changes in S. biloba in the presence of Cr(VI) and metal removal The experimental design and Cr(VI) exposure conditions were based on those described in Martínez Saucedo et al. ( 2025 ), where morphological parameters (fresh weight (FW), number of total and damaged leaves and root length) were analyzed. Individual plants were exposed to varying Cr(VI) concentrations in glass containers, each containing 500 ml of nutrient solution and 7.42 ± 0.36 g of FW. Before treatment, the plants were briefly placed on absorbent paper to remove excess water. Potassium dichromate (K 2 Cr 2 O 7 ) stock solution (1000 ppm Cr(VI)) was added to each container to achieve the target Cr(VI) concentrations: 1, 3 and 5 ppm. The treatments were incubated for 15 days in a controlled culture chamber under previously described conditions. Each treatment was performed in triplicate in three independent experiments, including a control group (0 ppm Cr(VI)). Vegetation cover was analyzed using the ImageJ software (version 1.53s) through photographic documentation at the beginning and end of the experiment. To determine residual Cr concentrations in the solution, total Cr was quantified by atomic absorption spectroscopy and Cr(VI) was quantified by colorimetry, using 30 ml aliquots from each treatment, preserved with nitric acid until analysis, as described below. At the end of the experiment, distilled water was added to restore the initial volume, compensating for evaporative losses. 2.2.2. Determination of photosynthetic pigments The content of photosynthetic pigments (chlorophyll a , b and carotenoids) was determined after 15 days of exposure to Cr(VI) using the methodology described by Emiliani et al. ( 2021 ). 1 g of fresh biomass was homogenized in 10 ml of 96% (v/v) ethanol and incubated in the dark for 24 h at room temperature. The resulting solution was centrifuged at 2500 rpm for 20 minutes, and the absorbance of the supernatant was measured at 470, 649, and 665 nm using a UV-Vis spectrophotometer (UV-1280, Shimadzu). The concentrations of chlorophyll a , chlorophyll b , and total carotenoids were calculated according to the equations proposed by Lichtenthaler and Wellburn ( 1983 ) and expressed in µg/g FW. 2.3. Isolation, maintenance and cultivation of microorganisms A total of 25 microorganisms isolated from soil, water, and sediment samples collected in Entre Ríos, Argentina (33°29'32''S 58°43'44''W) were used. This region is part of the Paraná Delta and Islands Ecoregion and was selected due to its high level of contamination with various pollutants, including heavy metals, resulting from intensive anthropogenic activities in the surrounding area (Avigliano et al. 2019 ; Peluso et al. 2020 ). To obtain cultivable microorganisms, 1 ml or 1 g of each sample (depending on the sample type) was inoculated into liquid LB medium and incubated under agitation at 30°C for 24–48 h. Subsequently, aliquots were plated onto Petri dishes containing agarized LB medium (Sambrook 2001 ) to assess the cultivable microbial diversity present in the samples. Individual bacterial isolates were obtained by successive streaking using the depletion streak technique. Morphological differentiation and biochemical assays (LIA, TSI, IMViC) were conducted to characterize the metabolic diversity of the isolated bacteria and to exclude the presence of potential pathogens. The 25 isolates were labeled with the acronym ER (representing Entre Ríos, the collection site) followed by a unique letter identifier and were maintained on LB agar plates at 4°C, with periodic subculturing to preserve viability. These bacterial isolates were preserved in LB medium supplemented with 20% glycerol at -80°C. 2.4. Identification of plant growth-promoting characteristics in vitro The bacterial isolates were characterized for their plant growth-promoting activities by evaluating the production of hydrolytic enzymes (proteases, cellulases, and lipases), phosphate solubilization, siderophores production, biological nitrogen fixation and phytohormones (auxins) production. The results were visually assessed and recorded as either presence or absence of activity, with some degree of qualitative differentiation when possible. Protease activity : The capacity to produce protease was evaluated by spot inoculation of colonies on skim milk agar plates (skim milk 50 g/l; agar 10 g/l), followed by incubation at 37°C for four days (Walsh et al. 1995 ). Cellulase activity : Cellulase production was tested on plates containing carboxymethylcellulose (CMC) medium (NaNO 3 2 g/l; K 2 HPO 4 1 g/l; MgSO 4 0.5 g/l; KCl 0.5 g/l; peptone 0.2 g/l; CMC 2 g/l; agar 17 g/l). The plates were incubated at 37°C for four days. To visualize activity, the plates were stained with 0.1% Congo Red, washed with 1 M NaCl, and fixed with 0.5% acetic acid (Teather and Wood 1982 ). Lipase activity : Lipase production was determined using the method described by Samad et al. ( 1989 ). Colonies were inoculated on plates containing medium composed of NaCl 5 g/l, CaCl 2 0.1 g/l, peptone 10 g/l, Tween-80 10 g/l, and agar 20 g/l, and incubated at 37°C for four days. Phosphate solubilization : Phosphate solubilization was assessed by plating isolates on NBRIP medium (glucose 10 g/l; (NH 4 ) 2 SO 4 0.5 g/l; NaCl 0.3 g/l; KCl 0.3 g/l; FeSO 4 .7H 2 O 0.03 g/l; MnSO 4 .H 2 O 0.03 g/l; Ca 3 (PO 4 ) 2 1 g/l; agar 15 g/l) (Nautiyal 1999 ). Plates were incubated at 37°C for four days, and solubilization was visually identified by the formation of clear halos around colonies. Siderophore production : The ability to produce siderophores was assessed using the modified Chrome Azurol S (CAS) method described by Pérez-Miranda et al. ( 2007 ). Colonies were inoculated on R2A medium (yeast extract 0.5 g/l; peptone 0.5 g/l; glucose 0.5 g/l; pyruvate 0.3 g/l; K 2 HPO 4 0.3 g/l; MgSO 4 0.05 g/l; starch 0.5 g/l; agar 15 g/l; pH 6.5 ± 0.2) and incubated at 37°C for six days. After incubation, a CAS overlay (CAS 15.12 mg; FeCl 3 2.5 mg; CTAB 18.23 mg; PIPES 7.50 mg; agar 2.25 g in 250 ml; pH 6.8) was applied and allowed to develop at room temperature. Biological nitrogen fixation : Nitrogen fixation capacity was evaluated using deep inoculation in semisolid LGI medium (glucose 5 g/l; K 2 HPO 4 0.2 g/l; KH 2 PO 4 0.6 g/l; MgSO 4 .7H 2 O 0.2 g/l; CaCl 2 .2H 2 O 0.02 g/l; Na 2 MoO 4 .2H 2 O 0.002 g/l; FeCl 3 .6H 2 O 0.01 g/l; agar 1.8 g/l) (Baldani et al. 2014 ). Colonies were resuspended in 200 µl of sterile physiological solution (NaCl 0.85% w/v) and inoculated into the medium by puncture. The tubes were incubated at 37°C for five days, and nitrogen fixation was identified by the appearance of a pellicle near the surface of the medium. Auxin production : Indole-3-acetic acid (IAA) production was quantified using the Salkowski colorimetric method (Glickmann and Dessaux 1995 ). Bacteria were cultured in 24-well plates containing 1 ml of BT liquid medium (glucose 5 g/l; K 2 HPO 4 1 g/l; NH 4 NO 3 0.4 g/l; NaCl 0.2 g/l; MgSO 4 ·7H 2 O 0.2 g/l; tryptone 20 g/l; pH 7.0 ± 0.2) under agitation (150 rpm) at 37°C for four days. After incubation, cultures were centrifuged, and 1 ml of supernatant was mixed with 1 ml of modified Salkowski reagent (98 ml of 35% perchloric acid and 2 ml of 0.5 M ferric chloride). Results were recorded after 20 minutes. 2.5. Chromium tolerance analysis of selected microorganisms The Cr(VI) tolerance of the selected PGPB was evaluated by exposing the isolates to different Cr(VI) concentrations. Inocula were obtained by culturing the bacterial isolates in liquid LB medium under agitation (120 rpm) at 30°C for 48 h. After incubation, the cultures were centrifuged at 4000 rpm for 10 minutes, and the resulting pellets were resuspended in sterile distilled water. The bacterial suspensions were adjusted to an initial optical density (OD) of 0.1 at 560 nm. Four treatments were established by adding the appropriate volume of the stock solution of K 2 Cr 2 O 7 to the LB medium to achieve final concentrations of 0, 1, 3 and 5 ppm of Cr(VI). Each treatment was set up in triplicate in 24-well plates, with 1 ml of culture per well. The plates were incubated at 30°C under agitation (120 rpm) for 48 h. Following incubation, the OD at 560 nm was measured to assess the effect of Cr(VI) on bacterial growth. Each treatment was performed in triplicate in three independent assays. 2.6. In vivo analysis of plant growth-promotion of selected microorganisms To assess the interaction between the selected bacterial isolates and S. biloba , aquatic systems were established by placing individual plants with a FW of approximately 1.6 ± 0.3 g in 100 ml of nutrient solution. The systems were inoculated with the bacterial suspensions under evaluation and incubated in a controlled culture chamber with regulated temperature and light conditions, as previously described, for 15 days. Each treatment was performed in triplicate, including an uninoculated control group. Inocula were prepared by culturing microorganisms in LB medium under agitation at 30°C for 48 h. After incubation, cultures were centrifuged and the supernatant was discarded. The resulting pellet was resuspended in sterile distilled water, and the OD at 560 nm was measured. The inoculum volume was adjusted to ensure an initial OD at 560 nm of 0.1 in each treatment. At the beginning and end of the experimental period, plant growth parameters, including FW, total number of leaves, and root length were recorded. Additionally, photographic documentation of each treatment was carried out to monitor morphological changes and total plant cover. Each treatment was performed in triplicate in three independent assays. 2.7. Inoculation of Salvinia biloba exposed to 5 ppm Cr(VI) To assess whether inoculation with the selected microorganisms enhances the Cr(VI) tolerance of S. biloba , aquatic systems containing 500 ml of nutrient solution and approximately 8.3 ± 0.06 g FW were prepared. A stock solution of K 2 Cr 2 O 7 was added to achieve a final Cr(VI) concentration of 5 ppm. The systems were inoculated with the bacterial isolates under study, including an uninoculated control group. Inocula were prepared as previously described and adjusted to an initial OD of 0.1 at 560 nm before being added to the systems. The treatments were incubated in culture chambers with controlled temperature and light conditions for 15 days. At the initial and final time, the FW, total number of leaves and root length of each treatment were recorded. Vegetation cover was analyzed as previously described, at the beginning and the final time. Additionally, a 30 ml aliquot of the solution was collected for subsequent Cr analysis. To compensate for liquid loss due to evaporation, distilled water was added at the end of the experiment to restore the initial volume. Each treatment was performed in triplicate in three independent assays. 2.8. Determination of chromium concentration in solution To evaluate the Cr removal capacity of S. biloba at different Cr(VI) concentrations and determine whether inoculation with the studied microorganisms influences removal efficiency, 30 ml aliquots of the culture solution were collected at the start and end of the experiments. The concentration of total Cr and Cr(VI) was analyzed. Cr(VI) was measured immediately after sampling using 10 ml of the aliquot, while the remaining 20 ml were stored under refrigeration for later total Cr analysis. Total Cr determination : The concentration of total Cr in the samples was determined by flame atomic absorption spectroscopy (Perkin Elmer AAnalyst 100). A calibration curve was constructed using a stock solution of Cr(NO 3 ) 3 (1000 ppm) and standard solutions of 1, 2, 4, and 6 ppm Cr prepared in 25 ml of nitric acid (HNO 3 ). Measurements were taken at a wavelength of 357.9 nm, with HNO 3 as blank. Cr(VI) determination : The Cr(VI) concentration was determined colorimetrically according to the method described in the 22nd edition of Standard Methods for the Analysis of Drinking Water and Wastewater 3500-Cr B (American Public Health Association 2012 ). A 10 ml sample was used for determination, and a calibration curve was prepared using standard solutions of 1, 2, 4, 5, and 6 ppm Cr derived from a K 2 Cr 2 O 7 stock solution (1000 X). Both samples and standards were treated with 0.1 ml of 50% (v/v) sulfuric acid, followed by the addition of 0.2 ml of a 0.5% (w/v) diphenylcarbazide (DPC) solution prepared by dissolving 5 mg of DPC in 1 ml of acetone. The reaction mixture was left to stand for 5–10 minutes to allow color development. A reaction blank using distilled water was prepared following the same procedure. After the resting period, absorbance was measured at 540 nm using a UV-vis spectrophotometer. 2.9. Identification of bacteria by partial 16S rRNA gene sequencing The microorganism that displayed positive results across the various assays was taxonomically identified via partial sequencing of the 16S rRNA gene, performed by Macrogen Inc. (Seoul, South Korea). Amplification of the 16S rRNA gene was carried out by PCR using the universal primers 27F (5'-AGA GTT TGA TCM TGG CTC AG-3') and 1492R (5'-TAC GGY TAC CTT GTT ACG ACT T-3'). The resulting PCR product was purified and sequenced using the internal primers 785F (5'-GGA TTA GAT ACC ACC CTG GTA GTA-3') and 907R (5'-CCG TCA ATT ATT CMT TTR AGT TT-3'). Identification was conducted by comparing the obtained sequence against the National Center for Biotechnology Information (NCBI) database using the BLASTn algorithm (Basic Local Alignment Search Tool). The List of Prokaryotic Names with Standing in Nomenclature (LPSN) was consulted for the genus and species-level identification of the analyzed microorganism and its comparison with the reference strain. 2.10. Statistical analysis All experiments were conducted in triplicate for each treatment, and three independent assays were performed. Results are presented as mean values with standard error of the mean (SEM). Differences between treatments were analyzed using one-way ANOVA or two-way ANOVA, followed by Tukey’s multiple comparison test. Changes between initial and final time points were assessed using a paired Student’s t -test. Prior to the analysis, data normality was assessed using the Shapiro-Wilk test ( p > 0.05). Statistical significance was set at p < 0.05. Statistical analyses were performed using GraphPad Prism 8 (version 5.01), with a significance threshold set at p < 0.05. 3. Results 3.1. Tolerance of Salvinia biloba to different Cr(VI) concentrations In a previous study (Martínez Saucedo et al. 2025 ), we reported the morphological effects of Cr(VI) toxicity on S. biloba , including reductions in fresh weight, root length, and total leaf number. These findings provided key insights into the visible damage caused by metal exposure. However, physiological and structural responses to Cr(VI) may occur earlier or independently from morphological changes. Vegetation cover may reflect alterations in plant structure and water balance, particularly in free-floating species like S. biloba , where turgor loss and leaf folding can increase apparent surface area without indicating actual growth. Likewise, photosynthetic pigments (such as chlorophyll a , chlorophyll b , and carotenoids) are sensitive biochemical markers of stress. In this context, the present study complements our earlier morphological assessment by incorporating vegetation cover analysis and pigment quantification, offering a more comprehensive understanding of the plant's responses to Cr(VI) stress. Vegetation cover measurements revealed a significant increase in surface area occupied by S. biloba at the end of the assay in treatments with 3 and 5 ppm Cr(VI) (Fig. 1 ). While no significant changes were observed at 0 and 1 ppm, higher concentrations led to a marked expansion of the plant mat, despite previous evidence of growth inhibition under the same conditions (Martínez Saucedo et al. 2025 ). This apparent increase in coverage is likely not attributable to biomass accumulation, but rather to structural alterations such as turgor loss and leaf displacement, which cause the plants to spread horizontally. The content of photosynthetic pigments (chlorophyll a , chlorophyll b and carotenoids) in leaves at the final time was determined as a physiological parameter to evaluate the toxicity of Cr(VI) in the plant species analyzed. No significant differences in photosynthetic pigment content were found in the treatments analyzed (Fig. 2 ). The results obtained are consistent with the effects of heavy metal exposure previously reported in studies on plants of the Salvinia genus (Das and Goswami 2017 ; Emiliani et al. 2020 , 2021 ; Loría et al. 2019 ). Cr(VI) exposure induced signs of toxicity, including chlorosis, loss of turgor, and necrosis, as well as reduced growth and development. These phytotoxic effects occurred progressively with increasing metal concentration and exposure time. When evaluating the range of Cr(VI) concentrations tolerated by S. biloba , it was concluded that concentrations of 1, 3, and 5 ppm were tolerated by the plants, as they survived exposure to the metal despite exhibiting toxicity symptoms. 3.2. Chromium removal capacity by Salvinia biloba After determining the Cr(VI) concentrations tolerated by S. biloba , the metal removal capacity was evaluated for the analyzed concentrations. S. biloba plants were exposed to 1, 3, and 5 ppm of Cr(VI) for 15 days as previously described, and measurements of the Cr concentration in the culture solution were taken at the initial and final time. At the initial time, all Cr present in the solution corresponded to Cr(VI). For the 1, 3, and 5 ppm treatments, the initial measured Cr concentrations were 1.48 ± 0.05, 3.93 ± 0.11, and 6.15 ± 0.10 ppm, respectively. Table 1 shows the results of total Cr and Cr(VI) concentrations in the culture solution after 15 days of plant exposure to the different treatments. At the end of the experiment, a reduction of 32%, 26%, and 21% in total Cr concentration was observed in the 1, 3, and 5 ppm treatments, respectively. These differences were statistically significant only for the 1 and 3 ppm treatments ( p < 0.01). In the different treatments, the percentage of Cr removal decreased as the initial metal concentration increased. This effect has also been reported with other metals (Emiliani et al. 2021 ). The differences observed between the initial and final total Cr concentrations were attributed to the removal of Cr by S. biloba plants through metal absorption within plant tissues and/or adsorption on the plant surface in contact with the solution. Table 1 Removal capacity of total Cr and Cr(VI) by S. biloba plants exposed to 1, 3 and 5 ppm Cr(VI) for 15 days. The mean and standard deviation of the concentration of total Cr and Cr(VI) in solution (ppm) are shown. Means indicated with asterisks are significantly different from the initial concentration ( p < 0.01). Treatment Initial total Cr (ppm) Final total Cr (ppm) % Total Cr removal 1 ppm 1.48 ± 0.05 1.01 ± 0.04 * 32% 3 ppm 3.93 ± 0.11 2.89 ± 0.15 * 26% 5 ppm 6.15 ± 0.10 4.85 ± 0.17 21% Treatment Initial Cr(VI) (ppm) Final Cr(VI) (ppm) % Cr(VI) removal 1 ppm 1.48 ± 0.05 0.27 ± 0.07 * 82% 3 ppm 3.93 ± 0.11 2.15 ± 0.26 * 45% 5 ppm 6.15 ± 0.10 4.14 ± 0.14 33% On the other hand, for Cr(VI), a reduction of 82%, 45%, and 33% in its concentration was observed in the 1, 3, and 5 ppm treatments, respectively, with the differences being statistically significant in the 1 and 3 ppm treatments ( p < 0.01). When comparing total Cr and Cr(VI) at the final time point, a greater reduction in Cr(VI) concentration can be observed. This is particularly notable in the 1 ppm treatment, where the decrease in Cr(VI) concentration is 50% greater than that of total Cr, while in the 3 and 5 ppm treatments, the difference with total Cr is 19% and 12%, respectively. These differences between total Cr and Cr(VI) concentrations at the final time point correspond to the presence of Cr in a different oxidation state. Since Cr(III) is one of the most stable Cr states (Tchounwou et al. 2012 ), it was considered that the difference between total Cr and Cr(VI) concentrations at the final time point is due to the presence of Cr(III). At the final point, a Cr(III) concentration of 0.74 ppm was observed in the 1 and 3 ppm treatments, and 0.71 ppm in the 5-ppm treatment. As mentioned earlier, a Cr(VI) solution was used to contaminate the systems at the beginning of the experiment, so the presence of the metal in other oxidation states could be attributed to the action of the plants and their associated microorganisms. For example, the native microbiota present on the plants could be capable of reducing Cr(VI) to Cr(III), or the plants themselves could have the ability to perform this reduction, as has been reported for other species in the Salvinia genus (Chocobar-Ponce et al. 2022 ). This would contribute to the detoxification of the contaminated solution, as Cr(VI) is more toxic than its trivalent form. In summary, Fig. 3 shows the percentage of Cr removal and the remaining amounts of Cr(VI) and Cr(III) in solution. 3.3. Characterization of isolated microorganisms according to their in vitro plant growth promoting characteristics PGPB can promote plant growth through direct and indirect mechanisms. Direct mechanisms result in the promotion of plant growth and development by increasing nutrient availability and producing phytohormones, while indirect mechanisms inhibit the growth of phytopathogens such as fungi and bacteria (Kong and Glick 2017 ). To characterize the 25 microorganisms isolated from soil, water, and sediment samples from the Paraná River Delta (Entre Ríos, Argentina) in terms of their plant growth-promoting traits, the presence of the following enzymatic activities was evaluated in vitro : lytic enzymes production (lipases, cellulases, proteases), phosphate solubilization, siderophore production, biological nitrogen fixation and auxins production. The results of each evaluated activity will be described below (Fig. 6 ). Production of lytic enzymes : To detect lipase production, a medium containing Tween 80 was used. The formation of a precipitate around the colonies indicates a positive result, as the hydrolysis of Tween 80 produces a precipitation reaction of free fatty acids with the CaCl 2 present in the medium (Plou et al., 1998 ) (Fig. 4 A). Cellulase production was evidenced by the formation of a decolorized halo around the colonies due to the degradation of CMC present in the medium (Teather and Wood 1982 ) (Fig. 4 B). Finally, the presence of proteases was detected by the formation of a transparent halo around the colony due to the hydrolysis of proteins in the culture medium (Fig. 4 C). Phosphate solubilization : To detect this activity, a culture medium with calcium phosphate as the phosphorus source was used. The formation of a transparent halo around the colony due to the production of organic acids indicates a positive result (Nautiyal 1999 ) (Fig. 4 D). Siderophore production : To identify siderophore production, a culture medium with an iron-dye complex (CAS-FeCl 3 ) was used. When a strong iron chelator, such as a siderophore, removes iron from the dye complex, a color change is observed (Schwyn and Neilands 1987 ). Therefore, this color change in the culture medium around the colony indicates a positive result (Fig. 4 E). Biological nitrogen fixation : For the detection of biological nitrogen fixation, a semisolid medium without nitrogen sources was used. The nitrogenase complex (a set of enzymes responsible for reducing atmospheric nitrogen to ammonia) is highly sensitive to oxygen. Therefore, the use of semisolid media and deep puncture inoculation allows the bacteria to grow while protecting the nitrogenase from inhibition by elevated oxygen concentrations (Baldani et al. 2014 ). By using a nitrogen-free medium, a microorganism is positive for biological nitrogen fixation if it is able to grow (Fig. 4 F). Auxins production : Bacteria that produce auxins were identified by the development of a pink color due to the reaction of the Salkowski reagent with the phytohormones. The intensity of the color developed corresponds to different levels of auxin production, with the intensity increasing as the concentration in the solution rises (Abeed et al. 2022 ). As shown in Fig. 4 G, a marked change to an orange/pink coloration was considered a positive result for auxin production. Although it was possible in some cases to distinguish between different levels of compound production, the results were analyzed qualitatively and recorded as either positive or negative. Of the 25 isolates tested (results provided in Supplementary Information), 3 microorganisms with the highest number of positive results were selected to proceed with further testing. The selected isolates were named ER-C, ER-G and ER-Y. The results for these isolates are presented in Table 2 . Table 2 Results of the three bacterial isolates (ER-C, ER-G and ER-Y) with the highest number of positive plant growth promotion (PGP) activities. (+) positive activity; (-) negative activity. PGP Activity ER-C ER-G ER-Y Lipases production + - + Cellulases production - - - Proteases production + + + Phosphate solubilization - + + Siderophore production + + + Biological nitrogen fixation + + + Auxins production + + - Total positives 5 5 5 The selected microorganisms were used as PGPB in the following experiments in order to evaluate their potential to promote the growth of S. biloba plants and to analyze the effect of their application on the phytoremediation process. 3.4. Chromium tolerance of selected microorganisms Figure 5 . Tolerance of the microorganisms ER-C, ER-G and ER-Y to 0, 1, 3 and 5 ppm Cr(VI) expressed as ΔOD at 560 nm. The mean and standard deviations are shown. Means marked with asterisks are significantly different (*** p = 0.0003; **** p < 0.0001). When analyzing the growth of the 3 selected isolates (Fig. 5 ), different variations in the final OD were observed. The ER-C microorganism showed no significant changes in growth, except in the 3-ppm treatment, where the final OD increased by 11.29% compared to the control group. The growth of ER-G decreased significantly by 4.44% in the 5-ppm treatment compared to the control. Lastly, a progressive reduction in the growth of the ER-Y microorganism was observed as Cr(VI) concentration increased. The 5-ppm treatment differed significantly from the 1 ppm treatment, showing a 4.07% reduction compared to the control. Although the presence of Cr(VI) affected the growth of the microorganisms, none of the concentrations tested were lethal. Notably, ER-C exhibited lower growth in all treatments compared to the other microorganisms. This could be attributed to its lower growth rate, which may impact its ability to effectively colonize plant tissues and promote plant growth in subsequent experiments. 3.5. In vivo plant growth promotion by selected microorganisms Figure 6 shows the results of the development of the plants when inoculated with the different microorganisms, expressed as the variation (Δ) of the measurements of FW (Fig. 6 A), total leaves (Fig. 6 B) and root length (Fig. 6 C) between the final and initial time. No significant differences were observed in any of the cases. This is likely attributable to the high variability inherent in biological systems. Regarding FW (Fig. 6 A), all plants showed an increase in weight compared to the initial condition. The uninoculated group increased an average its FW by 6.85%, while the other treatments exhibited increases of 1.15%, 7.83% and 11.58% for plants inoculated with the microorganisms ER-C, ER-G and ER-Y, respectively. However, the observed differences were not statistically significant. On the other hand, when analyzing the total number of leaves (Fig. 6 B), it was observed that in the uninoculated plants, the increase in the number of leaves was 22% over the 15 days of the experiment. Although no significant differences were found between the treatments and the control, it was observed that inoculation with ER-C tended to decrease the number of leaves compared to the uninoculated group, showing a variation of 19.23%. In contrast, inoculation with ER-G and ER-Y showed a tendency to increase the number of leaves by 32% and 33.33%, respectively. Finally, regarding the variation in root length (Fig. 6 C), the uninoculated plants showed an average increase of 7.95%. None of the treatments with microorganisms showed significant differences compared to this value. However, inoculation with ER-G tended to decrease this parameter relative to the uninoculated group, with a 6.67% increase, while inoculation with ER-Y showed a root length increase similar to that of the uninoculated treatment, at 7.50%. Inoculation with ER-C resulted in a greater root length variation compared to the uninoculated treatment, with an increase of 12.94%. 3.6. Phytoremediation of 5 ppm Cr(VI) by Salvinia biloba inoculated with the selected microorganisms 3.6.1. Evaluation of the improvement in the tolerance to Cr(VI) by inoculated S. biloba Considering the range of Cr(VI) concentrations analyzed in this study, 5 ppm of Cr(VI) was selected, as it is the highest concentration tolerated by both the microorganisms under study and the plants, and it has also been evaluated for other species of the genus Salvinia (Prado et al. 2010 ). Figure 7 shows the results of the variation of FW (Fig. 7 A), total leaves (Fig. 7 B) and root length (Fig. 7 C) between initial and final time (15 days) in inoculated S. biloba plants exposed to 5 ppm Cr(VI). Additionally, the morphology of the plants was visually analyzed between the initial and final time. When observing the results of the FW variation (Fig. 7 A), it was found that, although there were no significant differences between any treatment, inoculation with ER-C resulted in a smaller weight increase compared to the uninoculated treatment, with 3.72% and 7.72%, respectively. Inoculation with ER-G resulted in an average variation of 11.75%, while the greatest increase was found when inoculating with ER-Y, which showed a 17.93% FW increase. In the case of the number of total leaves (Fig. 7 B), no notable differences were observed, but a positive trend was found when inoculating with ER-G and ER-Y in comparison to the uninoculated control (increases of 31.39%, 26.92% and 21.81%, respectively). Ultimately, when analyzing root length (Fig. 7 C), the uninoculated control showed a negative trend in their development (a decrease of 2.09%). A positive trend in root development was observed in the presence of 5 ppm of Cr(VI) when inoculated with ER-C (5.64%), ER-G (15.32%), and ER-Y (10.48%). Regarding plant cover, an increase of 11.03%, 14.18%, and 15.27% was observed in the systems inoculated with ER C, ER G, and ER Y, respectively (Fig. 8 ). These differences between the initial and final time points were statistically significant for the treatments with ER-G and ER-Y ( p < 0.05). However, unlike the findings reported by Martínez Saucedo et al. ( 2025 ) for uninoculated plants and the observations shown in Fig. 1 , the increase in plant cover observed in Fig. 8 is attributed to actual plant growth rather than turgor loss, as demonstrated in Fig. 9 . 3.6.2. Evaluation of the improvement in Cr phytoremediation by inoculated S. biloba To determine whether the inoculation of S. biloba plants with the selected microorganisms enhances their Cr removal capacity, the Cr(VI) concentration in solution was measured at both the initial and final times (Table 3 ). In uninoculated plants, the Cr(VI) concentration decreased by 35.54%, a value similar to the reduction percentage obtained in the previous assay for the treatment with 5 ppm of Cr(VI) (33%). Regarding the plants inoculated with the microorganisms under study, the Cr concentration reduction was 4.14% lower in the treatments inoculated with ER-C, compared to the uninoculated group. Plants inoculated with ER-G and ER-Y showed a greater reduction compared to the uninoculated group, with reductions of 1.09% and 8.46%, respectively. The differences between the initial and final times were significant in all treatments ( p < 0.05); however, no statistically significant differences were found between the treatments at the final time. Table 3 Cr(VI) removal capacity by S. biloba plants inoculated with the selected microorganisms and the uninoculated treatment (U) exposed to 5 ppm for 15 days. The means marked with asterisks are significantly different from the initial concentration ( p < 0.01). Initial Cr(VI) (ppm) Final Cr(VI) (ppm) Cr(VI) removal (%) Difference with U U 5.43 ± 0.14 3.50 ± 0.21 * 35.54 - ER-C 5.40 ± 0.08 3.56 ± 0.26 * 34.07 − 4.14% ER-G 5.26 ± 0.07 3.37 ± 0.30 * 35.93 + 1.09% ER-Y 5.24 ± 0.14 3.22 ± 0.01 * 38.55 + 8.46% 3.7. Identification of the most promising microorganism for assisted phytoremediation of Cr(VI) by Salvinia biloba Based on the results obtained, the ER-Y microorganism demonstrated promising outcomes in both plant growth promotion and enhancing the efficiency of the Cr(VI) phytoremediation process mediated by S. biloba . Upon analyzing the 16S rRNA gene sequence, it was identified that the ER-Y microorganism belongs to the species Serratia nematodiphila , with a 99.85% similarity with the reference strain, S. nematodiphila DSM21420 T . This species corresponds to Gram-negative, aerobic, rod-shaped, motile bacteria with a single lateral flagellum, isolated from the intestine of the nematode Heterorhabditidoides chongmingensis (Zhang et al. 2009 ). The partial 16S rRNA gene sequence for the microorganism S. nematodiphila ER-Y was deposited in the NCBI Sequence Read Archive (SRA) database under the BioSample Accession SAMN46231118. 4. Discussions and conclusion Phytoremediation is a sustainable alternative for the remediation of sites contaminated with various pollutants, including heavy metals. However, the efficiency of the process depends on the ability of the plants used to tolerate and accumulate contaminants, as well as on biomass production and the bioavailability of contaminants (Abbaszadeh-Dahaji et al. 2016 ). One strategy to increase the efficiency of the process is the application of PGPB, given their ability to promote plant growth, increase plant tolerance to stress, and/or enhance the solubility and mobility of metals (Ma et al. 2011 ). Based on the above, the hypothesis was proposed that the inoculation of S. biloba with PGPB isolated from contaminated site samples improves the Cr phytoremediation process in aquatic systems; and various experiments were conducted to test this hypothesis. The observed increase in vegetation cover of S. biloba at 3 and 5 ppm Cr(VI) suggests a complex response to metal exposure that challenges traditional interpretations of surface area expansion as a proxy for plant growth. Despite the significant increase in surface coverage, particularly at the highest Cr(VI) concentrations tested, previous reports have indicated that S. biloba exhibits growth inhibition and physiological stress under similar conditions (Martínez Saucedo et al. 2025 ). This apparent contradiction highlights the need to interpret cover expansion with caution, especially in floating macrophytes where horizontal spread may result not from active growth but from morphological alterations. In this case, structural changes such as leaf displacement, reduced turgor pressure, or alterations in leaf angle and buoyancy likely contributed to the increased surface area. These modifications may reflect stress responses rather than enhanced performance, reinforcing the importance of integrating physiological and morphological metrics when assessing phytotoxicity and plant behavior in contaminated environments. The lack of significant differences in photosynthetic pigment content (chlorophyll a , chlorophyll b , and carotenoids) across treatments further supports the notion that increased cover did not correspond to improved physiological status. Similar results have been reported for aquatic macrophytes exposed to Cr(VI) and other metals, where pigment stability was observed despite growth inhibition (Dhir et al., 2009 ; Prado et al., 2010 ). One possible explanation is the limited translocation of Cr to photosynthetically active tissues, a mechanism described in several aquatic plant species as a means of protecting chloroplasts from oxidative damage (Srivastava et al., 2021 ). However, studies have demonstrated that S. biloba is capable of translocating metals to aerial tissues, including leaves (Emiliani et al., 2020 ), indicating that the Cr concentrations used in this study may not have reached thresholds sufficient to impair pigment biosynthesis. Alternatively, pigment stability may reflect a tolerance strategy in S. biloba, enabling maintenance of photosynthetic function under moderate metal stress. Together, these results underscore the importance of a multidimensional approach when evaluating plant responses to contaminants. Relying solely on surface area or pigment content as indicators may lead to misinterpretation of plant health and performance. In particular, morphological spread under metal stress may mask underlying physiological impairments, complicating assessments of phytoremediation efficiency. Future studies should combine measurements of pigment degradation, antioxidant enzyme activity, and biomass accumulation to more accurately characterize the impact of Cr(VI) on S. biloba and other floating macrophytes. Regarding the Cr removal capacity, S. biloba plants removed 32%, 26%, and 21% of the total Cr in treatments with 1, 3, and 5 ppm, respectively. This reduction could be attributed to the absorption and accumulation of the metal inside the plants and/or to the adsorption on the surface of the plant tissues in contact with the contaminated solution. However, since the Cr content in the plant tissues was not measured in this study, the mechanism of the phytoremediation process cannot be determined. However, Emiliani et al. ( 2020 ) reported that this plant exhibited the ability to both adsorb certain metals on its surface and absorb and accumulate them in its biomass, the latter being the main phytoremediation mechanism of S. biloba , since the metal concentrations found inside the plant were higher than those found on its surface. On the other hand, a greater reduction in the Cr(VI) concentration was observed compared to total Cr, suggesting that Cr(VI) was reduced to other oxidation states, likely due to the action of native microorganisms associated with the plants or the plants themselves, as has been reported for other species of this genus (Chocobar-Ponce et al. 2022 ). Cr(VI) is the most toxic form of Cr due to the ease with which it diffuses through the cell membrane, so its reduction to other oxidation states, such as Cr(III), contributes to the detoxification process of the contaminated solution (Deepa et al. 2025 ). On the other hand, from soil, water, and sediment samples collected from areas contaminated with heavy metals due to anthropogenic activity in the Paraná River Delta, 25 morphologically distinct microorganisms were isolated. These microorganisms were characterized in vitro based on their plant growth-promoting activities, from which the ER-C, ER- G and ER-Y isolates were selected due to their highest number of positive results. The feasibility of applying the selected microorganisms in the Cr phytoremediation process carried out by S. biloba depends on the microorganisms' tolerance to the metal. Therefore, their growth in the presence of the different concentrations of Cr used in the assays was evaluated. The three selected isolates were able to grow at all Cr(VI) concentrations analyzed, exhibiting slight alterations in growth; thus, no microorganism was discarded for subsequent experiments. For PGPB to interact with plants and produce a beneficial effect on plant growth in aquatic systems, they must be able to colonize plant tissue and compete with the native microbiota associated with the plants (Ishizawa et al. 2017 ). It is also important to consider that microorganisms isolated from the soil may not be able to survive in hydroponic cultures (Stegelmeier et al. 2022 ). Therefore, to determine the ability of the selected microorganisms to interact with S. biloba plants in aquatic systems, plant growth promotion was evaluated in vivo . The three microorganisms had positive effects on various morphological parameters indicative of plant development compared to uninoculated plants. In treatments inoculated with ER-G and ER-Y, an increase in the number of leaves and FW of the plants was observed, while root length increased in plants inoculated with ER-C, which was the microorganism that showed the highest production of auxins, phytohormones associated with root tissue development (Vanneste et al. 2025 ). Based on these results, it was concluded that the three selected microorganisms were able to interact with the plants and produce a plant growth-promoting effect on hydroponic cultures. Finally, it was evaluated whether the inoculation of S. biloba plants with the selected microorganisms affects the plant morphology and the Cr(VI) phytoremediation process. Although no statistically significant differences in FW were detected across treatments, the observed trends suggest that bacterial inoculation influenced plant performance under Cr(VI) exposure. In particular, S. biloba inoculated with ER-Y exhibited the greatest FW increase, nearly doubling the response seen in the uninoculated control. This trend was also reflected in the number of total leaves, where treatments with ER-G and ER-Y resulted in higher percentages of leaf increase compared to the control. While not statistically significant, these morphological indicators suggest a potential growth-promoting effect of both isolates, particularly ER-Y. Root length responses further support the positive role of bacterial inoculation in mitigating Cr(VI) stress. Plants in the uninoculated control displayed a reduction in root development, consistent with the known inhibitory effects of Cr(VI) on root elongation (Dhir et al., 2009 ). In contrast, inoculated plants showed positive trends in root length, especially those treated with ER-G and ER-Y. These findings indicate that the selected bacterial strains may alleviate Cr(VI) toxicity through plant growth-promoting mechanisms such as phytohormone production, improved nutrient uptake, or Cr detoxification at the rhizoplane. Vegetation cover measurements provide further evidence for the beneficial effects of inoculation. In contrast to previous findings where increases in cover were associated with loss of turgor and horizontal spread under Cr(VI) stress (Martínez Saucedo et al. 2025 ), the present results demonstrate significant increases in cover for ER-G and ER-Y treatments that are attributable to true biomass accumulation and vertical growth, as confirmed by morphological observations. These results reinforce the utility of combining vegetation cover data with visual and quantitative morphological assessments to avoid misinterpretations in phytoremediation studies. These results suggest that the bacterial strains ER-G and ER-Y enhance S. biloba growth under moderate Cr(VI) exposure, likely through mechanisms typical of PGPB. While the absence of statistical significance in some parameters limits definitive conclusions, the consistency of positive trends across multiple growth indicators highlights the potential of these strains for application in assisted phytoremediation strategies. Further studies involving larger sample sizes, longer exposure periods, and mechanistic assays (e.g., quantification of IAA production or Cr reduction capacity) are warranted to elucidate the functional roles of these bacterial partners. Regarding the effect of inoculation on Cr removal, a greater decrease in Cr(VI) concentration was observed in treatments inoculated with ER-Y compared to the uninoculated group. This decrease could be due to either the absorption and/or adsorption of the metal by the plants (Maine et al. 2016 ; Oliveira et al. 2019 ), or its reduction to other oxidation states by the action of the inoculated microorganisms, the plants' native microbiota, and/or the plants themselves (Ahemad 2015 ; Chocobar-Ponce et al. 2022 ). The plants inoculated with the ER-Y microorganism showed one of the greatest increases in plant biomass compared to the uninoculated plants, and also a greater decrease in Cr(VI) concentration in the effluent compared to the uninoculated group. This difference in Cr removal could be attributed to a higher capacity for metal accumulation in the plant tissues due to the increased plant biomass. Considering all of this, ER-Y, identified as Serratia nematodiphila , was selected as a promising microorganism to continue with the assisted phytoremediation studies. While some species of the Serratia genus, primarily S. marcescens , are of clinical importance due to their pathogenicity to humans, S. nematodiphila has not been described as a pathogen (Tang et al. 2020 ). On the other hand, this microorganism has been previously reported as a PGPB, exhibiting various plant growth-promoting activities such as siderophore production, phosphate solubilization (results also found in this work), ACC deaminase production, as well as antimicrobial activity, which could be related to the enzymatic activities found in this study (Basharat et al. 2018 ; Chen et al. 2010 ; Sanchez-Cruz et al. 2019 ). This species also shows tolerance to the presence of several contaminants, including metals (Sanchez-Cruz et al. 2019 ), which contributes to its application as a PGPB in phytoremediation processes. For example, inoculation of S. nematodiphila in Solanum nigrum L. plants growing in soils contaminated with various concentrations of Cd not only stimulated plant growth but also influenced metal accumulation in different plant tissues (Chen et al. 2010 ). On the other hand, S. nematodiphila isolated from chromite mine soils has been reported to reduce Cr(VI) to Cr(III) (Leonard and Mishra 2022 ), suggesting its potential application in bioreduction processes for this contaminant. Moreover, a proteomic analysis of S. nematodiphila sp. MB307 revealed the production of chaperones, membrane integrity proteins, mobility, and transporter proteins. Comparative analysis with other metal-resistant bacteria provided evidence that these proteins play a crucial role in tolerance to high metal concentrations (Basharat et al. 2022 ). Genomic analysis of S. nematodiphila MB307 demonstrated the presence of genes associated with plant growth promotion, antimicrobial activity, bioremediation, metal tolerance, azo dye degradation, and ibuprofen degradation, among others (Basharat et al. 2018 ). Therefore, this microorganism represents a promising candidate for further evaluation in these processes. In conclusion, a positive effect was observed on the Cr(VI) phytoremediation process performed by S. biloba plants when inoculated with the microorganism ER-Y, isolated from heavy metal-contaminated sites in the Paraná River Delta, Argentina. Notably, this microorganism, identified as S. nematodiphila , demonstrated promising results both as a PGPB and in assisted phytoremediation when applied to S. biloba . Future studies focusing on optimizing microorganism dosage and application methods are necessary to enhance the process. The use of PGPB could represent a promising strategy for improving the efficiency of phytoremediation; however, further research is required to optimize its application. Declarations Acknowledgements The authors gratefully acknowledge the support provided by the Universidad Argentina de la Empresa (UADE) for the development of this study. Funding This work was supported by Universidad Argentina de la Empresa (UADE) (Project numbers P21T08). Author Pamela Romina Bernabeu has received research support from UADE. Author Contributions All authors contributed to the study conception and design. Material preparation, data collection and analysis were performed by María de los Ángeles Martínez Saucedo and Pamela Romina Bernabeu. The first draft of the manuscript was written by Pamela Romina Bernabeu and all authors commented on previous versions of the manuscript. All authors read and approved the final manuscript. Ethical Approval This is not applicable. Consent to Participate This is not applicable. Consent to Publish This is not applicable. Competing Interests The authors have no relevant financial or non-financial interests to disclose. Data Availability Statement Data will be made available on reasonable request. References Abbaszadeh-Dahaji P, Omidvari M, Ghorbanpour M (2016) Increasing phytoremediation efficiency of heavy metal-contaminated soil using PGPR for sustainable agriculture. Plant-microbe interaction: An approach to sustainable agriculture. 187–204. https://doi.org/10.1007/978-981-10-2854-0_9 Abeed AH, Mahdy RE, Alshehri D, Hammami I, Eissa MA, Abdel Latef AAH, Mahmoud GAE (2022) Induction of resilience strategies against biochemical deteriorations prompted by severe cadmium stress in sunflower plant when Trichoderma and bacterial inoculation were used as biofertilizers. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-7178802","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":494730529,"identity":"8867c684-871f-4aaa-b0e2-c43d42972dfb","order_by":0,"name":"María de los Ángeles Martínez Saucedo","email":"","orcid":"","institution":"Universidad Argentina de la Empresa","correspondingAuthor":false,"prefix":"","firstName":"María","middleName":"de los Ángeles Martínez","lastName":"Saucedo","suffix":""},{"id":494730530,"identity":"a788798a-3cb8-4df8-801d-1546b3598764","order_by":1,"name":"Pamela Romina Bernabeu","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA6ElEQVRIiWNgGAWjYDACCTB5gIGBvYGBgQfKJlILzwGStUgkEKnFXLr52OeCmjtyBjffGH54U8Egx3cjgfFzBR4tlnOOJc+eceyZscHtHGPJOWcYjCVvJDBLnsGjxeBGjjEzD9vhxA23cwykedsYEjfcSGCQbMCrJf8zM8+/w/Ubbp4x/s37j6EeqIX5J34tOczMvG2HEwxu8JhJ8zYwABkJbHhtAfrFmJm375nhzDNpZUCOBJDxsM0SnxZgiD1m5vl2R57v+OHNN97U2AAZyYdv4nUYGh8UTYz4NGBqGQWjYBSMglGACQACxVJ/Fv+YdAAAAABJRU5ErkJggg==","orcid":"https://orcid.org/0009-0008-9323-1225","institution":"Instituto de Tecnología (INTEC), Universidad Argentina de la Empresa (UADE)","correspondingAuthor":true,"prefix":"","firstName":"Pamela","middleName":"Romina","lastName":"Bernabeu","suffix":""}],"badges":[],"createdAt":"2025-07-21 15:00:15","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-7178802/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-7178802/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1007/s11356-025-37186-6","type":"published","date":"2025-11-14T15:58:28+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":88658290,"identity":"ba76aa21-1145-4b65-be43-a9c2ce4af634","added_by":"auto","created_at":"2025-08-08 20:07:39","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":563994,"visible":true,"origin":"","legend":"\u003cp\u003eVegetation cover (cm\u003csup\u003e2\u003c/sup\u003e) of \u003cem\u003eS. biloba\u003c/em\u003e at the initial and final time (15 days) under different Cr(VI) concentrations (0, 1, 3, and 5 ppm). ***\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.0001.\u003c/p\u003e","description":"","filename":"Fig.1.png","url":"https://assets-eu.researchsquare.com/files/rs-7178802/v1/7303eca95e62b00403add2a1.png"},{"id":88658287,"identity":"9a5b19ff-84ef-44a3-9652-21f061e46d6d","added_by":"auto","created_at":"2025-08-08 20:07:39","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":523705,"visible":true,"origin":"","legend":"\u003cp\u003ePhotosynthetic pigment content (chlorophyll \u003cem\u003ea\u003c/em\u003e, chlorophyll \u003cem\u003eb\u003c/em\u003e and carotenoids, expressed as µg/g) of plants exposed to 0, 1, 3 and 5 ppm Cr(VI) for 15 days.\u003c/p\u003e","description":"","filename":"Fig.2.png","url":"https://assets-eu.researchsquare.com/files/rs-7178802/v1/e63f74478526b12fdbcd8aed.png"},{"id":88658962,"identity":"40f3cb7c-e8c4-4b8a-8f24-11fec83be381","added_by":"auto","created_at":"2025-08-08 20:15:39","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":1246878,"visible":true,"origin":"","legend":"\u003cp\u003ePercentage of Cr(VI) and Cr(III) remaining in solution and percentage of removal in the treatments with \u003cem\u003eS. biloba\u003c/em\u003e exposed to 1, 3 and 5 ppm Cr(VI) for 15 days.\u003c/p\u003e","description":"","filename":"Fig.3.png","url":"https://assets-eu.researchsquare.com/files/rs-7178802/v1/eae08d0952447f74aef9423a.png"},{"id":88658969,"identity":"18f9c603-aebd-46b6-8391-60d6caed50ad","added_by":"auto","created_at":"2025-08-08 20:15:39","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":24680242,"visible":true,"origin":"","legend":"\u003cp\u003eExamples of positive results for lipases (A), cellulases (B) and proteases production (C), phosphate solubilization (D), siderophore production (E), biological nitrogen fixation (F) and auxins production (G). Arrows indicate positive results. In the case of auxin production, the intensity of the color developed depends on the auxin concentration (shown in increasing order).\u003c/p\u003e","description":"","filename":"Fig.4.png","url":"https://assets-eu.researchsquare.com/files/rs-7178802/v1/87a2a584da6a3041991eeee0.png"},{"id":88658251,"identity":"98859a95-87eb-4d77-a456-bad670beda44","added_by":"auto","created_at":"2025-08-08 20:07:37","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":478705,"visible":true,"origin":"","legend":"\u003cp\u003eTolerance of the microorganisms ER-C, ER-G and ER-Y to 0, 1, 3 and 5 ppm Cr(VI) expressed as ΔOD at 560 nm. The mean and standard deviations are shown. Means marked with asterisks are significantly different (***\u003cem\u003ep\u003c/em\u003e = 0.0003; ****\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.0001).\u003c/p\u003e","description":"","filename":"Fig.5.png","url":"https://assets-eu.researchsquare.com/files/rs-7178802/v1/cc765497b0dbc61f8a079d29.png"},{"id":88658256,"identity":"f7f9104e-73bb-4d83-8293-80f10132acd0","added_by":"auto","created_at":"2025-08-08 20:07:38","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":1905389,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003eIn vivo\u003c/em\u003e plant growth promotion of the microorganisms evaluated in \u003cem\u003eS. biloba\u003c/em\u003eplants and the uninoculated (U) treatment. Variation (Δ) of fresh weight (FW) (A), number of total leaves (B) and root length (C) between initial and final time (15 days).\u003c/p\u003e","description":"","filename":"Fig.6.png","url":"https://assets-eu.researchsquare.com/files/rs-7178802/v1/d203b9aed57471a428f492d9.png"},{"id":88658270,"identity":"3230d877-10d8-4df8-9e93-c24d49ea3d72","added_by":"auto","created_at":"2025-08-08 20:07:38","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":1974605,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003eIn vivo\u003c/em\u003e plant growth promotion of microorganisms (ER-C, ER-G and ER-Y) evaluated in \u003cem\u003eS. biloba\u003c/em\u003e plants and the uninoculated (U) treatment in the presence of 5 ppm Cr(VI). Variation (Δ) of fresh weight (A), number of leaves (B) and root length (C) at initial and final time (15 days). *\u003cem\u003ep \u003c/em\u003e\u0026lt; 0.05.\u003c/p\u003e","description":"","filename":"Fig.7.png","url":"https://assets-eu.researchsquare.com/files/rs-7178802/v1/997404ba8ff418e40176f502.png"},{"id":88658288,"identity":"d5fc8cec-1494-4c2b-9772-83da7aa3e31e","added_by":"auto","created_at":"2025-08-08 20:07:39","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":342643,"visible":true,"origin":"","legend":"\u003cp\u003eChanges in vegetation cover (cm\u003csup\u003e2\u003c/sup\u003e) in \u003cem\u003eS. biloba\u003c/em\u003e plants inoculated with ER-C, ER-G, and ER-Y under exposure to 5 ppm of Cr(VI). Each treatment was analyzed independently using a paired \u003cem\u003et\u003c/em\u003e-test to compare initial and final time points. * \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05.\u003c/p\u003e","description":"","filename":"Fig.8.png","url":"https://assets-eu.researchsquare.com/files/rs-7178802/v1/611f3081b101433feed57083.png"},{"id":88658254,"identity":"8f9df1a2-ba15-4ef8-8022-fca920f3013e","added_by":"auto","created_at":"2025-08-08 20:07:37","extension":"jpeg","order_by":9,"title":"Figure 9","display":"","copyAsset":false,"role":"figure","size":442288,"visible":true,"origin":"","legend":"\u003cp\u003eMorphological changes observed in \u003cem\u003eS. biloba\u003c/em\u003eplants inoculated with microorganisms ER-C, ER-G and ER-Y exposed to 5 ppm of Cr(VI) for 15 days.\u003c/p\u003e","description":"","filename":"Fig.9.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-7178802/v1/0c2c9ba9e4c26db7432fbc7c.jpeg"},{"id":96105125,"identity":"672f9d2e-065e-42e5-8644-5ac5e561ccae","added_by":"auto","created_at":"2025-11-17 16:09:00","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":29707957,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7178802/v1/a171ca24-b681-486a-ae02-7cc331dfe1bd.pdf"},{"id":88658271,"identity":"eff5687c-dcae-4731-b35d-3a2aeb797201","added_by":"auto","created_at":"2025-08-08 20:07:38","extension":"pdf","order_by":17,"title":"","display":"","copyAsset":false,"role":"supplement","size":188960,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryInformation.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7178802/v1/6e9fb64eb8b1433e21984870.pdf"}],"financialInterests":"","formattedTitle":"Use of plant growth-promoting bacteria to enhance chromium phytoremediation by Salvinia biloba","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eHeavy metals are a group of metals and metalloids characterized by high atomic weight (greater than 40.04 g/mol), high relative density (greater than 5 g/cm\u003csup\u003e3\u003c/sup\u003e), and toxicity, even at low concentrations in some cases (Haldar and Ghosh \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Yadav et al. \u003cspan citationid=\"CR81\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). These metals naturally occur in the environment; however, the primary source of heavy metal pollution comes from human activities, such as mining, industrial processes, industrial and domestic wastewater discharge, and excessive use of pesticides and fertilizers in agriculture (Ali et al. \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). Consequently, heavy metal concentrations in the environment have risen to dangerous levels (Vareda et al. \u003cspan citationid=\"CR78\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). Common examples of heavy metal pollutants include arsenic (As), lead (Pb), mercury (Hg), chromium (Cr), zinc (Zn), cadmium (Cd), copper (Cu), and nickel (Ni) (Pratush et al. \u003cspan citationid=\"CR62\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). While some of these heavy metals are essential for life, acting as cofactors in metabolic and enzymatic pathways, they can become toxic when present in high concentrations (Briffa et al. \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Mishra et al. \u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e2019\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eThe toxicity of heavy metals depends on their chemical nature, concentration, oxidation state, and bioavailability (Haldar and Ghosh \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). In the case of Cr, toxicity is primarily determined by its oxidation state and solubility (Tchounwou et al. \u003cspan citationid=\"CR72\" class=\"CitationRef\"\u003e2012\u003c/span\u003e). Cr can exist in various oxidation states in the environment, with trivalent chromium (Cr(III)) and hexavalent chromium (Cr(VI)) being the most prevalent forms. Cr(VI) predominantly exists as chromate (CrO\u003csub\u003e4\u003c/sub\u003e\u003csup\u003e2\u0026minus;\u003c/sup\u003e), dichromate (Cr\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e7\u003c/sub\u003e\u003csup\u003e2\u0026minus;\u003c/sup\u003e), and chromium trioxide (CrO\u003csub\u003e3\u003c/sub\u003e) (Pratush et al. \u003cspan citationid=\"CR62\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). These two oxidation states exhibit distinct physicochemical properties, chemical behavior, mobility, bioavailability, and toxicity (Malaviya et al. \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). Cr(VI) is more toxic than Cr(III) due to its higher redox potential, greater solubility, and ability to easily diffuse across cell membranes. Once inside the cell, Cr(VI) is reduced to Cr(III), generating free radicals and reactive oxygen species (ROS) (Ahemad \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2015\u003c/span\u003e; Briffa et al. \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Prado et al. \u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). High oxygen levels in the environment can reconvert Cr(III) into Cr(VI), making Cr a significant water pollutant (Malaviya et al. \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e2020\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eWater pollution by heavy metals is considered one of the most severe environmental problems, affecting not only aquatic organisms but also plants, since metals are transported to sediments and soils where they accumulate (Aziz et al. \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). Once heavy metals enter aquatic environments, they tend to bind with iron, manganese oxides, sulfides, organic compounds, and clay minerals in sediments, where they are quickly deposited. Depending on environmental conditions, these metals can be released back into the water (Zhao et al. \u003cspan citationid=\"CR83\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Industrial wastewater discharges are one of the main sources of heavy metals in the environment, and thus many rivers located near industries that use heavy metals are heavily polluted (Briffa et al. \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). To comply with regulations, both water intended for human consumption and wastewater must undergo treatment (Vareda et al. \u003cspan citationid=\"CR78\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). However, many water treatment techniques used in developing countries are ineffective at removing heavy metals (Joseph et al. \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). Various physical and chemical methods exist for removing heavy metals from liquid media, such as membrane filtration, ion exchange, adsorption, and chemical precipitation (Fei and Hu \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). However, these methods are not always efficient, sustainable, or cost-effective (Ali et al. \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Emiliani et al. \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). For this reason, alternative methods that are more environmentally friendly and economical for remediating contaminated sites have been investigated, such as phytoremediation. This is a biotechnological technique that uses plant biomass to absorb and accumulate contaminants from soil, air, and water through different mechanisms (Kafle et al. \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Tiwari et al. \u003cspan citationid=\"CR74\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). A wide range of organic and inorganic contaminants, such as insecticides, chlorinated solvents, hydrocarbons, surfactants, and heavy metals, can be remediated through phytoremediation (Ali et al. \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Tufail et al. \u003cspan citationid=\"CR76\" class=\"CitationRef\"\u003e2022\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eTo be effectively used in phytoremediation, a plant must be native to the contaminated site, have rapid growth, and be capable of absorbing and tolerating contaminants (Ali et al. \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). The main objective of most phytoremediation studies is to extract contaminants through plant roots, translocate them to photosynthetically active biomass (leaves), and concentrate them for subsequent processing (Malaviya et al. \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). Aquatic plants, which often use rhizofiltration as a mechanism for removing heavy metals, are considered for treating contaminated waters due to their rapid growth rate, high biomass production, and potential for contaminant removal, as well as their ability to develop an extensive root system (Ali et al. \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Bora and Sarma \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). They play an important role in aquatic ecosystems as they act as natural filters for contaminants transported by water and serve as bioindicators of various stressors in ecosystems (Eid et al. \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Emiliani et al. \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Furthermore, many of the aquatic plants studied for their phytoremediation ability are typically invasive species that are resistant to nutrient deficiencies and environmental variations (Newete and Byrne \u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e2016\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eSeveral species of aquatic plants, such as \u003cem\u003eEichhornia\u003c/em\u003e spp., \u003cem\u003eSalvinia\u003c/em\u003e spp., \u003cem\u003ePistia stratiotes\u003c/em\u003e, \u003cem\u003eLemna minor\u003c/em\u003e, \u003cem\u003eAzolla\u003c/em\u003e spp., among others, have been shown to be capable of removing heavy metals from wastewater (Rezania et al. \u003cspan citationid=\"CR63\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). The \u003cem\u003eSalviniaceae\u003c/em\u003e family is one of the most studied in terms of its phytoremediation capabilities (Delgado-Gonz\u0026aacute;lez et al. \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). \u003cem\u003eSalviniaceae\u003c/em\u003e is a family of floating aquatic ferns consisting of the genera \u003cem\u003eAzolla\u003c/em\u003e and \u003cem\u003eSalvinia\u003c/em\u003e, with around 20 species distributed in tropical to temperate regions (Xu and Deng \u003cspan citationid=\"CR80\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). The genus \u003cem\u003eSalvinia\u003c/em\u003e includes 10 species that inhabit slow-moving freshwater bodies such as lakes, ponds, and lagoons (Contreras and Robledo \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). These species lack true roots and have a stem that grows horizontally across the water surface, where three leaves develop at each node: two upper floating leaves folded over each other along the central vein, and one submerged lower leaf, highly divided and structurally similar to roots (hereafter referred to as \u0026ldquo;roots\u0026rdquo; for simplicity), where sporocarps develop (Nagalingum et al. \u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e2006\u003c/span\u003e). In Argentina, the most abundant \u003cem\u003eSalvinia\u003c/em\u003e species are \u003cem\u003eS. auriculata\u003c/em\u003e, \u003cem\u003eS. minima\u003c/em\u003e, \u003cem\u003eS. natans\u003c/em\u003e, and \u003cem\u003eS\u003c/em\u003e. \u003cem\u003ebiloba\u003c/em\u003e (Emiliani et al. \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). Particularly, \u003cem\u003eS.biloba\u003c/em\u003e, native to the Paran\u0026aacute; River region, exhibits a high growth rate, can survive adverse conditions, and is capable of absorbing heavy metals such as Cd, Pb, Cu, Zn, Ni and Cr (Emiliani et al. \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Generally, \u003cem\u003eSalvinia\u003c/em\u003e populations are sterile and expand through vegetative multiplication due to the ease with which they fragment, with each node capable of generating new individuals (Miranda and Schwartsburd \u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). In stagnant or slow-flowing waters, they can form large communities that cover the entire water surface where they grow, and in some cases, they can become invasive weeds (Motitsoe et al., \u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e2020\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eAlthough phytoremediation is considered an economic and ecological technique for the \u003cem\u003ein-situ\u003c/em\u003e remediation of contaminated environments compared to conventional methods (Newete and Byrne \u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e2016\u003c/span\u003e), the main disadvantage is the required time. The use of plants does not allow for complete cleanup, as the absorption rate decreases as the concentration of the contaminant diminishes (Phieler et al. \u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e2014\u003c/span\u003e). Efficient phytoremediation within a reasonable timeframe can be achieved by increasing the plant\u0026rsquo;s yield and the accumulation of contaminants (Rezania et al. \u003cspan citationid=\"CR63\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). To achieve this, the use of plant growth-promoting bacteria (PGPB) has been proposed due to their ability to improve plant growth and stress tolerance (Mesa-Mar\u0026iacute;n et al. \u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). PGPB are microorganisms associated with plant tissues or free-living that have a beneficial effect on plant growth and protect plants from pathogens and abiotic stress.\u003c/p\u003e\u003cp\u003eFor PGPB to have a beneficial effect on plants, they must be able to colonize the rhizosphere or root surface for an extended period in a competitive environment with other present microorganisms (Zvinavashe et al. \u003cspan citationid=\"CR84\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Colonization is related to exudation, as microorganisms exhibit chemotaxis toward the exudates, attracting them to the plant. Some exudates can have negative effects by acting as antimicrobial agents against certain microorganisms while also serving as stimuli for establishing beneficial interactions with other microorganisms (Compant et al. \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2010\u003c/span\u003e; Souza et al. \u003cspan citationid=\"CR68\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). Others may activate genes in PGPB involved in root adhesion and colonization, such as genes that encode secretion systems, fimbriae, flagella, lytic enzymes (cellulases, xylanases, pectinases, endoglucanases, proteases, lipases), and quorum-sensing systems (G\u0026oacute;mez-God\u0026iacute;nez et al. \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). On the other hand, due to the high competitiveness present in the rhizosphere, the production of siderophores, lytic enzymes, and antibiotics by PGPB contributes to the colonization process by reducing the growth of phytopathogens (Compant et al. \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2010\u003c/span\u003e).\u003c/p\u003e\u003cp\u003ePGPB can promote plant growth through direct and indirect mechanisms. Direct mechanisms result in plant growth and development, such as increasing available nutrients and/or regulating phytohormone levels. Indirect mechanisms, on the other hand, act by inhibiting plant pathogens like fungi and bacteria (Kong and Glick \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e2017\u003c/span\u003e; Olanrewaju et al. \u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). The main mechanisms involved in nutrient provision are phosphate solubilization, biological nitrogen fixation, and iron chelation through siderophore production. As for the regulation of phytohormone levels, there is the production of auxins, cytokinins, gibberellins and the modulation of ethylene levels through ACC deaminase activity (Gamalero and Glick \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2011\u003c/span\u003e). These phytohormones can stimulate plant growth and developmental stages, such as cell elongation, cell division, root development, shoot initiation, and tissue differentiation (Ajijah et al. \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). The production of antibiotics, lytic enzymes, degradation of toxins produced by pathogens, and competition for nutrients and space are some of the mechanisms involved in disease inhibition (Chepsergon and Moleleki \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). Different PGPB may use one or more mechanisms to promote plant growth, and the mechanism employed can vary depending on the environmental situation (Olanrewaju et al. \u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e2017\u003c/span\u003e; Stegelmeier et al. \u003cspan citationid=\"CR70\" class=\"CitationRef\"\u003e2022\u003c/span\u003e).\u003c/p\u003e\u003cp\u003ePGPB have been used to promote plant growth while simultaneously mitigating the level of toxicity or damage caused by exposure to various contaminants, such as heavy metals, in a process known as microorganism-assisted phytoremediation (Ahemad \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). The efficiency of heavy metal phytoremediation is limited by metal availability in the soil, root system development, production of photosynthetically active biomass, and the plant\u0026rsquo;s tolerance to each specific metal (Gamalero et al. \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2009\u003c/span\u003e). For this reason, PGPB play a crucial role in heavy metal phytoremediation by enhancing the process\u0026rsquo;s efficiency through promoting plant growth, increasing plant tolerance to metal toxicity, increasing metal solubility and mobility, and/or biotransforming metals into less toxic compounds. Most studies on the application of PGPB have focused on terrestrial plants; however, research has recently started investigating their application in hydroponic crops and aquatic plants (Ishizawa et al. \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2017\u003c/span\u003e, \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Jewell et al. \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e2023\u003c/span\u003e; Makino et al. \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Stegelmeier et al. \u003cspan citationid=\"CR70\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Toyama et al. \u003cspan citationid=\"CR75\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). The application of PGPB in hydroponic crops presents some differences compared to terrestrial plants. Since an aquatic system is different from the soil surrounding the rhizosphere, the same plant may have different microbial communities depending on whether it grows in soil or water. Many microorganisms that promote plant growth in soil do not survive the transition to a hydroponic medium (Stegelmeier et al. \u003cspan citationid=\"CR70\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). On the other hand, the inoculation of PGPB in aquatic systems does not always produce the desired effects due to failure in colonization or competition with the native microorganisms in the inoculated plant (Ishizawa et al. \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2017\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eConsidering these findings and the inherent capacity of PGPB to stimulate plant growth, their integration into phytoremediation processes has emerged as a promising strategy to enhance both remediation efficiency and plant health under stress conditions, a concept known as assisted phytoremediation.\u003c/p\u003e"},{"header":"2. Materials and methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\u003ch2\u003e2.1. Procurement and maintenance of Salvinia biloba plants\u003c/h2\u003e\u003cp\u003e\u003cem\u003eS. biloba\u003c/em\u003e specimens used in the experiments were collected from Reserva Ecol\u0026oacute;gica Costanera Sur, Ciudad Aut\u0026oacute;noma de Buenos Aires, Argentina (34\u0026deg;36\u0026prime;14\u0026Prime;S 58\u0026deg;21\u0026prime;09\u0026Prime;O), which belongs to the Paran\u0026aacute; Delta and Islands Ecoregion. To remove potential contaminants adhered to the plants, they were thoroughly rinsed several times with running water. After washing, the plants were transferred to containers filled with a nutrient solution (CaCl\u003csub\u003e2\u003c/sub\u003e 0.588 mg/l, MgSO\u003csub\u003e4\u003c/sub\u003e 0.246 mg/l, NaHCO\u003csub\u003e3\u003c/sub\u003e 0.126 mg/l, KCl 0.055 mg/l; pH 7.0) (Mendes et al. \u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). The containers were then placed in a controlled culture chamber under regulated temperature (26\u0026deg;C) and light conditions (photoperiod of 14 h of light and 10 h of darkness) (Prado et al. \u003cspan citationid=\"CR61\" class=\"CitationRef\"\u003e2010\u003c/span\u003e) until the plants were used in the assays.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec4\" class=\"Section2\"\u003e\u003ch2\u003e2.2. Tolerance of Salvinia biloba to different Cr(VI) concentrations\u003c/h2\u003e\u003cdiv id=\"Sec5\" class=\"Section3\"\u003e\u003ch2\u003e2.2.1. Evaluation of morphological changes in S. biloba in the presence of Cr(VI) and metal removal\u003c/h2\u003e\u003cp\u003eThe experimental design and Cr(VI) exposure conditions were based on those described in Mart\u0026iacute;nez Saucedo et al. (\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e2025\u003c/span\u003e), where morphological parameters (fresh weight (FW), number of total and damaged leaves and root length) were analyzed. Individual plants were exposed to varying Cr(VI) concentrations in glass containers, each containing 500 ml of nutrient solution and 7.42\u0026thinsp;\u0026plusmn;\u0026thinsp;0.36 g of FW. Before treatment, the plants were briefly placed on absorbent paper to remove excess water. Potassium dichromate (K\u003csub\u003e2\u003c/sub\u003eCr\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e7\u003c/sub\u003e) stock solution (1000 ppm Cr(VI)) was added to each container to achieve the target Cr(VI) concentrations: 1, 3 and 5 ppm. The treatments were incubated for 15 days in a controlled culture chamber under previously described conditions. Each treatment was performed in triplicate in three independent experiments, including a control group (0 ppm Cr(VI)).\u003c/p\u003e\u003cp\u003eVegetation cover was analyzed using the ImageJ software (version 1.53s) through photographic documentation at the beginning and end of the experiment. To determine residual Cr concentrations in the solution, total Cr was quantified by atomic absorption spectroscopy and Cr(VI) was quantified by colorimetry, using 30 ml aliquots from each treatment, preserved with nitric acid until analysis, as described below. At the end of the experiment, distilled water was added to restore the initial volume, compensating for evaporative losses.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec6\" class=\"Section3\"\u003e\u003ch2\u003e2.2.2. Determination of photosynthetic pigments\u003c/h2\u003e\u003cp\u003eThe content of photosynthetic pigments (chlorophyll \u003cem\u003ea\u003c/em\u003e, \u003cem\u003eb\u003c/em\u003e and carotenoids) was determined after 15 days of exposure to Cr(VI) using the methodology described by Emiliani et al. (\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). 1 g of fresh biomass was homogenized in 10 ml of 96% (v/v) ethanol and incubated in the dark for 24 h at room temperature. The resulting solution was centrifuged at 2500 rpm for 20 minutes, and the absorbance of the supernatant was measured at 470, 649, and 665 nm using a UV-Vis spectrophotometer (UV-1280, Shimadzu). The concentrations of chlorophyll \u003cem\u003ea\u003c/em\u003e, chlorophyll \u003cem\u003eb\u003c/em\u003e, and total carotenoids were calculated according to the equations proposed by Lichtenthaler and Wellburn (\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e1983\u003c/span\u003e) and expressed in \u0026micro;g/g FW.\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv id=\"Sec7\" class=\"Section2\"\u003e\u003ch2\u003e2.3. Isolation, maintenance and cultivation of microorganisms\u003c/h2\u003e\u003cp\u003eA total of 25 microorganisms isolated from soil, water, and sediment samples collected in Entre R\u0026iacute;os, Argentina (33\u0026deg;29'32''S 58\u0026deg;43'44''W) were used. This region is part of the Paran\u0026aacute; Delta and Islands Ecoregion and was selected due to its high level of contamination with various pollutants, including heavy metals, resulting from intensive anthropogenic activities in the surrounding area (Avigliano et al. \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Peluso et al. \u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). To obtain cultivable microorganisms, 1 ml or 1 g of each sample (depending on the sample type) was inoculated into liquid LB medium and incubated under agitation at 30\u0026deg;C for 24\u0026ndash;48 h. Subsequently, aliquots were plated onto Petri dishes containing agarized LB medium (Sambrook \u003cspan citationid=\"CR65\" class=\"CitationRef\"\u003e2001\u003c/span\u003e) to assess the cultivable microbial diversity present in the samples. Individual bacterial isolates were obtained by successive streaking using the depletion streak technique. Morphological differentiation and biochemical assays (LIA, TSI, IMViC) were conducted to characterize the metabolic diversity of the isolated bacteria and to exclude the presence of potential pathogens. The 25 isolates were labeled with the acronym ER (representing Entre R\u0026iacute;os, the collection site) followed by a unique letter identifier and were maintained on LB agar plates at 4\u0026deg;C, with periodic subculturing to preserve viability. These bacterial isolates were preserved in LB medium supplemented with 20% glycerol at -80\u0026deg;C.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e\u003ch2\u003e2.4. Identification of plant growth-promoting characteristics in vitro\u003c/h2\u003e\u003cp\u003eThe bacterial isolates were characterized for their plant growth-promoting activities by evaluating the production of hydrolytic enzymes (proteases, cellulases, and lipases), phosphate solubilization, siderophores production, biological nitrogen fixation and phytohormones (auxins) production. The results were visually assessed and recorded as either presence or absence of activity, with some degree of qualitative differentiation when possible.\u003c/p\u003e\u003cp\u003e\u003col\u003e\u003cspan\u003e\u003cli\u003e\u003cp\u003e\u003cem\u003eProtease activity\u003c/em\u003e: The capacity to produce protease was evaluated by spot inoculation of colonies on skim milk agar plates (skim milk 50 g/l; agar 10 g/l), followed by incubation at 37\u0026deg;C for four days (Walsh et al. \u003cspan citationid=\"CR79\" class=\"CitationRef\"\u003e1995\u003c/span\u003e).\u003c/p\u003e\u003c/li\u003e\u003c/span\u003e\u003cspan\u003e\u003cli\u003e\u003cp\u003e\u003cem\u003eCellulase activity\u003c/em\u003e: Cellulase production was tested on plates containing carboxymethylcellulose (CMC) medium (NaNO\u003csub\u003e3\u003c/sub\u003e 2 g/l; K\u003csub\u003e2\u003c/sub\u003eHPO\u003csub\u003e4\u003c/sub\u003e 1 g/l; MgSO\u003csub\u003e4\u003c/sub\u003e 0.5 g/l; KCl 0.5 g/l; peptone 0.2 g/l; CMC 2 g/l; agar 17 g/l). The plates were incubated at 37\u0026deg;C for four days. To visualize activity, the plates were stained with 0.1% Congo Red, washed with 1 M NaCl, and fixed with 0.5% acetic acid (Teather and Wood \u003cspan citationid=\"CR73\" class=\"CitationRef\"\u003e1982\u003c/span\u003e).\u003c/p\u003e\u003c/li\u003e\u003c/span\u003e\u003cspan\u003e\u003cli\u003e\u003cp\u003e\u003cem\u003eLipase activity\u003c/em\u003e: Lipase production was determined using the method described by Samad et al. (\u003cspan citationid=\"CR64\" class=\"CitationRef\"\u003e1989\u003c/span\u003e). Colonies were inoculated on plates containing medium composed of NaCl 5 g/l, CaCl\u003csub\u003e2\u003c/sub\u003e 0.1 g/l, peptone 10 g/l, Tween-80 10 g/l, and agar 20 g/l, and incubated at 37\u0026deg;C for four days.\u003c/p\u003e\u003c/li\u003e\u003c/span\u003e\u003cspan\u003e\u003cli\u003e\u003cp\u003e\u003cem\u003ePhosphate solubilization\u003c/em\u003e: Phosphate solubilization was assessed by plating isolates on NBRIP medium (glucose 10 g/l; (NH\u003csub\u003e4\u003c/sub\u003e)\u003csub\u003e2\u003c/sub\u003eSO\u003csub\u003e4\u003c/sub\u003e 0.5 g/l; NaCl 0.3 g/l; KCl 0.3 g/l; FeSO\u003csub\u003e4\u003c/sub\u003e.7H\u003csub\u003e2\u003c/sub\u003eO 0.03 g/l; MnSO\u003csub\u003e4\u003c/sub\u003e.H\u003csub\u003e2\u003c/sub\u003eO 0.03 g/l; Ca\u003csub\u003e3\u003c/sub\u003e(PO\u003csub\u003e4\u003c/sub\u003e)\u003csub\u003e2\u003c/sub\u003e 1 g/l; agar 15 g/l) (Nautiyal \u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e1999\u003c/span\u003e). Plates were incubated at 37\u0026deg;C for four days, and solubilization was visually identified by the formation of clear halos around colonies.\u003c/p\u003e\u003c/li\u003e\u003c/span\u003e\u003cspan\u003e\u003cli\u003e\u003cp\u003e\u003cem\u003eSiderophore production\u003c/em\u003e: The ability to produce siderophores was assessed using the modified Chrome Azurol S (CAS) method described by P\u0026eacute;rez-Miranda et al. (\u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e2007\u003c/span\u003e). Colonies were inoculated on R2A medium (yeast extract 0.5 g/l; peptone 0.5 g/l; glucose 0.5 g/l; pyruvate 0.3 g/l; K\u003csub\u003e2\u003c/sub\u003eHPO\u003csub\u003e4\u003c/sub\u003e 0.3 g/l; MgSO\u003csub\u003e4\u003c/sub\u003e 0.05 g/l; starch 0.5 g/l; agar 15 g/l; pH 6.5\u0026thinsp;\u0026plusmn;\u0026thinsp;0.2) and incubated at 37\u0026deg;C for six days. After incubation, a CAS overlay (CAS 15.12 mg; FeCl\u003csub\u003e3\u003c/sub\u003e 2.5 mg; CTAB 18.23 mg; PIPES 7.50 mg; agar 2.25 g in 250 ml; pH 6.8) was applied and allowed to develop at room temperature.\u003c/p\u003e\u003c/li\u003e\u003c/span\u003e\u003cspan\u003e\u003cli\u003e\u003cp\u003e\u003cem\u003eBiological nitrogen fixation\u003c/em\u003e: Nitrogen fixation capacity was evaluated using deep inoculation in semisolid LGI medium (glucose 5 g/l; K\u003csub\u003e2\u003c/sub\u003eHPO\u003csub\u003e4\u003c/sub\u003e 0.2 g/l; KH\u003csub\u003e2\u003c/sub\u003ePO\u003csub\u003e4\u003c/sub\u003e 0.6 g/l; MgSO\u003csub\u003e4\u003c/sub\u003e.7H\u003csub\u003e2\u003c/sub\u003eO 0.2 g/l; CaCl\u003csub\u003e2\u003c/sub\u003e.2H\u003csub\u003e2\u003c/sub\u003eO 0.02 g/l; Na\u003csub\u003e2\u003c/sub\u003eMoO\u003csub\u003e4\u003c/sub\u003e.2H\u003csub\u003e2\u003c/sub\u003eO 0.002 g/l; FeCl\u003csub\u003e3\u003c/sub\u003e.6H\u003csub\u003e2\u003c/sub\u003eO 0.01 g/l; agar 1.8 g/l) (Baldani et al. \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2014\u003c/span\u003e). Colonies were resuspended in 200 \u0026micro;l of sterile physiological solution (NaCl 0.85% w/v) and inoculated into the medium by puncture. The tubes were incubated at 37\u0026deg;C for five days, and nitrogen fixation was identified by the appearance of a pellicle near the surface of the medium.\u003c/p\u003e\u003c/li\u003e\u003c/span\u003e\u003cspan\u003e\u003cli\u003e\u003cp\u003e\u003cem\u003eAuxin production\u003c/em\u003e: Indole-3-acetic acid (IAA) production was quantified using the Salkowski colorimetric method (Glickmann and Dessaux \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e1995\u003c/span\u003e). Bacteria were cultured in 24-well plates containing 1 ml of BT liquid medium (glucose 5 g/l; K\u003csub\u003e2\u003c/sub\u003eHPO\u003csub\u003e4\u003c/sub\u003e 1 g/l; NH\u003csub\u003e4\u003c/sub\u003eNO\u003csub\u003e3\u003c/sub\u003e 0.4 g/l; NaCl 0.2 g/l; MgSO\u003csub\u003e4\u003c/sub\u003e\u0026middot;7H\u003csub\u003e2\u003c/sub\u003eO 0.2 g/l; tryptone 20 g/l; pH 7.0\u0026thinsp;\u0026plusmn;\u0026thinsp;0.2) under agitation (150 rpm) at 37\u0026deg;C for four days. After incubation, cultures were centrifuged, and 1 ml of supernatant was mixed with 1 ml of modified Salkowski reagent (98 ml of 35% perchloric acid and 2 ml of 0.5 M ferric chloride). Results were recorded after 20 minutes.\u003c/p\u003e\u003c/li\u003e\u003c/span\u003e\u003c/ol\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec9\" class=\"Section2\"\u003e\u003ch2\u003e2.5. Chromium tolerance analysis of selected microorganisms\u003c/h2\u003e\u003cp\u003eThe Cr(VI) tolerance of the selected PGPB was evaluated by exposing the isolates to different Cr(VI) concentrations. Inocula were obtained by culturing the bacterial isolates in liquid LB medium under agitation (120 rpm) at 30\u0026deg;C for 48 h. After incubation, the cultures were centrifuged at 4000 rpm for 10 minutes, and the resulting pellets were resuspended in sterile distilled water. The bacterial suspensions were adjusted to an initial optical density (OD) of 0.1 at 560 nm. Four treatments were established by adding the appropriate volume of the stock solution of K\u003csub\u003e2\u003c/sub\u003eCr\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e7\u003c/sub\u003e to the LB medium to achieve final concentrations of 0, 1, 3 and 5 ppm of Cr(VI). Each treatment was set up in triplicate in 24-well plates, with 1 ml of culture per well. The plates were incubated at 30\u0026deg;C under agitation (120 rpm) for 48 h. Following incubation, the OD at 560 nm was measured to assess the effect of Cr(VI) on bacterial growth. Each treatment was performed in triplicate in three independent assays.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec10\" class=\"Section2\"\u003e\u003ch2\u003e2.6. In vivo analysis of plant growth-promotion of selected microorganisms\u003c/h2\u003e\u003cp\u003eTo assess the interaction between the selected bacterial isolates and \u003cem\u003eS. biloba\u003c/em\u003e, aquatic systems were established by placing individual plants with a FW of approximately 1.6\u0026thinsp;\u0026plusmn;\u0026thinsp;0.3 g in 100 ml of nutrient solution. The systems were inoculated with the bacterial suspensions under evaluation and incubated in a controlled culture chamber with regulated temperature and light conditions, as previously described, for 15 days. Each treatment was performed in triplicate, including an uninoculated control group. Inocula were prepared by culturing microorganisms in LB medium under agitation at 30\u0026deg;C for 48 h. After incubation, cultures were centrifuged and the supernatant was discarded. The resulting pellet was resuspended in sterile distilled water, and the OD at 560 nm was measured. The inoculum volume was adjusted to ensure an initial OD at 560 nm of 0.1 in each treatment. At the beginning and end of the experimental period, plant growth parameters, including FW, total number of leaves, and root length were recorded. Additionally, photographic documentation of each treatment was carried out to monitor morphological changes and total plant cover. Each treatment was performed in triplicate in three independent assays.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e\u003ch2\u003e2.7. Inoculation of Salvinia biloba exposed to 5 ppm Cr(VI)\u003c/h2\u003e\u003cp\u003eTo assess whether inoculation with the selected microorganisms enhances the Cr(VI) tolerance of \u003cem\u003eS. biloba\u003c/em\u003e, aquatic systems containing 500 ml of nutrient solution and approximately 8.3\u0026thinsp;\u0026plusmn;\u0026thinsp;0.06 g FW were prepared. A stock solution of K\u003csub\u003e2\u003c/sub\u003eCr\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e7\u003c/sub\u003e was added to achieve a final Cr(VI) concentration of 5 ppm. The systems were inoculated with the bacterial isolates under study, including an uninoculated control group. Inocula were prepared as previously described and adjusted to an initial OD of 0.1 at 560 nm before being added to the systems. The treatments were incubated in culture chambers with controlled temperature and light conditions for 15 days. At the initial and final time, the FW, total number of leaves and root length of each treatment were recorded. Vegetation cover was analyzed as previously described, at the beginning and the final time. Additionally, a 30 ml aliquot of the solution was collected for subsequent Cr analysis. To compensate for liquid loss due to evaporation, distilled water was added at the end of the experiment to restore the initial volume. Each treatment was performed in triplicate in three independent assays.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e\u003ch2\u003e2.8. Determination of chromium concentration in solution\u003c/h2\u003e\u003cp\u003eTo evaluate the Cr removal capacity of \u003cem\u003eS. biloba\u003c/em\u003e at different Cr(VI) concentrations and determine whether inoculation with the studied microorganisms influences removal efficiency, 30 ml aliquots of the culture solution were collected at the start and end of the experiments. The concentration of total Cr and Cr(VI) was analyzed. Cr(VI) was measured immediately after sampling using 10 ml of the aliquot, while the remaining 20 ml were stored under refrigeration for later total Cr analysis.\u003c/p\u003e\u003cp\u003e\u003col\u003e\u003cspan\u003e\u003cli\u003e\u003cp\u003e\u003cem\u003eTotal Cr determination\u003c/em\u003e: The concentration of total Cr in the samples was determined by flame atomic absorption spectroscopy (Perkin Elmer AAnalyst 100). A calibration curve was constructed using a stock solution of Cr(NO\u003csub\u003e3\u003c/sub\u003e)\u003csub\u003e3\u003c/sub\u003e (1000 ppm) and standard solutions of 1, 2, 4, and 6 ppm Cr prepared in 25 ml of nitric acid (HNO\u003csub\u003e3\u003c/sub\u003e). Measurements were taken at a wavelength of 357.9 nm, with HNO\u003csub\u003e3\u003c/sub\u003e as blank.\u003c/p\u003e\u003c/li\u003e\u003c/span\u003e\u003cspan\u003e\u003cli\u003e\u003cp\u003e\u003cem\u003eCr(VI) determination\u003c/em\u003e: The Cr(VI) concentration was determined colorimetrically according to the method described in the 22nd edition of Standard Methods for the Analysis of Drinking Water and Wastewater 3500-Cr B (American Public Health Association \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2012\u003c/span\u003e). A 10 ml sample was used for determination, and a calibration curve was prepared using standard solutions of 1, 2, 4, 5, and 6 ppm Cr derived from a K\u003csub\u003e2\u003c/sub\u003eCr\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e7\u003c/sub\u003e stock solution (1000 X). Both samples and standards were treated with 0.1 ml of 50% (v/v) sulfuric acid, followed by the addition of 0.2 ml of a 0.5% (w/v) diphenylcarbazide (DPC) solution prepared by dissolving 5 mg of DPC in 1 ml of acetone. The reaction mixture was left to stand for 5\u0026ndash;10 minutes to allow color development. A reaction blank using distilled water was prepared following the same procedure. After the resting period, absorbance was measured at 540 nm using a UV-vis spectrophotometer.\u003c/p\u003e\u003c/li\u003e\u003c/span\u003e\u003c/ol\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec13\" class=\"Section2\"\u003e\u003ch2\u003e2.9. Identification of bacteria by partial 16S rRNA gene sequencing\u003c/h2\u003e\u003cp\u003eThe microorganism that displayed positive results across the various assays was taxonomically identified via partial sequencing of the 16S rRNA gene, performed by Macrogen Inc. (Seoul, South Korea). Amplification of the 16S rRNA gene was carried out by PCR using the universal primers 27F (5'-AGA GTT TGA TCM TGG CTC AG-3') and 1492R (5'-TAC GGY TAC CTT GTT ACG ACT T-3'). The resulting PCR product was purified and sequenced using the internal primers 785F (5'-GGA TTA GAT ACC ACC CTG GTA GTA-3') and 907R (5'-CCG TCA ATT ATT CMT TTR AGT TT-3'). Identification was conducted by comparing the obtained sequence against the National Center for Biotechnology Information (NCBI) database using the BLASTn algorithm (Basic Local Alignment Search Tool). The List of Prokaryotic Names with Standing in Nomenclature (LPSN) was consulted for the genus and species-level identification of the analyzed microorganism and its comparison with the reference strain.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec14\" class=\"Section2\"\u003e\u003ch2\u003e2.10. Statistical analysis\u003c/h2\u003e\u003cp\u003eAll experiments were conducted in triplicate for each treatment, and three independent assays were performed. Results are presented as mean values with standard error of the mean (SEM). Differences between treatments were analyzed using one-way ANOVA or two-way ANOVA, followed by Tukey\u0026rsquo;s multiple comparison test. Changes between initial and final time points were assessed using a paired Student\u0026rsquo;s \u003cem\u003et\u003c/em\u003e-test. Prior to the analysis, data normality was assessed using the Shapiro-Wilk test (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026gt;\u0026thinsp;0.05). Statistical significance was set at \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05. Statistical analyses were performed using GraphPad Prism 8 (version 5.01), with a significance threshold set at \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05.\u003c/p\u003e\u003c/div\u003e"},{"header":"3. Results","content":"\u003cdiv id=\"Sec16\" class=\"Section2\"\u003e\u003ch2\u003e3.1. Tolerance of Salvinia biloba to different Cr(VI) concentrations\u003c/h2\u003e\u003cp\u003eIn a previous study (Mart\u0026iacute;nez Saucedo et al. \u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e2025\u003c/span\u003e), we reported the morphological effects of Cr(VI) toxicity on \u003cem\u003eS. biloba\u003c/em\u003e, including reductions in fresh weight, root length, and total leaf number. These findings provided key insights into the visible damage caused by metal exposure. However, physiological and structural responses to Cr(VI) may occur earlier or independently from morphological changes. Vegetation cover may reflect alterations in plant structure and water balance, particularly in free-floating species like \u003cem\u003eS. biloba\u003c/em\u003e, where turgor loss and leaf folding can increase apparent surface area without indicating actual growth. Likewise, photosynthetic pigments (such as chlorophyll \u003cem\u003ea\u003c/em\u003e, chlorophyll \u003cem\u003eb\u003c/em\u003e, and carotenoids) are sensitive biochemical markers of stress. In this context, the present study complements our earlier morphological assessment by incorporating vegetation cover analysis and pigment quantification, offering a more comprehensive understanding of the plant's responses to Cr(VI) stress.\u003c/p\u003e\u003cp\u003eVegetation cover measurements revealed a significant increase in surface area occupied by \u003cem\u003eS. biloba\u003c/em\u003e at the end of the assay in treatments with 3 and 5 ppm Cr(VI) (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). While no significant changes were observed at 0 and 1 ppm, higher concentrations led to a marked expansion of the plant mat, despite previous evidence of growth inhibition under the same conditions (Mart\u0026iacute;nez Saucedo et al. \u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e2025\u003c/span\u003e). This apparent increase in coverage is likely not attributable to biomass accumulation, but rather to structural alterations such as turgor loss and leaf displacement, which cause the plants to spread horizontally.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eThe content of photosynthetic pigments (chlorophyll \u003cem\u003ea\u003c/em\u003e, chlorophyll \u003cem\u003eb\u003c/em\u003e and carotenoids) in leaves at the final time was determined as a physiological parameter to evaluate the toxicity of Cr(VI) in the plant species analyzed. No significant differences in photosynthetic pigment content were found in the treatments analyzed (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eThe results obtained are consistent with the effects of heavy metal exposure previously reported in studies on plants of the \u003cem\u003eSalvinia\u003c/em\u003e genus (Das and Goswami \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2017\u003c/span\u003e; Emiliani et al. \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2020\u003c/span\u003e, \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Lor\u0026iacute;a et al. \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). Cr(VI) exposure induced signs of toxicity, including chlorosis, loss of turgor, and necrosis, as well as reduced growth and development. These phytotoxic effects occurred progressively with increasing metal concentration and exposure time. When evaluating the range of Cr(VI) concentrations tolerated by \u003cem\u003eS. biloba\u003c/em\u003e, it was concluded that concentrations of 1, 3, and 5 ppm were tolerated by the plants, as they survived exposure to the metal despite exhibiting toxicity symptoms.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec17\" class=\"Section2\"\u003e\u003ch2\u003e3.2. Chromium removal capacity by Salvinia biloba\u003c/h2\u003e\u003cp\u003eAfter determining the Cr(VI) concentrations tolerated by \u003cem\u003eS. biloba\u003c/em\u003e, the metal removal capacity was evaluated for the analyzed concentrations. \u003cem\u003eS. biloba\u003c/em\u003e plants were exposed to 1, 3, and 5 ppm of Cr(VI) for 15 days as previously described, and measurements of the Cr concentration in the culture solution were taken at the initial and final time. At the initial time, all Cr present in the solution corresponded to Cr(VI). For the 1, 3, and 5 ppm treatments, the initial measured Cr concentrations were 1.48\u0026thinsp;\u0026plusmn;\u0026thinsp;0.05, 3.93\u0026thinsp;\u0026plusmn;\u0026thinsp;0.11, and 6.15\u0026thinsp;\u0026plusmn;\u0026thinsp;0.10 ppm, respectively.\u003c/p\u003e\u003cp\u003eTable\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e shows the results of total Cr and Cr(VI) concentrations in the culture solution after 15 days of plant exposure to the different treatments. At the end of the experiment, a reduction of 32%, 26%, and 21% in total Cr concentration was observed in the 1, 3, and 5 ppm treatments, respectively. These differences were statistically significant only for the 1 and 3 ppm treatments (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01). In the different treatments, the percentage of Cr removal decreased as the initial metal concentration increased. This effect has also been reported with other metals (Emiliani et al. \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). The differences observed between the initial and final total Cr concentrations were attributed to the removal of Cr by \u003cem\u003eS. biloba\u003c/em\u003e plants through metal absorption within plant tissues and/or adsorption on the plant surface in contact with the solution.\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eRemoval capacity of total Cr and Cr(VI) by \u003cem\u003eS. biloba\u003c/em\u003e plants exposed to 1, 3 and 5 ppm Cr(VI) for 15 days. The mean and standard deviation of the concentration of total Cr and Cr(VI) in solution (ppm) are shown. Means indicated with asterisks are significantly different from the initial concentration (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01).\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\u003eTreatment\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003eInitial total Cr (ppm)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003eFinal total Cr (ppm)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e\u003cp\u003e% Total Cr removal\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e1 ppm\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e1.48\u0026thinsp;\u0026plusmn;\u0026thinsp;0.05\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e1.01\u0026thinsp;\u0026plusmn;\u0026thinsp;0.04\u003csup\u003e*\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e32%\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e3 ppm\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e3.93\u0026thinsp;\u0026plusmn;\u0026thinsp;0.11\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e2.89\u0026thinsp;\u0026plusmn;\u0026thinsp;0.15\u003csup\u003e*\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e26%\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e5 ppm\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e6.15\u0026thinsp;\u0026plusmn;\u0026thinsp;0.10\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e4.85\u0026thinsp;\u0026plusmn;\u0026thinsp;0.17\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e21%\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eTreatment\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u003cb\u003eInitial Cr(VI) (ppm)\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e\u003cb\u003eFinal Cr(VI) (ppm)\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e\u003cb\u003e% Cr(VI) removal\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e1 ppm\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e1.48\u0026thinsp;\u0026plusmn;\u0026thinsp;0.05\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e0.27\u0026thinsp;\u0026plusmn;\u0026thinsp;0.07\u003csup\u003e*\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e82%\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e3 ppm\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e3.93\u0026thinsp;\u0026plusmn;\u0026thinsp;0.11\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e2.15\u0026thinsp;\u0026plusmn;\u0026thinsp;0.26\u003csup\u003e*\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e45%\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e5 ppm\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e6.15\u0026thinsp;\u0026plusmn;\u0026thinsp;0.10\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e4.14\u0026thinsp;\u0026plusmn;\u0026thinsp;0.14\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e33%\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003eOn the other hand, for Cr(VI), a reduction of 82%, 45%, and 33% in its concentration was observed in the 1, 3, and 5 ppm treatments, respectively, with the differences being statistically significant in the 1 and 3 ppm treatments (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01). When comparing total Cr and Cr(VI) at the final time point, a greater reduction in Cr(VI) concentration can be observed. This is particularly notable in the 1 ppm treatment, where the decrease in Cr(VI) concentration is 50% greater than that of total Cr, while in the 3 and 5 ppm treatments, the difference with total Cr is 19% and 12%, respectively. These differences between total Cr and Cr(VI) concentrations at the final time point correspond to the presence of Cr in a different oxidation state.\u003c/p\u003e\u003cp\u003eSince Cr(III) is one of the most stable Cr states (Tchounwou et al. \u003cspan citationid=\"CR72\" class=\"CitationRef\"\u003e2012\u003c/span\u003e), it was considered that the difference between total Cr and Cr(VI) concentrations at the final time point is due to the presence of Cr(III). At the final point, a Cr(III) concentration of 0.74 ppm was observed in the 1 and 3 ppm treatments, and 0.71 ppm in the 5-ppm treatment. As mentioned earlier, a Cr(VI) solution was used to contaminate the systems at the beginning of the experiment, so the presence of the metal in other oxidation states could be attributed to the action of the plants and their associated microorganisms. For example, the native microbiota present on the plants could be capable of reducing Cr(VI) to Cr(III), or the plants themselves could have the ability to perform this reduction, as has been reported for other species in the \u003cem\u003eSalvinia\u003c/em\u003e genus (Chocobar-Ponce et al. \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). This would contribute to the detoxification of the contaminated solution, as Cr(VI) is more toxic than its trivalent form.\u003c/p\u003e\u003cp\u003eIn summary, Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e shows the percentage of Cr removal and the remaining amounts of Cr(VI) and Cr(III) in solution.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec18\" class=\"Section2\"\u003e\u003ch2\u003e3.3. Characterization of isolated microorganisms according to their in vitro plant growth promoting characteristics\u003c/h2\u003e\u003cp\u003ePGPB can promote plant growth through direct and indirect mechanisms. Direct mechanisms result in the promotion of plant growth and development by increasing nutrient availability and producing phytohormones, while indirect mechanisms inhibit the growth of phytopathogens such as fungi and bacteria (Kong and Glick \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). To characterize the 25 microorganisms isolated from soil, water, and sediment samples from the Paran\u0026aacute; River Delta (Entre R\u0026iacute;os, Argentina) in terms of their plant growth-promoting traits, the presence of the following enzymatic activities was evaluated \u003cem\u003ein vitro\u003c/em\u003e: lytic enzymes production (lipases, cellulases, proteases), phosphate solubilization, siderophore production, biological nitrogen fixation and auxins production. The results of each evaluated activity will be described below (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e).\u003c/p\u003e\u003cp\u003e\u003col\u003e\u003cspan\u003e\u003cli\u003e\u003cp\u003e\u003cem\u003eProduction of lytic enzymes\u003c/em\u003e: To detect lipase production, a medium containing Tween 80 was used. The formation of a precipitate around the colonies indicates a positive result, as the hydrolysis of Tween 80 produces a precipitation reaction of free fatty acids with the CaCl\u003csub\u003e2\u003c/sub\u003e present in the medium (Plou et al., \u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e1998\u003c/span\u003e) (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA). Cellulase production was evidenced by the formation of a decolorized halo around the colonies due to the degradation of CMC present in the medium (Teather and Wood \u003cspan citationid=\"CR73\" class=\"CitationRef\"\u003e1982\u003c/span\u003e) (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB). Finally, the presence of proteases was detected by the formation of a transparent halo around the colony due to the hydrolysis of proteins in the culture medium (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC).\u003c/p\u003e\u003c/li\u003e\u003c/span\u003e\u003cspan\u003e\u003cli\u003e\u003cp\u003e\u003cem\u003ePhosphate solubilization\u003c/em\u003e: To detect this activity, a culture medium with calcium phosphate as the phosphorus source was used. The formation of a transparent halo around the colony due to the production of organic acids indicates a positive result (Nautiyal \u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e1999\u003c/span\u003e) (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eD).\u003c/p\u003e\u003c/li\u003e\u003c/span\u003e\u003cspan\u003e\u003cli\u003e\u003cp\u003e\u003cem\u003eSiderophore production\u003c/em\u003e: To identify siderophore production, a culture medium with an iron-dye complex (CAS-FeCl\u003csub\u003e3\u003c/sub\u003e) was used. When a strong iron chelator, such as a siderophore, removes iron from the dye complex, a color change is observed (Schwyn and Neilands \u003cspan citationid=\"CR67\" class=\"CitationRef\"\u003e1987\u003c/span\u003e). Therefore, this color change in the culture medium around the colony indicates a positive result (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eE).\u003c/p\u003e\u003c/li\u003e\u003c/span\u003e\u003cspan\u003e\u003cli\u003e\u003cp\u003e\u003cem\u003eBiological nitrogen fixation\u003c/em\u003e: For the detection of biological nitrogen fixation, a semisolid medium without nitrogen sources was used. The nitrogenase complex (a set of enzymes responsible for reducing atmospheric nitrogen to ammonia) is highly sensitive to oxygen. Therefore, the use of semisolid media and deep puncture inoculation allows the bacteria to grow while protecting the nitrogenase from inhibition by elevated oxygen concentrations (Baldani et al. \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2014\u003c/span\u003e). By using a nitrogen-free medium, a microorganism is positive for biological nitrogen fixation if it is able to grow (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eF).\u003c/p\u003e\u003c/li\u003e\u003c/span\u003e\u003cspan\u003e\u003cli\u003e\u003cp\u003e\u003cem\u003eAuxins production\u003c/em\u003e: Bacteria that produce auxins were identified by the development of a pink color due to the reaction of the Salkowski reagent with the phytohormones. The intensity of the color developed corresponds to different levels of auxin production, with the intensity increasing as the concentration in the solution rises (Abeed et al. \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eG, a marked change to an orange/pink coloration was considered a positive result for auxin production.\u003c/p\u003e\u003c/li\u003e\u003c/span\u003e\u003c/ol\u003e\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eAlthough it was possible in some cases to distinguish between different levels of compound production, the results were analyzed qualitatively and recorded as either positive or negative. Of the 25 isolates tested (results provided in Supplementary Information), 3 microorganisms with the highest number of positive results were selected to proceed with further testing. The selected isolates were named ER-C, ER-G and ER-Y. The results for these isolates are presented in 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\u003eResults of the three bacterial isolates (ER-C, ER-G and ER-Y) with the highest number of positive plant growth promotion (PGP) activities. (+) positive activity; (-) negative activity.\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\u003ePGP Activity\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003eER-C\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003eER-G\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e\u003cp\u003eER-Y\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eLipases production\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u003cb\u003e+\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e\u003cb\u003e-\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e\u003cb\u003e+\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eCellulases production\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u003cb\u003e-\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e\u003cb\u003e-\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e\u003cb\u003e-\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eProteases production\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u003cb\u003e+\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e\u003cb\u003e+\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e\u003cb\u003e+\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003ePhosphate solubilization\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u003cb\u003e-\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e\u003cb\u003e+\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e\u003cb\u003e+\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eSiderophore production\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u003cb\u003e+\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e\u003cb\u003e+\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e\u003cb\u003e+\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eBiological nitrogen fixation\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u003cb\u003e+\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e\u003cb\u003e+\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e\u003cb\u003e+\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eAuxins production\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u003cb\u003e+\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e\u003cb\u003e+\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e\u003cb\u003e-\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eTotal positives\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e5\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e5\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e5\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003eThe selected microorganisms were used as PGPB in the following experiments in order to evaluate their potential to promote the growth of \u003cem\u003eS. biloba\u003c/em\u003e plants and to analyze the effect of their application on the phytoremediation process.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec19\" class=\"Section2\"\u003e\u003ch2\u003e3.4. Chromium tolerance of selected microorganisms\u003c/h2\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eFigure\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e. Tolerance of the microorganisms ER-C, ER-G and ER-Y to 0, 1, 3 and 5 ppm Cr(VI) expressed as ΔOD at 560 nm. The mean and standard deviations are shown. Means marked with asterisks are significantly different (***\u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.0003; ****\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.0001).\u003c/p\u003e\u003cp\u003eWhen analyzing the growth of the 3 selected isolates (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e), different variations in the final OD were observed. The ER-C microorganism showed no significant changes in growth, except in the 3-ppm treatment, where the final OD increased by 11.29% compared to the control group. The growth of ER-G decreased significantly by 4.44% in the 5-ppm treatment compared to the control. Lastly, a progressive reduction in the growth of the ER-Y microorganism was observed as Cr(VI) concentration increased. The 5-ppm treatment differed significantly from the 1 ppm treatment, showing a 4.07% reduction compared to the control.\u003c/p\u003e\u003cp\u003eAlthough the presence of Cr(VI) affected the growth of the microorganisms, none of the concentrations tested were lethal. Notably, ER-C exhibited lower growth in all treatments compared to the other microorganisms. This could be attributed to its lower growth rate, which may impact its ability to effectively colonize plant tissues and promote plant growth in subsequent experiments.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec20\" class=\"Section2\"\u003e\u003ch2\u003e3.5. In vivo plant growth promotion by selected microorganisms\u003c/h2\u003e\u003cp\u003eFigure\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e shows the results of the development of the plants when inoculated with the different microorganisms, expressed as the variation (Δ) of the measurements of FW (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA), total leaves (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eB) and root length (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eC) between the final and initial time. No significant differences were observed in any of the cases. This is likely attributable to the high variability inherent in biological systems.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eRegarding FW (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA), all plants showed an increase in weight compared to the initial condition. The uninoculated group increased an average its FW by 6.85%, while the other treatments exhibited increases of 1.15%, 7.83% and 11.58% for plants inoculated with the microorganisms ER-C, ER-G and ER-Y, respectively. However, the observed differences were not statistically significant. On the other hand, when analyzing the total number of leaves (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eB), it was observed that in the uninoculated plants, the increase in the number of leaves was 22% over the 15 days of the experiment. Although no significant differences were found between the treatments and the control, it was observed that inoculation with ER-C tended to decrease the number of leaves compared to the uninoculated group, showing a variation of 19.23%. In contrast, inoculation with ER-G and ER-Y showed a tendency to increase the number of leaves by 32% and 33.33%, respectively. Finally, regarding the variation in root length (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eC), the uninoculated plants showed an average increase of 7.95%. None of the treatments with microorganisms showed significant differences compared to this value. However, inoculation with ER-G tended to decrease this parameter relative to the uninoculated group, with a 6.67% increase, while inoculation with ER-Y showed a root length increase similar to that of the uninoculated treatment, at 7.50%. Inoculation with ER-C resulted in a greater root length variation compared to the uninoculated treatment, with an increase of 12.94%.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec21\" class=\"Section2\"\u003e\u003ch2\u003e3.6. Phytoremediation of 5 ppm Cr(VI) by Salvinia biloba inoculated with the selected microorganisms\u003c/h2\u003e\u003cdiv id=\"Sec22\" class=\"Section3\"\u003e\u003ch2\u003e3.6.1. Evaluation of the improvement in the tolerance to Cr(VI) by inoculated S. biloba\u003c/h2\u003e\u003cp\u003eConsidering the range of Cr(VI) concentrations analyzed in this study, 5 ppm of Cr(VI) was selected, as it is the highest concentration tolerated by both the microorganisms under study and the plants, and it has also been evaluated for other species of the genus \u003cem\u003eSalvinia\u003c/em\u003e (Prado et al. \u003cspan citationid=\"CR61\" class=\"CitationRef\"\u003e2010\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eFigure \u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e shows the results of the variation of FW (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eA), total leaves (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eB) and root length (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eC) between initial and final time (15 days) in inoculated \u003cem\u003eS. biloba\u003c/em\u003e plants exposed to 5 ppm Cr(VI). Additionally, the morphology of the plants was visually analyzed between the initial and final time.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eWhen observing the results of the FW variation (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eA), it was found that, although there were no significant differences between any treatment, inoculation with ER-C resulted in a smaller weight increase compared to the uninoculated treatment, with 3.72% and 7.72%, respectively. Inoculation with ER-G resulted in an average variation of 11.75%, while the greatest increase was found when inoculating with ER-Y, which showed a 17.93% FW increase. In the case of the number of total leaves (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eB), no notable differences were observed, but a positive trend was found when inoculating with ER-G and ER-Y in comparison to the uninoculated control (increases of 31.39%, 26.92% and 21.81%, respectively). Ultimately, when analyzing root length (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eC), the uninoculated control showed a negative trend in their development (a decrease of 2.09%). A positive trend in root development was observed in the presence of 5 ppm of Cr(VI) when inoculated with ER-C (5.64%), ER-G (15.32%), and ER-Y (10.48%).\u003c/p\u003e\u003cp\u003eRegarding plant cover, an increase of 11.03%, 14.18%, and 15.27% was observed in the systems inoculated with ER C, ER G, and ER Y, respectively (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003e). These differences between the initial and final time points were statistically significant for the treatments with ER-G and ER-Y (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05). However, unlike the findings reported by Mart\u0026iacute;nez Saucedo et al. (\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e2025\u003c/span\u003e) for uninoculated plants and the observations shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e, the increase in plant cover observed in Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003e is attributed to actual plant growth rather than turgor loss, as demonstrated in Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003e.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec23\" class=\"Section3\"\u003e\u003ch2\u003e3.6.2. Evaluation of the improvement in Cr phytoremediation by inoculated S. biloba\u003c/h2\u003e\u003cp\u003eTo determine whether the inoculation of \u003cem\u003eS. biloba\u003c/em\u003e plants with the selected microorganisms enhances their Cr removal capacity, the Cr(VI) concentration in solution was measured at both the initial and final times (Table\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). In uninoculated plants, the Cr(VI) concentration decreased by 35.54%, a value similar to the reduction percentage obtained in the previous assay for the treatment with 5 ppm of Cr(VI) (33%). Regarding the plants inoculated with the microorganisms under study, the Cr concentration reduction was 4.14% lower in the treatments inoculated with ER-C, compared to the uninoculated group. Plants inoculated with ER-G and ER-Y showed a greater reduction compared to the uninoculated group, with reductions of 1.09% and 8.46%, respectively. The differences between the initial and final times were significant in all treatments (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05); however, no statistically significant differences were found between the treatments at the final time.\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab3\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 3\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eCr(VI) removal capacity by \u003cem\u003eS. biloba\u003c/em\u003e plants inoculated with the selected microorganisms and the uninoculated treatment (U) exposed to 5 ppm for 15 days. The means marked with asterisks are significantly different from the initial concentration (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01).\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"5\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003eInitial Cr(VI) (ppm)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003eFinal Cr(VI) (ppm)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e\u003cp\u003eCr(VI) removal (%)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c5\"\u003e\u003cp\u003eDifference with U\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eU\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e\u003cp\u003e5.43\u0026thinsp;\u0026plusmn;\u0026thinsp;0.14\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e\u003cp\u003e3.50\u0026thinsp;\u0026plusmn;\u0026thinsp;0.21\u003csup\u003e*\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e35.54\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eER-C\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e\u003cp\u003e5.40\u0026thinsp;\u0026plusmn;\u0026thinsp;0.08\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e\u003cp\u003e3.56\u0026thinsp;\u0026plusmn;\u0026thinsp;0.26\u003csup\u003e*\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e34.07\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e\u0026minus;\u0026thinsp;4.14%\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eER-G\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e\u003cp\u003e5.26\u0026thinsp;\u0026plusmn;\u0026thinsp;0.07\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e\u003cp\u003e3.37\u0026thinsp;\u0026plusmn;\u0026thinsp;0.30\u003csup\u003e*\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e35.93\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e+\u0026thinsp;1.09%\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eER-Y\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e\u003cp\u003e5.24\u0026thinsp;\u0026plusmn;\u0026thinsp;0.14\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e\u003cp\u003e3.22\u0026thinsp;\u0026plusmn;\u0026thinsp;0.01\u003csup\u003e*\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e38.55\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e+\u0026thinsp;8.46%\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\u003c/div\u003e\u003cdiv id=\"Sec24\" class=\"Section2\"\u003e\u003ch2\u003e3.7. Identification of the most promising microorganism for assisted phytoremediation of Cr(VI) by Salvinia biloba\u003c/h2\u003e\u003cp\u003eBased on the results obtained, the ER-Y microorganism demonstrated promising outcomes in both plant growth promotion and enhancing the efficiency of the Cr(VI) phytoremediation process mediated by \u003cem\u003eS. biloba\u003c/em\u003e. Upon analyzing the 16S rRNA gene sequence, it was identified that the ER-Y microorganism belongs to the species \u003cem\u003eSerratia nematodiphila\u003c/em\u003e, with a 99.85% similarity with the reference strain, \u003cem\u003eS. nematodiphila\u003c/em\u003e DSM21420\u003csup\u003eT\u003c/sup\u003e. This species corresponds to Gram-negative, aerobic, rod-shaped, motile bacteria with a single lateral flagellum, isolated from the intestine of the nematode \u003cem\u003eHeterorhabditidoides chongmingensis\u003c/em\u003e (Zhang et al. \u003cspan citationid=\"CR82\" class=\"CitationRef\"\u003e2009\u003c/span\u003e). The partial 16S rRNA gene sequence for the microorganism \u003cem\u003eS. nematodiphila\u003c/em\u003e ER-Y was deposited in the NCBI Sequence Read Archive (SRA) database under the BioSample Accession SAMN46231118.\u003c/p\u003e\u003c/div\u003e"},{"header":"4. Discussions and conclusion","content":"\u003cp\u003ePhytoremediation is a sustainable alternative for the remediation of sites contaminated with various pollutants, including heavy metals. However, the efficiency of the process depends on the ability of the plants used to tolerate and accumulate contaminants, as well as on biomass production and the bioavailability of contaminants (Abbaszadeh-Dahaji et al. \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). One strategy to increase the efficiency of the process is the application of PGPB, given their ability to promote plant growth, increase plant tolerance to stress, and/or enhance the solubility and mobility of metals (Ma et al. \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e2011\u003c/span\u003e). Based on the above, the hypothesis was proposed that the inoculation of \u003cem\u003eS. biloba\u003c/em\u003e with PGPB isolated from contaminated site samples improves the Cr phytoremediation process in aquatic systems; and various experiments were conducted to test this hypothesis.\u003c/p\u003e\u003cp\u003eThe observed increase in vegetation cover of \u003cem\u003eS. biloba\u003c/em\u003e at 3 and 5 ppm Cr(VI) suggests a complex response to metal exposure that challenges traditional interpretations of surface area expansion as a proxy for plant growth. Despite the significant increase in surface coverage, particularly at the highest Cr(VI) concentrations tested, previous reports have indicated that S. biloba exhibits growth inhibition and physiological stress under similar conditions (Mart\u0026iacute;nez Saucedo et al. \u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e2025\u003c/span\u003e). This apparent contradiction highlights the need to interpret cover expansion with caution, especially in floating macrophytes where horizontal spread may result not from active growth but from morphological alterations. In this case, structural changes such as leaf displacement, reduced turgor pressure, or alterations in leaf angle and buoyancy likely contributed to the increased surface area. These modifications may reflect stress responses rather than enhanced performance, reinforcing the importance of integrating physiological and morphological metrics when assessing phytotoxicity and plant behavior in contaminated environments.\u003c/p\u003e\u003cp\u003eThe lack of significant differences in photosynthetic pigment content (chlorophyll \u003cem\u003ea\u003c/em\u003e, chlorophyll \u003cem\u003eb\u003c/em\u003e, and carotenoids) across treatments further supports the notion that increased cover did not correspond to improved physiological status. Similar results have been reported for aquatic macrophytes exposed to Cr(VI) and other metals, where pigment stability was observed despite growth inhibition (Dhir et al., \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2009\u003c/span\u003e; Prado et al., \u003cspan citationid=\"CR61\" class=\"CitationRef\"\u003e2010\u003c/span\u003e). One possible explanation is the limited translocation of Cr to photosynthetically active tissues, a mechanism described in several aquatic plant species as a means of protecting chloroplasts from oxidative damage (Srivastava et al., \u003cspan citationid=\"CR69\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). However, studies have demonstrated that \u003cem\u003eS. biloba\u003c/em\u003e is capable of translocating metals to aerial tissues, including leaves (Emiliani et al., \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2020\u003c/span\u003e), indicating that the Cr concentrations used in this study may not have reached thresholds sufficient to impair pigment biosynthesis. Alternatively, pigment stability may reflect a tolerance strategy in S. biloba, enabling maintenance of photosynthetic function under moderate metal stress.\u003c/p\u003e\u003cp\u003eTogether, these results underscore the importance of a multidimensional approach when evaluating plant responses to contaminants. Relying solely on surface area or pigment content as indicators may lead to misinterpretation of plant health and performance. In particular, morphological spread under metal stress may mask underlying physiological impairments, complicating assessments of phytoremediation efficiency. Future studies should combine measurements of pigment degradation, antioxidant enzyme activity, and biomass accumulation to more accurately characterize the impact of Cr(VI) on \u003cem\u003eS. biloba\u003c/em\u003e and other floating macrophytes.\u003c/p\u003e\u003cp\u003eRegarding the Cr removal capacity, \u003cem\u003eS. biloba\u003c/em\u003e plants removed 32%, 26%, and 21% of the total Cr in treatments with 1, 3, and 5 ppm, respectively. This reduction could be attributed to the absorption and accumulation of the metal inside the plants and/or to the adsorption on the surface of the plant tissues in contact with the contaminated solution. However, since the Cr content in the plant tissues was not measured in this study, the mechanism of the phytoremediation process cannot be determined. However, Emiliani et al. (\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2020\u003c/span\u003e) reported that this plant exhibited the ability to both adsorb certain metals on its surface and absorb and accumulate them in its biomass, the latter being the main phytoremediation mechanism of \u003cem\u003eS. biloba\u003c/em\u003e, since the metal concentrations found inside the plant were higher than those found on its surface. On the other hand, a greater reduction in the Cr(VI) concentration was observed compared to total Cr, suggesting that Cr(VI) was reduced to other oxidation states, likely due to the action of native microorganisms associated with the plants or the plants themselves, as has been reported for other species of this genus (Chocobar-Ponce et al. \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). Cr(VI) is the most toxic form of Cr due to the ease with which it diffuses through the cell membrane, so its reduction to other oxidation states, such as Cr(III), contributes to the detoxification process of the contaminated solution (Deepa et al. \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2025\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eOn the other hand, from soil, water, and sediment samples collected from areas contaminated with heavy metals due to anthropogenic activity in the Paran\u0026aacute; River Delta, 25 morphologically distinct microorganisms were isolated. These microorganisms were characterized \u003cem\u003ein vitro\u003c/em\u003e based on their plant growth-promoting activities, from which the ER-C, ER- G and ER-Y isolates were selected due to their highest number of positive results. The feasibility of applying the selected microorganisms in the Cr phytoremediation process carried out by \u003cem\u003eS. biloba\u003c/em\u003e depends on the microorganisms' tolerance to the metal. Therefore, their growth in the presence of the different concentrations of Cr used in the assays was evaluated. The three selected isolates were able to grow at all Cr(VI) concentrations analyzed, exhibiting slight alterations in growth; thus, no microorganism was discarded for subsequent experiments.\u003c/p\u003e\u003cp\u003eFor PGPB to interact with plants and produce a beneficial effect on plant growth in aquatic systems, they must be able to colonize plant tissue and compete with the native microbiota associated with the plants (Ishizawa et al. \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). It is also important to consider that microorganisms isolated from the soil may not be able to survive in hydroponic cultures (Stegelmeier et al. \u003cspan citationid=\"CR70\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). Therefore, to determine the ability of the selected microorganisms to interact with \u003cem\u003eS. biloba\u003c/em\u003e plants in aquatic systems, plant growth promotion was evaluated \u003cem\u003ein vivo\u003c/em\u003e. The three microorganisms had positive effects on various morphological parameters indicative of plant development compared to uninoculated plants. In treatments inoculated with ER-G and ER-Y, an increase in the number of leaves and FW of the plants was observed, while root length increased in plants inoculated with ER-C, which was the microorganism that showed the highest production of auxins, phytohormones associated with root tissue development (Vanneste et al. \u003cspan citationid=\"CR77\" class=\"CitationRef\"\u003e2025\u003c/span\u003e). Based on these results, it was concluded that the three selected microorganisms were able to interact with the plants and produce a plant growth-promoting effect on hydroponic cultures.\u003c/p\u003e\u003cp\u003eFinally, it was evaluated whether the inoculation of \u003cem\u003eS. biloba\u003c/em\u003e plants with the selected microorganisms affects the plant morphology and the Cr(VI) phytoremediation process. Although no statistically significant differences in FW were detected across treatments, the observed trends suggest that bacterial inoculation influenced plant performance under Cr(VI) exposure. In particular, \u003cem\u003eS. biloba\u003c/em\u003e inoculated with ER-Y exhibited the greatest FW increase, nearly doubling the response seen in the uninoculated control. This trend was also reflected in the number of total leaves, where treatments with ER-G and ER-Y resulted in higher percentages of leaf increase compared to the control. While not statistically significant, these morphological indicators suggest a potential growth-promoting effect of both isolates, particularly ER-Y. Root length responses further support the positive role of bacterial inoculation in mitigating Cr(VI) stress. Plants in the uninoculated control displayed a reduction in root development, consistent with the known inhibitory effects of Cr(VI) on root elongation (Dhir et al., \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2009\u003c/span\u003e). In contrast, inoculated plants showed positive trends in root length, especially those treated with ER-G and ER-Y. These findings indicate that the selected bacterial strains may alleviate Cr(VI) toxicity through plant growth-promoting mechanisms such as phytohormone production, improved nutrient uptake, or Cr detoxification at the rhizoplane.\u003c/p\u003e\u003cp\u003eVegetation cover measurements provide further evidence for the beneficial effects of inoculation. In contrast to previous findings where increases in cover were associated with loss of turgor and horizontal spread under Cr(VI) stress (Mart\u0026iacute;nez Saucedo et al. \u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e2025\u003c/span\u003e), the present results demonstrate significant increases in cover for ER-G and ER-Y treatments that are attributable to true biomass accumulation and vertical growth, as confirmed by morphological observations. These results reinforce the utility of combining vegetation cover data with visual and quantitative morphological assessments to avoid misinterpretations in phytoremediation studies.\u003c/p\u003e\u003cp\u003eThese results suggest that the bacterial strains ER-G and ER-Y enhance \u003cem\u003eS. biloba\u003c/em\u003e growth under moderate Cr(VI) exposure, likely through mechanisms typical of PGPB. While the absence of statistical significance in some parameters limits definitive conclusions, the consistency of positive trends across multiple growth indicators highlights the potential of these strains for application in assisted phytoremediation strategies. Further studies involving larger sample sizes, longer exposure periods, and mechanistic assays (e.g., quantification of IAA production or Cr reduction capacity) are warranted to elucidate the functional roles of these bacterial partners.\u003c/p\u003e\u003cp\u003eRegarding the effect of inoculation on Cr removal, a greater decrease in Cr(VI) concentration was observed in treatments inoculated with ER-Y compared to the uninoculated group. This decrease could be due to either the absorption and/or adsorption of the metal by the plants (Maine et al. \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e2016\u003c/span\u003e; Oliveira et al. \u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e2019\u003c/span\u003e), or its reduction to other oxidation states by the action of the inoculated microorganisms, the plants' native microbiota, and/or the plants themselves (Ahemad \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2015\u003c/span\u003e; Chocobar-Ponce et al. \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). The plants inoculated with the ER-Y microorganism showed one of the greatest increases in plant biomass compared to the uninoculated plants, and also a greater decrease in Cr(VI) concentration in the effluent compared to the uninoculated group. This difference in Cr removal could be attributed to a higher capacity for metal accumulation in the plant tissues due to the increased plant biomass.\u003c/p\u003e\u003cp\u003eConsidering all of this, ER-Y, identified as \u003cem\u003eSerratia nematodiphila\u003c/em\u003e, was selected as a promising microorganism to continue with the assisted phytoremediation studies. While some species of the \u003cem\u003eSerratia\u003c/em\u003e genus, primarily \u003cem\u003eS. marcescens\u003c/em\u003e, are of clinical importance due to their pathogenicity to humans, \u003cem\u003eS. nematodiphila\u003c/em\u003e has not been described as a pathogen (Tang et al. \u003cspan citationid=\"CR71\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). On the other hand, this microorganism has been previously reported as a PGPB, exhibiting various plant growth-promoting activities such as siderophore production, phosphate solubilization (results also found in this work), ACC deaminase production, as well as antimicrobial activity, which could be related to the enzymatic activities found in this study (Basharat et al. \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; Chen et al. \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2010\u003c/span\u003e; Sanchez-Cruz et al. \u003cspan citationid=\"CR66\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). This species also shows tolerance to the presence of several contaminants, including metals (Sanchez-Cruz et al. \u003cspan citationid=\"CR66\" class=\"CitationRef\"\u003e2019\u003c/span\u003e), which contributes to its application as a PGPB in phytoremediation processes. For example, inoculation of \u003cem\u003eS. nematodiphila\u003c/em\u003e in \u003cem\u003eSolanum nigrum\u003c/em\u003e L. plants growing in soils contaminated with various concentrations of Cd not only stimulated plant growth but also influenced metal accumulation in different plant tissues (Chen et al. \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2010\u003c/span\u003e). On the other hand, \u003cem\u003eS. nematodiphila\u003c/em\u003e isolated from chromite mine soils has been reported to reduce Cr(VI) to Cr(III) (Leonard and Mishra \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e2022\u003c/span\u003e), suggesting its potential application in bioreduction processes for this contaminant. Moreover, a proteomic analysis of \u003cem\u003eS. nematodiphila\u003c/em\u003e sp. MB307 revealed the production of chaperones, membrane integrity proteins, mobility, and transporter proteins. Comparative analysis with other metal-resistant bacteria provided evidence that these proteins play a crucial role in tolerance to high metal concentrations (Basharat et al. \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). Genomic analysis of \u003cem\u003eS. nematodiphila\u003c/em\u003e MB307 demonstrated the presence of genes associated with plant growth promotion, antimicrobial activity, bioremediation, metal tolerance, azo dye degradation, and ibuprofen degradation, among others (Basharat et al. \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). Therefore, this microorganism represents a promising candidate for further evaluation in these processes.\u003c/p\u003e\u003cp\u003eIn conclusion, a positive effect was observed on the Cr(VI) phytoremediation process performed by \u003cem\u003eS. biloba\u003c/em\u003e plants when inoculated with the microorganism ER-Y, isolated from heavy metal-contaminated sites in the Paran\u0026aacute; River Delta, Argentina. Notably, this microorganism, identified as \u003cem\u003eS. nematodiphila\u003c/em\u003e, demonstrated promising results both as a PGPB and in assisted phytoremediation when applied to \u003cem\u003eS. biloba\u003c/em\u003e. Future studies focusing on optimizing microorganism dosage and application methods are necessary to enhance the process. The use of PGPB could represent a promising strategy for improving the efficiency of phytoremediation; however, further research is required to optimize its application.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors gratefully acknowledge the support provided by the Universidad Argentina de la Empresa (UADE) for the development of this study.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was supported by Universidad Argentina de la Empresa (UADE) (Project numbers P21T08). Author Pamela Romina Bernabeu has received research support from UADE.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor Contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll authors contributed to the study conception and design. Material preparation, data collection and analysis were performed by María\u0026nbsp;de los Ángeles\u0026nbsp;Martínez Saucedo and Pamela Romina Bernabeu. The first draft of the manuscript was written by Pamela Romina Bernabeu and all authors commented on previous versions of the manuscript. All authors read and approved the final manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthical Approval\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis is not applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent to Participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis is not applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent to Publish\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis is not applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting Interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp;The authors have no relevant financial or non-financial interests to disclose.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData Availability Statement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eData will be made available on reasonable request.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eAbbaszadeh-Dahaji P, Omidvari M, Ghorbanpour M (2016) Increasing phytoremediation efficiency of heavy metal-contaminated soil using PGPR for sustainable agriculture. 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J Agric Food Chem 69(45):13270\u0026ndash;13285. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1021/acs.jafc.1c00138\u003c/span\u003e\u003cspan address=\"10.1021/acs.jafc.1c00138\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eStatements \u0026amp; Declarations\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":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":true,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"environmental-science-and-pollution-research","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"espr","sideBox":"Learn more about [Environmental Science and Pollution Research](https://www.springer.com/journal/11356)","snPcode":"11356","submissionUrl":"https://submission.nature.com/new-submission/11356/3","title":"Environmental Science and Pollution Research","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"Biotechnology, Chromium, Phytoremediation, Plant growth-promoting bacteria, Salvinia biloba, Serratia nematodiphila","lastPublishedDoi":"10.21203/rs.3.rs-7178802/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7178802/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eHeavy metals, such as chromium (Cr), are toxic even at low concentrations and are a significant environmental concern. Phytoremediation is an emerging biotechnological method to clean contaminated environments, using plant biomass to absorb and accumulate contaminants. To enhance the phytoremediation capacity, plant growth-promoting bacteria (PGPB) have been proposed as a strategy. This study aimed to evaluate the effect of PGPB isolated from sediment samples of the Paran\u0026aacute; River Delta (Argentina) on the phytoremediation of Cr by \u003cem\u003eSalvinia biloba\u003c/em\u003e, a floating macrophyte native to the region.\u003c/p\u003e\u003cp\u003eThe plants were exposed to 1, 3, and 5 ppm of Cr, exhibiting phytotoxicity characterized by turgor loss, which resulted in an increased vegetation cover; however, the photosynthetic pigment content remained unaffected at these concentrations. The total Cr concentration decreased by 32%, 26%, and 21% in the 1, 3, and 5 ppm treatments, respectively, while hexavalent chromium (Cr(VI)) was reduced by 82%, 45%, and 33%.\u003c/p\u003e\u003cp\u003eThree microorganisms exhibiting the highest plant growth-promoting activity were selected, as they positively impacted plant growth and tolerated different Cr(VI) concentrations. To assess the effect of PGPB on the phytoremediation process, inoculated \u003cem\u003eS. biloba\u003c/em\u003e plants were exposed to 5 ppm of Cr(VI). Notably, inoculation with microorganism ER-Y, identified as \u003cem\u003eSerratia nematodiphila\u003c/em\u003e, a previously reported PGPB, enhanced plant growth in contaminated systems. Furthermore, inoculation significantly increased vegetation cover compared to the uninoculated control; however, unlike the increase observed in uninoculated plants, this expansion was not due to turgor loss but rather to actual plant growth stimulated by the PGPB under Cr(VI) exposure. Additionally, ER-Y contributed to an almost 9% greater Cr(VI) reduction compared to the uninoculated group, highlighting its potential role in improving both plant resilience and remediation efficiency.\u003c/p\u003e","manuscriptTitle":"Use of plant growth-promoting bacteria to enhance chromium phytoremediation by Salvinia biloba","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-08-08 20:07:32","doi":"10.21203/rs.3.rs-7178802/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Major Revision","date":"2025-08-28T04:14:06+00:00","index":"","fulltext":""},{"type":"reviewerAgreed","content":"","date":"2025-08-03T13:43:14+00:00","index":0,"fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-08-02T15:52:47+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"Environmental Science and Pollution Research","date":"2025-07-30T11:51:24+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-07-24T04:46:21+00:00","index":"","fulltext":""},{"type":"submitted","content":"Environmental Science and Pollution Research","date":"2025-07-22T07:57:06+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"environmental-science-and-pollution-research","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"espr","sideBox":"Learn more about [Environmental Science and Pollution Research](https://www.springer.com/journal/11356)","snPcode":"11356","submissionUrl":"https://submission.nature.com/new-submission/11356/3","title":"Environmental Science and Pollution Research","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"c50c23b7-3d2b-4ab4-a9e6-62fc1a0feecb","owner":[],"postedDate":"August 8th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2025-11-17T16:03:26+00:00","versionOfRecord":{"articleIdentity":"rs-7178802","link":"https://doi.org/10.1007/s11356-025-37186-6","journal":{"identity":"environmental-science-and-pollution-research","isVorOnly":false,"title":"Environmental Science and Pollution Research"},"publishedOn":"2025-11-14 15:58:28","publishedOnDateReadable":"November 14th, 2025"},"versionCreatedAt":"2025-08-08 20:07:32","video":"","vorDoi":"10.1007/s11356-025-37186-6","vorDoiUrl":"https://doi.org/10.1007/s11356-025-37186-6","workflowStages":[]},"version":"v1","identity":"rs-7178802","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7178802","identity":"rs-7178802","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}