Metals are considered to be one of the major pollutants of water resources around the world. They are used in various industrial processes, and several tons of waste per year end up in effluents that reach different water sources (Chakraborty et al., 2020; Bilal et al., 2021). Metals are found naturally in the Earth's crust and are essential in several biochemical processes. However, some of them, such as cadmium (Cd), lead (Pb), mercury (Hg), chromium (Cr), and copper (Cu), are toxic even at low concentrations and have no known specific biological role (Ballardo de la Cruz et al., 2015; Beltrán-Pineda and Gómez-Rodríguez, 2016; Mustapha and Halimoon, 2015). Metal toxicity leads to biological and chemical degradation within ecosystems, affecting environmental and public health (Suárez and Reyes, 2002; Bilal et al., 2021).

Pb is a persistent pollutant of environmental interest. It is biomagnified through the different links in the food chain and causes long-term damage (Bedoya-Vélez et al., 2019). It inhibits enzymatic activity and affects nucleic acid and protein conformation, as well as cell membrane functions (Jaros+awiecka and Piotrowska-Seget, 2014). It can replace metallic ions such as Zn, Ca, and Fe in enzymes that are important for microbial metabolism (Naik and Dubey, 2013). High concentrations of Pb have been found up to a value of 250 ppm (Jaros+awiecka and Piotrowska-Seget, 2014) in the effluents derived from industrial activities such as lead battery manufacturing, welding, metal refining, weapons manufacturing, the automotive industry, power plants, and waste incineration, among others (Fernández et al., 2020; Naik and Dubey, 2013).

On the other hand, Cr is a transition metal that is found in different oxidation states. The most common states are Cr (VI) and Cr (III). Hexavalent chromium, Cr (VI), is considered toxic and is soluble, but it is rare in nature given its high oxidation state. Trivalent chromium, Cr (III), is less mobile but is frequently present in soil and water samples (Cervantes et al., 2001; Mandina et al., 2013). For instance, concentrations of Cr (III) up to 450 ppm have been found in tanneries (Pinzón-Bedoya and Cardona-Tamayo, 2010), especially in small industries that lack physicochemical wastewater treatments. These levels exceed the maximum permissible limits established in national environmental legislation (Resolution 0631 of 2015) (Cardona-Guitiérrez et al., 2013). Cr (III) interacts electrostatically with anionic groups in DNA phosphates, producing DNA transcription errors and causing mutagenesis. Additionally, Cr (III) affects enzyme structures (Cervantes et al., 2001; Joutey et al., 2015; Viti et al., 2013).

The presence of toxic metals in water sources has driven the development of different water treatment methods. Conventional methods include membrane filtration, electrodialysis, reverse osmosis, nanofiltration, and ultrafiltration (Cervantes et al., 2001; Chakraborty et al., 2020; Joutey et al., 2015; Viti et al., 2013). However, electrochemical methods, ionic exchange, precipitation, and recovery by evaporation are expensive techniques and only remove 1 and 100 mg/L of metals (Özer and Özer, 2003). Some methods are costly due to equipment maintenance costs, and state-of-the-art technology is generally too expensive for companies that operate at a local scale. Thus, efforts have been made over the past several years to design alternative biotechnological processes that efficiently remove toxic metals and are easy to operate, widely available, and relatively cheap. One of the alternatives for industrial wastewater decontamination is the use of several types of biomass from plant-based agro-industrial waste as bio-adsorbents. Bioadsorption is defined as the ability to retain metallic ions (adsorbate) present in a liquid phase (solvent) on a solid surface (adsorbent) (Ballardo de la Cruz et al., 2015). For instance, elodea (Egeria densa, ELO) is a submerged freshwater plant that is found in lakes. This plant is able to take up nutrients from the body of water and perform photosynthesis even with low amounts of CO₂ and light, which makes it highly efficient at colonizing different ecosystems, to the point where it has been deemed an invasive species in many bodies of water (Rimac et al., 2018; Yarrow et al., 2009). Currently, there are few studies on the use of ELO for metal removal, but, in one study, it was able to remove nearly 91% of Pb (II) from an aqueous solution (Hernández-Gómez et al., 2017).

Other potential bio-adsorbents are coconut husk, saw dust-based biochar, and seed composite, which have been used to remove metallic ions from mining effluents and water bodies with an effectiveness between 75 and 96,36% (Fernández et al., 2020; Mahour et al., 2022; Manyuchi et al., 2022). Cassia fistula seeds and three different surfaces of modified adsorbent materials retained between 13,22 and 129,3 mg/g of Pb (II) at pH equal to 5,0 and 30 minutes of contact (Hemavathy et al., 2021). Orange peel (OP) has been used as a bioadsorbent for Pb, Fe, Cu, Zn, and Cr, with adsorption percentages between 67,2 and 99,5%, where pH and particle size play an important role in the metal retention of the adsorbent surface (Cardona-Guitiérrez et al., 2013; Fernández et al., 2020; Patiño-Saldivar et al., 2021). The removal of contaminants from solutions has also been investigated using tea waste, modified diatomite and bentonite, and the Metal Organic Framework (MOF) to adsorb dyes, metals, and pharmaceutical compounds. For example, carbon from tea waste was able to remove 120,13 mg/g of methylene blue, and UiO- 66 MOF adsorbed 30 mg/g of Cr (VI) and 485,4 mg/g of the pharmaceutical waste diclofenac sodium (Patil et al., 2022; Sriram et al., 2022a, 2022b; Rego, Sriram, et al., 2021a; Rego, Kuriya, et al., 2021b; Uthappa et al., 2020).

The use of biomaterials provides multiple benefits, as it repurposes solid waste, aids in the management of invasive species, and eliminates pollutants of environmental concern. Furthermore, bioadsorbents are efficient materials in the removal of metals and have a high biodegradation capacity, which is an advantage when they must be disposed of after use (Ojuederie and Babalola, 2017). When considering the use of biomaterials within water treatment systems, it is important to consider their biodegradation and stability in the design features.

During biodegradation, the materials' intrinsic biodegradability behaves as a function of time due to the occurrence of reactions dependent on the interaction with microorganism communities, temperature, and humidity, among other factors (Pischedda et al., 2019). Microorganisms responsible for the biodegradation process can be present in plant matrices, as well as in the effluents to be treated. Through different mechanisms, the microorganisms fragment biomaterials into simpler compounds that are necessary for their metabolic processes (Polman et al., 2019). During biodegradation, the carbon present in a material is transformed or mineralized, aerobically or anaerobically, into products such as biomass, water, carbon residues, and carbon dioxide (CO₂), as shown in Equation (1) (Chinaglia et al., 2018).

Respiration involves an electron transfer chain from one carrier to another, in which organic compounds (bioadsorbents) act as electron donors to the final electron acceptor (O₂). This chain yields adenosine triphosphate (ATP) and CO₂, among other substances. ATP provides the necessary energy to produce molecular compounds involved in growth, maintenance, and reproduction (Vanrolleghem et al., 1999). Biomass is the result of long-term microbial reproduction (Chinaglia et al., 2018), while CO₂ and H₂O tend to be the final products of mineralization. Aerobic respiration generates the chemical energy used by microorganisms for their vital functions through the use of organic substrates as electron donors and oxygen as the last electron acceptor (Vanrolleghem et al., 1999).

Respirometry techniques can therefore be used to calculate the cumulative production of CO₂ over time, using traps with alkaline solutions such as sodium hydroxide (NaOH) and potassium hydroxide (KOH). The amount of CO₂ generated via microorganism respiration is quantified by titrating the quantity of base that has not reacted with carbon dioxide (Castro-Aguirre et al., 2017; Pellizzari-Wielewski et al., 2014). This provides a quantification of the materials' biodegradability, which is linked to the ability of organic materials to maintain their physical and chemical properties over time (or biostability). Inevitable changes in the adsorbents' structure lead to a loss of the material's active function (Lemaire, 1997), which highlights the importance of considering these two aspects (biodegradability and biostability) for the scaling of real polluted water treatment systems using biomaterials.

Additionally, bacteria, depending on their morphology, have different mechanisms that influence pollutant absorption (Verma et al., 2021 ). Gram-positive bacteria interact with metals at the extracellular level through electrostatic attraction (Joutey et al., 2015; Suárez and Reyes, 2002) to exopolysaccharides conformed of macromolecules as polysaccharides, proteins, and nucleic acids (Naik and Dubey, 2013; Sajna et al., 2021). Functional groups such as peptidoglycan carboxylate and teichoic acid phosphate, components of the cell wall, act as anionic sites that bond with the metal cations Pb (II) and Cr (III) (Diep et al., 2018; Pérez-Bou et al., 2018; Ojuederie and Babalola, 2017; Yin et al., 2019). Gram-negative bacteria also have mechanisms of extracellular interaction with metals via through electrostatic attraction (Joutey et al., 2015; Suárez and Reyes, 2002), in which functional groups such as phosphoryl in lipopolysaccharide and the components of the external membrane are responsible for the union with metal cations (Pb (II), Cr (III)) (Jaros+awiecka and Piotrowska-Seget, 2014; Kadukova, 2016; Naik and Dubey, 2013).

The biostability of bioadsorbents is a poorly studied but important aspect of the design of wastewater treatment with alternative materials. Thereupon, the objectives of this study were 1) to isolate native microorganisms that were tolerant of metals such as Pb (II) and Cr (III), 2) to determine the biostability and biodegradation of plant-based ELO and OP bioadsorbents, and 3) to evaluate the contribution of tolerant microorganisms to metal removal from aqueous solutions.

Obtaining the bioadsorbents

Orange peel (OP) was obtained from a local market, and elodea (ELO) was collected from Simón Bolivar Metropolitan Park. Both plant-based materials were washed with abundant water and dried at room temperature. The bioadsorbents were chopped into pieces no larger than 1 cm² and dried again at room temperature. Then, the bioadsorbents were dried in an oven at 50 °C to constant weight. Each of the plant-based materials was ground in a hand crank mill and sifted to obtain a particle size between 2 000 and 500 um. The bioadsorbents were then stored in sealed plastic bags at room temperature (Castañeda-Figueredo et al., 2022).

Microbial characterization of the bioadsorbents

Microbial characterization of the plant-based materials was performed in order to identify native microorganisms in each bioadsorbent and establish whether they contained microbial strains resistant to Pb (II) and Cr (III). The microorganism concentration in OP and ELO was quantified using the plate count method. Macroscopic morphological characterization (morphotypes) was based on the shape and color of the colony. Subsequently, a test of the microorganisms' resistance to metals in the bioadsorbents over time was done. In this assay, a Pb (II) and Cr (III) solution at 450 mg/L was added to OP and ELO. After 2, 600, and 1 104 hours of contact between the bioadsorbents and metallic ions, the microbial concentration was determined using serial dilutions (from 1x10^-2 to 1x10^-6) and cultures via the plate spread method. Bacteria and fungi were isolated with nutrient agar (NA) (Ben-David and Davidson, 2014) and Sabouraud agar or potato dextrose agar (PDA), respectively. Cultured agars were incubated at 30±1 °C for 24 hours for bacteria and at 25 °C for 3 to 5 days for fungi. All tests were performed in duplicate.

The morphotypes that were most dominant and tolerant of Pb (II) and Cr (III) were selected to be individually isolated in NA using the streaking method, and they were preserved at 4 °C.

Biodegradation and stability study of the bioadsorbents

An incubation test was used to determine the biodegradation of ELO and OP as a function of the microorganisms' metabolic activity by quantifying CO₂ production (respirometry tests). The bio-adsorbents were incubated separately in hermetic glass jars with a 500 mL capacity. At the bottom of each jar, two smaller jars or vials were placed: one (amber) with 10 mL of sterile distilled water to maintain the humidity of the medium, and another one containing 2 g of dried bioadsorbent and 8 mL of a Pb (II) and Cr (III) solution at 450 mg/L (M1) (Figure 1). Controls consisted of 2 g of sterilized plant-based material and 8 mL of distilled water (M2), an empty glass jar (M3), and 8 mL of the Pb (II) and Cr (III) solution (M4). The CO₂ that evolved during the incubation test was trapped with 20 mL of NaOH 1 N placed in 30-mL vials on the upper part of the jar. All setups were evaluated in duplicate. The M1 setup was incubated for 46 days for ELO and 60 days for OP. Control tests M2, M3, and M4 were analyzed three times during the assay (beginning of the test, middle of the test, and end of the test), according to the time established for each matrix. All incubations were performed at room temperature (20±1 °C).

Figure 1 Source: Authors

The CO₂ that evolved during the incubation test was quantified by mixing 2 mL of NaOH from each sample with 5 mL of barium chloride BaCl₂ 0,5 M. The NaOH that did not react to the CO₂ was titrated with hydrochloric acid (HCl) 0,5N (Equations (2)-(4)).

The stability of bioadsorbents in terms of the product lifespan was established according to Leejarkpai et al. (2011)). The theoretical amount of CO₂ (ThCO₂) in grams was computed according to Equation (5), and the biodegradation percentage for ELO and OP was obtained from the produced amount of CO₂, as shown in Equation (6):

where ThCO ₂ is the theoretical amount of CO₂ that each bio-adsorbent can produce (g); ^M _TOT is the amount of dried solids (g) added at the beginning of the test; ^C TOT is the ratio of total organic carbon present in the samples' dried solids; 44 and 12 are the molecular weights of CO₂ and carbon, respectively; (CO ₂ ) _T is the accumulated amount of CO₂ (g) released from each setup; and (CO ₂ ) _B is the accumulated amount of CO₂ from the blank (g).

Biostability was estimated through a zero-order kinetic equation, as shown in Equation (7) (Pinchao-Pinchao et al., 2019).

where ( B ) is the percent biodegradation at time t, ( B ₀ ) is the initial percent biodegradation, and k ₀ is the constant velocity.

Pb (II) and Cr (III) bioadsorption

The bioadsorption of Pb (II) and Cr (III) over time was evaluated in ELO with and without the predominant and metal-tolerant morphotypes (B1, B2, and B3). ELO was used for this test because it had more microorganisms than OP. The bioadsorption of Pb (II) and Cr (III) was tested using three treatments (T1, T2, and T3) in 100 mL Erlenmeyer flasks. Treatment 1 (T1) consisted of 1 g of sterilized ELO with 45 mL of a Pb (II) and Cr (III) solution at 50 mg/L, and an aliquot (5 mL) of each bacterium with a cell concentration of 0,550 of absorbance -approximately 5,8x10⁸ cells according to the McFarland scale (EB1, EB2, and EB3). The bacteria were isolated separately through colony suspension in peptone water, followed by a reading in the spectrophotometer at a wavelength of 600 nm. Treatment 2 (T2) consisted of 1 g of sterilized bioadsorbent with 50 mL of the metal solution (MEL). Treatment 3 (T3) was prepared with an aliquot (5 mL) of each bacterium and 45 mL of the metal solution (MB1, MB2, and MB3). In T3, the nutrient broth was provided as a carbon source to support the bacteria's metabolism. All of the treatments were kept at room temperature (20±1 °C) under constant agitation at 120 rpm for 5 days. Every 12 hours, two tests were retrieved for each treatment, and the solution was filtered through 0,45 um membranes. The filtrates were preserved with nitric acid (HNO₃) at 65%, and the metals were quantified via atomic absorption spectrophotometry. For each assay, a microbial growth control was performed which consisted of a streaking culture in AN and Sabouraud agar or PDA. The control was verified every 24 hours throughout the test. The adsorbed quantity and removal percentages were calculated using the following Equations:

where q _t is the amount adsorbed (mg/kg), m ₀ is the initial mass of the metal ions, and m _f is the final mass of metal ions.

The adsorption kinetics of Pb (II) and Cr (III) in ELO were fit to two linear forms of kinetic reaction models (i.e., pseudo-first-order and pseudo-second-order equations) (Tran et al., 2017).

where q _e is the amount of adsorbate uptake per mass of adsorbent at equilibrium, k ₁ (1/time) is the rate constant of the pseudo-first-order equation, and k ₂ (mass/mass x time) is the rate constant of the pseudo-second-order model.

Statistical analysis

A one-way ANOVA (analysis of variance) was used to determine whether there were any statistically significant differences between the means of Pb (II) and Cr (III) bioadsorption and the results of plant-based materials biodegradation. The normality, homogeneity, and multiple comparisons of means were evaluated with Shapiro-Wilk, Levene, and Tukey tests. All statistical analyses were performed with the OriginPro software and considered an alpha-value of 0,05.

The study confirmed the potential of ELO and OP as bioadsorbents for metallic ion removal. Biostability and microbial characterizations demonstrated that OP had a longer lifespan than ELO (7,05 months vs. 2,61 months) and less microbial growth. Meanwhile, ELO contained a high abundance of native microorganisms that were resistant to Pb (II) and Cr (III). The abundant microbes in ELO degrade the plant-based material faster (higher CO₂ production in the respirometry test). Furthermore, the microorganisms present in ELO did not contribute significantly to metal retention, meaning that it is mainly the plant material itself that is responsible for metal adsorption, except for the bacteria EB1, which led to a small but significant increase in Pb (II) adsorption. The pseudo-second-order model was observed as the appropriate fittest one for Pb (II) and Cr (III) adsorption in ELO. Finally, it is our opinion that OP is the better material for filter development to be applied in polluted water treatment systems.

Figure 5 Source: Authors