microorganisms Article Heavy Metal Tolerance of Microorganisms Isolated from Coastal Marine Sediments and Their Lead Removal Potential Katleen L. Alvarado-Campo 1,† , Marynes Quintero 1,† , Bernarda Cuadrado-Cano 2 , Manuela Montoya-Giraldo 1,‡ , Elver Luis Otero-Tejada 1 , Lina Blandón 1 , Olga Sánchez 3 , Ana Zuleta-Correa 1, * and Javier Gómez-León 1 1 Marine Bioprospecting Line, Evaluation and Use of Marine and Coastal Resources Program–VAR, Marine and Coastal Research Institute–INVEMAR, Santa Marta 470006, Magdalena, Colombia; katleen.alvarado@invemar.org.co (K.L.A.-C.); marynes.quintero@invemar.org.co (M.Q.); elverotero@sophicol.org (E.L.O.-T.); lmblando@unal.edu.co (L.B.); javier.gomez@invemar.org.co (J.G.-L.) 2 Master’s Program in Microbiology, College of Medicine, Universidad de Cartagena, Cartagena de Indias 130014, Bolívar, Colombia; bcuadradoc@unicartagena.edu.co 3 Department of Genetics and Microbiology, Faculty of Biosciences, Universitat Autònoma de Barcelona, 08193 Bellaterra, Spain; olga.sanchez@uab.cat * Correspondence: ana.zuleta@invemar.org.co † These authors contributed equally to this work. ‡ Current address: Department of Biological Sciences, University at Albany, State University of New York, Albany, NY 12222, USA. Abstract: In this study, 338 microorganisms, comprising 271 bacteria and 67 fungi, were isolated from sediment samples collected from underexplored Pacific and Caribbean regions of Colombia. Screen- ing trials were conducted on selected strains (n = 276) to assess their tolerance to cadmium (Cd2+ ), lead (Pb2+ ), and zinc (Zn2+ ), leading to the identification of six bacteria capable of withstanding 750 mg·L−1 of each heavy metal ion. Three promising microorganisms, identified as Enterobacter sp. Citation: Alvarado-Campo, K.L.; INV PRT213, Pseudomonas sp. INV PRT215, and Stenotrophomonas sp. INV PRT216 were selected for Quintero, M.; Cuadrado-Cano, B.; lead removal experiments using LB broth medium supplemented with 400 mg·L−1 Pb2+ . Among Montoya-Giraldo, M.; Otero-Tejada, these, Pseudomonas sp. INV PRT215 exhibited significant potential, removing 49% of initial Pb2+ after E.L.; Blandón, L.; Sánchez, O.; 240 min of exposure (16.7 g wet biomass·L−1 , pH 5, 30 ◦ C). Infrared spectra of Pb-exposed biomass Zuleta-Correa, A.; Gómez-León, J. showed changes in functional groups, including carbonyl groups of amides, carboxylate, phosphate, Heavy Metal Tolerance of hydroxyl, and amine groups, compared to the not-exposed control. These changes suggested interac- Microorganisms Isolated from Coastal Marine Sediments and Their tions between the metal and functional groups in the biomass. The findings of this study highlight Lead Removal Potential. the potential of microorganisms derived from coastal marine environments as promising candidates Microorganisms 2023, 11, 2708. for future applications in bioremediation of polluted environments contaminated with heavy metals. https://doi.org/10.3390/ microorganisms11112708 Keywords: marine bacteria; marine fungi; cadmium; zinc; Enterobacter sp.; Stenotrophomonas sp.; Pseudomonas sp. Academic Editor: Giovanni Vallini Received: 19 September 2023 Revised: 23 October 2023 Accepted: 25 October 2023 1. Introduction Published: 4 November 2023 Metal ions reach ecosystems through various natural processes and anthropogenic activities [1]. The leading causes of their natural release from the lithosphere and sub- sequent accumulation in ecosystems include erosion, volcanic activity, and forest fires, Copyright: © 2023 by the authors. from which these metals undergo accumulation cycles without causing adverse effects on Licensee MDPI, Basel, Switzerland. the environment [2]. On the other hand, several human activities extensively use metals This article is an open access article such as lead, cadmium, molybdenum, vanadium, and chromium to manufacture multiple distributed under the terms and products, including batteries [3], plastic [4], projectile lubricants, insecticides, ceramics, conditions of the Creative Commons paint and glass pigments [5], photography [6], welding, wood conservation agents, and Attribution (CC BY) license (https:// tanning agents, and in the chemical industry as a whole [7]. The frequent use of met- creativecommons.org/licenses/by/ als in industrial settings is closely linked to environmental pollution due to inadequate 4.0/). Microorganisms 2023, 11, 2708. https://doi.org/10.3390/microorganisms11112708 https://www.mdpi.com/journal/microorganisms Microorganisms 2023, 11, 2708 2 of 24 residue management that accelerates metal accumulation in sediments and water-receiving bodies [1]. The development of suitable alternatives to mitigate heavy metal pollution and en- vironmental deterioration is the focus of different research fields [8,9]. The presence of these toxic agents in the environment generally induces morphological and physiological changes in microbial communities. Therefore, polluted environments are considered a source of promising metal-resistant strains helpful in the development of new bioremedi- ation methods, which are commonly regarded as cost-effective, ecological, and efficient technologies [10,11]. There is evidence of organisms and microorganisms that, due to their metabolic capabilities, can tolerate and/or transform metals for their benefit, accumulating them within themselves [12]. In this sense, microorganisms or their metabolic products have been employed to transform toxic compounds into less harmful forms, thus tackling environmental damage [13,14]. Microorganisms employ several mechanisms for bioremediation of soil and water polluted with heavy metals including the secretion of extracellular barriers, extracellular and intracellular sequestration, active transport of metal ions, production of metal chelators, and enzymatic detoxification [15,16]. The permeability barrier involves the production of exopolysaccharides and biofilm formation to physically sequester and chemically modify metal ions into less toxic molecules [17]. On the other hand, the extracellular sequestration process is a passive uptake mechanism that involves the accumulation of metal ions in the cellular periplasm or the complexation of metal ions into insoluble compounds, causing metal precipitation. Intracellular sequestration implicates their transport within the cell, and, therefore, it requires interaction of metal ions with cell-surface ligands [18]. Active transport of metal ions is based on either ATP hydrolysis or an electrochemical gradient comprising the participation of ABC transporter proteins, cation diffusion facilitator (CDF), and P-type ATPases, among other proteins involved in metal tolerance [19]. Several reports indicate the production of microbial metabolites acting as chelating agents, as well as the formation of molecules such as metallothioneins, phytochelatins, and biosurfactants during heavy metal detoxification. The production of enzymes that can biologically transform or chemically modify heavy metal ions in less toxic forms is also frequently reported [15]. The use of autochthonous bacteria and fungi for ecosystem remediation has become one of the most critical activities when designing biotechnological processes to restore affected environments [7]. This is especially significant for organisms from marine-coastal environments, which exhibit adaptability to a wide range of temperature, pressure, and high salinity gradients, allowing them to thrive under diverse conditions without cell damage [14,20]. The Northern Pacific and the southern area of the Atlantic (Gulf of Urabá) are consid- ered essential areas of Colombia, known for their high biodiversity and abundant water resources [21]. The Atrato River, on the Caribbean side, stands as one of the world’s fastest-flowing rivers, while the Pacific region boasts waterways of great importance for the country’s trade, as is the case of the Baudó and San Juan rivers. However, despite their economic and ecosystem significance, these rivers face concerning levels of heavy metal pollution attributed to illegal gold extraction and untreated wastewater dumping, which resulted in the accumulation of these toxic compounds into the sediments, particularly in the Colombian Pacific area [22]. For example, cadmium pollution is often linked with the use of agrochemicals, of battery manufacturing, pigments, anticorrosive agents, and contaminated irrigation water [23–25]; zinc contamination may result from discharge of dyes and detergents, while lead is mainly derived from the textile industry, battery produc- tion, paint manufacturing, insecticide use, fossil fuel consumption, waste incineration, and metallurgical processes [26,27]. In particular, lead represents one of the most significant heavy metal contaminants on a global scale, raising alarming concerns on both human health and biodiversity [17]. Distribution reports in Colombia indicate the presence of this metal across different environmental matrices, posing a public health risk [5,28]. In this context, there has been a growing interest in developing eco-friendly alternatives to treat context, there has been a growing interest in developing eco-friendly alternatives to tre lead-polluted environments, particularly microbial-based technologies that promote t sustainability of the treatment process. Microorganisms 2023, 11, 2708 Research focused on studying the microbial diversity of environments affected 3 of 24 heavy metal pollution is scarce, and even more so are studies that evaluate microbial versity associated with river mouth sediments in Colombia. Still, the few existing repo suggest an increased lead-polluted abundance environments, of metal-resistant particularly species microbial-based due tothat technologies challenging promote theconditio sustainability of the treatment process. [29]. Consequently, this study aimed to enhance the knowledge of Colombian microb Research focused on studying the microbial diversity of environments affected by diversity from underexplored environments such as coastal marine sediments. It a heavy metal pollution is scarce, and even more so are studies that evaluate microbial sought to evaluate diversity the with associated potential of isolated river mouth microorganisms sediments to withstand in Colombia. Still, various hea the few existing metals, withsuggest reports a special emphasis an increased on leadofremoval. abundance The results metal-resistant species of duethis work contribute to challenging globalconditions efforts to[29]. Consequently, broaden this study aimed our knowledge to enhance about the knowledge microbial of Colombian biodiversity from marine a microbial diversity from underexplored environments such as coastal marine sediments. coastal environments and evaluate its ability to bioremediate widespread pollutants su It also sought to evaluate the potential of isolated microorganisms to withstand various as heavy heavymetals. metals, with a special emphasis on lead removal. The results of this work contribute to global efforts to broaden our knowledge about microbial biodiversity from marine and 2. Materials and Methods coastal environments and evaluate its ability to bioremediate widespread pollutants such as heavy metals. 2.1. Study Area and Sample Collection 2. Materials Three and Methods sampling zones (eight total sampling sites) in the Chocó region of Colomb 2.1. Study Area and Sample Collection were selected for collecting sediment samples (Figure 1). These sites experience anthrop Three sampling zones (eight total sampling sites) in the Chocó region of Colombia were genic influence due to the discharge of untreated wastewater and alluvial gold mini selected for collecting sediment samples (Figure 1). These sites experience anthropogenic [30–32]. Threedue influence sampling sites were to the discharge locatedwastewater of untreated in estuarine andareas ingold alluvial the mining southern Atlantic p [30–32]. tion of thesampling Three Colombian Caribbean sites were located in Sea, i.e., inareas estuarine the in Atrato River Atlantic the southern mouth portion (Gulf of Urabá) (F of the ure 1,Colombian left). Sampling CaribbeansiteSea, details i.e., inare thegiven Atratoin Table River 1. The mouth remaining (Gulf sampling of Urabá) (Figure zones we 1, left). Sampling site details are given in Table 1. The remaining sampling located to the north of the Colombian Pacific Sea in the San Juan River mouth and Bau zones were located to the north of the Colombian Pacific Sea in the San Juan River mouth and Baudó River Rivermouth mouth (Figure 1, right). (Figure 1, right). FigureFigure 1. Sampling sites 1. Sampling sitesfor forthe the collection collection ofof estuarine estuarine sediments sediments in the Colombian in the Colombian South Atlan South Atlantic (left) and (left) North Pacific and North Sea Pacific Sea(right). Mapcreated (right). Map created in the in the Information Information Services Services Laboratory Laboratory (LABSIS) at(LABSIS Marine Marine and Coastal and Coastal ResearchInstitute Research Institute ‘José ‘JoséBenito Vives Benito de Andréis’ Vives (INVEMAR) de Andréis’ using the using (INVEMAR) ArcGISthe ArcG 10.8 software. 10.8 software. Microorganisms 2023, 11, 2708 4 of 24 Table 1. Sampling site location and physicochemical parameters of surface water. Sampling Site Salinity Locality Sampling Site Latitude Longitude Depth (m) Temperature (◦ C) pH Abbreviation (ppt) Margarita Atrato MARM N 8◦ 80 47.05200 W 76◦ 500 8.33700 2.3 27.7 0.2 6.2 river mouth Atrato Mangrove Paila MPAR N 8◦ 10 5100 W 76◦ 500 5100 1.1 28.0 7.5 6.5 river mouth Atrato river Mangrove Roto MRAR N 8◦ 120 42.35100 W 76◦ 560 33.55200 3.2 27.6 0.0 6.2 Atrato river Mangrove via Usaraga MVUBR N 4◦ 560 38.400 W 77◦ 210 48.600 0.5 27.1 7.2 6.2 Baudó Baudó river river mouth Out to sea OBR N 4◦ 560 19.900 W 77◦ 220 55.100 7.6 29.7 6.5 6.4 Baudó river Mangrove Choncho San Juan MCHSJ2 N 4◦ 20 56.200 W 77◦ 270 03.100 4.0 25.9 0.3 6.3 river two San Juan Mangrove San Juan river mouth MSJ2 N 4◦ 20 34.500 W 77◦ 260 05.100 4.0 26.2 3.1 6.4 river two Beach San Juan BSJM2 N 4◦ 10 31.200 W 77◦ 260 24.100 4.5 25.6 1.5 6.9 river mouth two A total of 200 g of sediments were collected from each sampling site using Van Veen dredges with a surface of 0.08 m2 . The sediments were stored in Whirl-Pak® sterile sample bags and maintained at 4 ◦ C until they were processed in the Marine Bioprospecting Laboratory of the Marine and Coastal Research Institute ‘José Benito Vives de Andréis’ (INVEMAR). Additionally, physicochemical parameters (temperature, salinity, and pH) were measured in the surface water of each sampling site. 2.2. Microorganism Isolation and Culture Microorganisms were isolated in Petri dishes using different culture media including starch casein agar (SCA), International Streptomyces project 2 (ISP2) medium, ISP4 medium, ISP3 medium, marine agar (MA), tryptic soy agar (TSA), Actinobacteria agar (AA) Boyd and Kohlmeyer (B&K) medium, malt extract agar, and Rose Bengal Chloramphenicol agar (RBC). Components of all culture media are shown in (Table S1). Three isolation methodologies were followed for sediment processing: (1) a selective pressure method using mercury chloride (HgCl2 ) to isolate mercury-resistant strains, (2) a phenol pretreatment to isolate sporulated microbes, and (3) a direct culture of non-pretreated sediments. For the first method, 5.0 g of each sediment sample was mixed with 10 mL of Luria Bertani-LB broth [33] and supplemented with 5.0 mg·L−1 of mercury chloride. The test tubes were incubated in a Scientific Industries SI™ Roto-Shaker for up to 60 min at room temperature (22 ◦ C) [34]. For the phenol pretreatment, a total of 20 g of each sediment sample was added to 100 mL of 1.5% (w/v) phenol [35]. The mixture was incubated in an orbital shaker at 140 rpm and 30 ◦ C for 60 min. Lastly, a sterile cotton swap was used to directly inoculate non-pretreated and pretreated sediments on the surface of different culture media (Table S1). After inoculation, plates were incubated at 25–30 ◦ C in the dark for three to five days. In order to obtain individual colonies, serial dilutions 1/10 and 1/102 were carried out in test tubes containing 9.0 mL of dilution water, and aliquots of 0.1 mL were inoculated via spread plating in the culture media. Obtained isolates were phenotypically characterized and the cultivable strains were quantified by means of the colony forming unit by gram (CFU·g−1 ). The obtained axenic cultures were cryopreserved at −80 ◦ C. All microorganisms were deposited in the culture collection of the Marine Natural History Museum of Colombia –Makuriwa located in INVEMAR, with consecutive numbers ranging from 76844 to 77182. Once microorganisms were identified, they were assigned a catalog number (i.e., INV PRT 211, INV PRT212, INV PRT213, INV PRT215, and INV PRT216). Microorganisms 2023, 11, 2708 5 of 24 2.3. Cadmium (Cd2+ ), Lead (Pb2+ ), and Zinc (Zn2+ ) Tolerance Screening A qualitative heavy metal tolerance screening was performed by exposing the mi- croorganisms to varying concentrations of Cadmium (Cd2+ ), Lead (Pb2+ ), and Zinc (Zn2+ ). Tolerance screening was performed using 276 selected microorganisms out of 338 isolated colonies. The selection was based on two criteria (a) microorganisms exhibited a rapid growth after 24 to 48 h of incubation, and (b) colonies had a creamy appearance that allowed for easy manipulation and culture. Screened microorganisms (n = 276) included 21 yeast and 255 bacteria. This tolerance screening was performed via the agar diffusion technique following the modified methodology previously reported [36]. Briefly, massive culturing of the microor- ganisms was carried out on Luria Bertani agar (LB) marked with non-perforated equidis- tant wells (diameter = 9 mm). Liquid standard solutions (Certipur® Merck, Darmstadt, Germany) of the heavy metals nitrate salts (i.e., Cd(NO3 )2 , Pb(NO3 )2 , and Zn(NO3 )2 , adjusted at a pH 6.0, 5.0 and 6.0, respectively) were placed on the marked wells at concen- trations of 50, 150, 250, and 350 mg·L−1 . Inoculated plates were incubated at 30 ◦ C for 48 h. All experiments were carried out in triplicate. A microorganism was catalogued as tolerant (+) if growth was observed after 24–48 h of culture; not tolerant (−) if growth was inhibited entirely around wells, or sensitive (±) if some colonies were formed [36]. Microorganisms exhibiting growth at the maximum concentration level (i.e., 350 mg·L−1) of all tested heavy metals were subjected to further tolerance screening trials at increased concentrations of Pb2+ , Cd2+ and Zn2+ ions. Lead and zinc were evaluated at 450, 550, 650, and 750 mg·L−1 , while Cd2+ ion was evaluated at 550 and 750 mg·L−1 . 2.4. Molecular Identification of Promising Bacteria Microorganisms (n = 6) that showed appropriate growth at a concentration of 750 mg·L−1 of cadmium, lead, and zinc during the qualitative tolerance screening were selected for taxonomic classification. Briefly, the bacterial DNA was extracted using the Ultraclean Tissue & Cells (MoBio Laboratories Inc., Carlsbad, CA, USA) and Wizard® Genomic DNA purification (Promega, Madison, WI, USA) kits while following the manufacturers’ instructions. DNA purity was determined via a NanoDrop 2000 UV-Vis spectrophotometer (ThermoFisher Scientific™, Waltham, MA, USA). The amplification of the bacterial 16S rRNA gene was carried out via PCR reactions in a BioRad T100™ thermal cycler (Hercules, CA, USA) with a final volume of 25 µL, which contained the universal primers (0.5 µL) 27F (5’ AGA GTT TGA TCM TGG CTC AG 3’)-(0.5 µL) 1492R (50 -GGT TAC CTT GTT ACG ACT T-30 ), a 1X polymerase buffer (5.0 µL), 12.0 µL of sterile MilliQ water, 0.5 µL of Taq polymerase (Bioline, Memphis, TN, USA), and 6.0 µL of DNA [37,38]. The PCR products amplification was verified by means of horizontal electrophoresis in 1% agar gel, using the GelRed® (Biotium, Inc., Hayward, CA, USA) revealing reagent and a molecular weight marker of 50 pb (HyperLadder™—Bioline, London, UK). The gels were documented via UV light exposure in the ENDURO™ GDS (Edison, NJ, USA)—LABNET digital capture system. The amplified products were sent to Macrogen (Seoul, Korea) for sequencing using the Sanger/capillary method. The final sequences were analyzed and compared with those available in the Refseq_rna (Reference RNA Sequences) database of the National Center for Biotechnology Information (NCBI, https://www.ncbi.nlm.nih.gov/ accessed on 8 September 2023). Alignment was performed using MUSCLE (Multiple Sequence Comparison by Log-Expectation). Phylogenetic analysis was conducted in the MEGA-X software, version 10.0.5, using the maximum likelihood test with 1000 Bootstrap replicates and based on the Kimura 2 evolution model, with a Gamma distribution (G) and six discrete Gamma categories. The 16S rRNA gene sequences were deposited in GenBank database with the accession numbers OR557202, OR557203, OR557204, OR557205 and OR557206. The bacteria with consecutive number 77050 could not be identified due to low-quality of the 16S rRNA gene sequence. Microorganisms 2023, 11, 2708 6 of 24 2.5. Viability Test of Promising Bacteria Three bacteria, identified as Enterobacter sp. INV PRT213, Pseudomonas sp. INV PRT215, and Stenotrophomonas sp. INV PRT216 were selected to verify their viability through a resazurin test. This test was performed in 96-well plates and evaluated concentrations up to 500 mg·L−1 of the three metal ions. To this effect, the bacteria were cultivated in a LB medium supplemented with Cd2+ , Pb2+ and Zn2+ salts at increasing concentrations of 10, 50, 100, 150, 200, 250, 350, 450, and 500 mg·L−1 . Growth controls were prepared, including (1) the microorganism without metal, (2) the no inoculated LB medium (abiotic control), and (3) the metal solutions (abiotic control). The plates were incubated at 30 ◦ C for 72 h in a wet chamber to avoid evaporation. A volume of 2.0 µL of resazurin was added to each well (0.4 mg·L−1 ), and spectrophotometric readings were performed at 570 and 603 nm [39,40]. The viability percentage was calculated following Equation (1): ( A LW − ( A HW × Ro) treated sample) Viability(%) = (1) ( A LW − ( A HW × Ro) growth control) where: ALW = Absorbance to the lowest wavelength minus the blank medium AHW = Absorbance to the highest wavelength minus the blank medium A LW Correction factor (Ro) = A HW 2.6. Bacterial Growth Curves of Promising Isolates at Different Pb2+ Concentrations Liquid cultures of three bacterial isolates (Enterobacter sp. INV PRT213, Pseudomonas sp. INV PRT215 and Stenotrophomonas sp. INV PRT216) that remained viable at 500 mg·L−1 of Cd2+ , Pb2+ y Zn2+ were completed in 100 mL flasks (25 mL working volume). Flasks contained LB broth at a pH of 5.0, supplemented with concentrations of 0 (control), 10, 100, 200, 300, and 400 mg·L−1 of the metal (Pb(NO3 )2 ). A volume of 1 mL of the inoculum adjusted to the 0.5 McFarland scale was added to each culture, and the flasks were incubated at 30 ◦ C in an orbital shaker-incubator (Thermo Scientific™, Waltham, MA, USA) at 140 rpm for 144 h [41]. Growth was measured at 0, 24, 48, 72, 96, 120, and 144 h, using 300 µL aliquots to measure the optical density (OD600nm ) with a Thermo Scientific™ spectrophotometer and the Multiskan Go software version 4.1. The assays were carried out in triplicate. 2.7. Pb2+ Removal Experiments Experiments were carried out to evaluate the potential of three bacterial isolates (Enterobacter sp. INV PRT213, Pseudomonas sp. INV PRT215, and Stenotrophomonas sp. INV PRT216) to remove lead from an aqueous solution. Lead (Pb2+ ) removal trials were performed at three initial wet bacterial biomass values (8.3, 16.7, and 25.0 g·L−1 ) and two ex- posure periods (120 and 240 min). Experiments were completed in 100 mL flasks containing 60 mL of LB broth (pH = 5.0, adjusted with 0.5 M nitric acid) and supplemented with 400 mg Pb2+ ·L−1 (as Pb(NO3 )2 ). Flasks were incubated in an orbital shaker (Thermo Scientific™) at 30 ◦ C and 160 rpm and sampled after 120 and 240 min of exposure. Abiotic controls of the solution (400 mg·L−1 Pb2+ ) were included and incubated under the same experimental conditions. All assays were carried out in triplicate. After incubation, flask contents were centrifuged at 4000 rpm and 4 ◦ C for 45 min (Hettich Universal 320 centrifuge) to separate biomass. The supernatants were acidified with a 65% HNO3 solution (Merck® ) until a pH ≤ 2 was reached and stored at 4 ◦ C until quantification of the total Pb2+ (mg Pb·L−1 ) in the liquid phase. The extent of Pb2+ metal ions removal was quantitatively evaluated via air-acetylene direct flame atomic absorption spectrophotometry (AAS) [41]. The samples were processed following the SM 3030E and SM 3111B methods from [42]. An AT Thermo S Series iCE-330 spectrophotometer was implemented to analyze the samples using a calibration curve Microorganisms 2023, 11, 2708 7 of 24 based on the standard addition method. The data were recorded as the mean plus the standard deviation [41]. The total Pb2+ removal percentage (R%) was calculated using Equation (2) [43]: Ci − Cf R (%) = × 100 (2) Ci where: Ci : Pb (II) concentration in the abiotic control Cf : final concentration of Pb (II) in cell-free supernatants 2.8. Fourier Transform Infrared (FT-IR) Spectroscopy Analysis of Bacterial Biomass A Fourier transform infrared (FT-IR) spectroscopy analysis aimed to determine the possible cell surface changes associated with Pb2+ removal capacity [44]. For this purpose, 2 mL aliquots were taken from experimental units showing the highest removal percentage. These samples were centrifuged at 4000 rpm and 4 ◦ C for 40 min. The resulting biomass was washed four times with a saline solution at 0.8% (w/v) and concentrated in a Speedvac Thermo Scientific™ (Waltham, MA, USA) SPD111V concentrator for 4 h at 80 ◦ C. The dry biomass was macerated in a mortar until a fine powder was obtained and mixed with anhydrous potassium bromide (KBr) at a proportion of 1:100 to obtain a pellet of the mix through a pellet press (PIKE). The biomass cultivated without Pb2+ salt was used as a negative control. A Shimadzu infrared spectrophotometer (IR-TRACER-100) (Kyoto, Japan) with a DLATGS detector (with temperature control for the far/medium infrared) was implemented to obtain spectra in the 400–4000 cm−1 wavenumber range, with 28 scans and a 4 cm−1 resolution. 2.9. Statistical Analysis The data are presented as average values, with bars representing their standard deviation. A descriptive analysis of the data was conducted. These data corresponded to the number of strains isolated in each sampling site and those that tolerated the different heavy metals, and they were presented as percentages (%). A nonlinear regression was performed using four parameters in the RStudio statistical software version 4.3.3 [45] to calculate the maximum concentration at which the strains remained 80% viable in the presence of the metals. The removal percentages were compared through a one- or two- way analysis of variance (ANOVA) following the methodology described by [46] and using the Statistics Kingdom online free software (https://www.statskingdom.com/ accessed on 31 May 2023). 3. Results 3.1. Study Area and Sample Collection Signs of pollution were observed in all locations, including the presence of foam and solid waste such as various plastics, food packaging, and textiles, among others. The environmental conditions of the sampling sites were influenced by the rainy season of the Chocó region. The shades of color in the surface waters were light brown, dark brown, and greyish green. However, surface water samples were odorless except for those at MVUBR and MCHSJ2 sites, which reported a slight sulfur and a woody odor, respectively. The physicochemical parameters of the surface water in the sampling sites are shown in Table 1. The salinity oscillated between 0 (below detection limit) and 7.5 ppt with the MPAR (7.5), MVUBR (7.2), and OBR (6.5) sites in the Atrato and Baudó river mouths exhibiting the highest values. In contrast, MRAR (0.0), MARM (0.2), and MCHSJ2 (0.3) collection sites in the Atrato and San Juan River mouths showed the lowest salinity values. Typical values for estuarine salinity range from 0.5 to 17 ppt. The salinity of brackish water exhibits a wide range since it can rapidly change depending on tides, weather, and other factors [47]. Most of the salinity values in this study fell within the typical range, while some of the observed lower values can be attributed to dilution events during the rainy season. The Microorganisms 2023, 11, 2708 8 of 24 surface water temperature oscillated between 25.6 and 29.7 ◦ C. Collected sediments from all sites were silt and clay with a dark brown color and collected from depths ranging from 0.5 to 7.6 m. 3.2. Microorganism Isolation and Culture A total of 338 strains were isolated from collected sediment samples, comprising 67 fungi and 271 bacteria. Phenotypical characterizations revealed bacterial colonies of various morphologies such as circular, filamentous, and irregular shapes, along with smooth, glistening, and rough surfaces. The elevations ranged from raised to convex, flat, umbonate, and crateriform. The edges of the colonies exhibited entire, undulate, curled, and lobate features, and they had creamy, mucilaginous, or dry consistencies. Additionally, some isolated samples showed pigmented colonies and the production of extracellular pigments, as well as iridescent colonies with pulverulent consistency. As for the isolated fungi, culture examinations showed cottony fluffy and filamentous colonies in addition to yeast forms (Figure S1). Regarding microscopic characteristics, three distinct bacteria shapes were observed: Gram-positive and Gram-negative bacilli, Gram-positive filamentous bacteria, and Gram-positive cocci. On the other hand, hyaline septate and non-septate fungal hyphae and yeast forms were observed for the fungal strains. Among the total isolated microorganisms, 137 strains (40.5%) were retrieved from the Atrato River mouth, 128 (37.9%) from the San Juan River mouth, and 73 (21.6%) from the Baudó River mouth (Figure 2a). It is important to note that the lower number of isolates in the Baudó region can be due to the fewer collection sites in this area (i.e., two sampling sites) compared to other collection stations (i.e., three sampling sites). Zone inaccessibility was the main reason for the discrepancy. The non-pretreated sediments allowed for the highest recovery of cultivable microbes, totaling 146 strains (43.2%). Among these, 62 were isolated from the San Juan River mouth, 52 from the Atrato River mouth, and 32 from the Baudó River mouth. These results were expected as no stressors restricted microbial growth, allowing for a higher proliferation rate of vegetative cells. On the other hand, challenging conditions, such as those imposed by the phenol pretreatment, resulted in lower isolate numbers (50 isolates, representing 14.8% of total strains). Phenol-treated sediments selectively supported sporulated slow-growth organisms like Actinomycetota, which are frequently outpaced by vegetative cells during solid culture. These microorganisms were mainly obtained from the Atrato and Baudó regions, as microbial growth was not detected (<1 CFU·g−1 ) in phenol-pretreated samples from two San Juan River mouth sites. Regarding mercury chloride pretreated samples, 142 cultivable microorganisms (42%) were obtained from the sampling sites: 63, 58, and 21 from the Atrato, San Juan, and Baudó river mouths, respectively. It is possible that the mercury pretreatment represented a milder pressure condition that may more closely resemble the natural environment from which these microorganisms were isolated, given that the sampling areas were characterized by the presence of mercury resulting from illegal mining processes [22,30]. Culture media such as SCA, ISP4, and MA strongly supported the isolation of microor- ganisms, accounting for 57.7% (n = 195) of the total isolates in this work. Notably, the SCA medium accounted for 25.4% (n = 86) of the recovered cultivable strains. The SCA culture media formulation contains complex carbon sources such as starch and a high salt content. This composition could promote the development of several microorganisms, which may explain the higher number of isolates obtained using this medium. Marine agar was the most suitable culture medium for colonies with a creamy consistency, pigmentation, or other interesting phenotypic characteristics, constituting 19.5% (n = 66) of the total isolates. The remaining 13.6% (n = 46) strains were isolated from ISP2, ISP3, Actinobacteria agar (AA), RBC, TSA, malt extract, and Boyd and Kohlmeyer (B&K) medium (Figure 2b). Microorganisms 2023, 11, x FOR PEER REVIEW Microorganisms 2023, 11, 2708 9 of 24 2. Number FigureFigure of isolates 2. Number from sediment of isolates samples samples from sediment collected collected from marine environments. from marine environm (a) Sediment processing method and (b) Culture medium. ment processing method and (b) Culture medium. 3.3. Cd, Pb, and Zn Tolerance Screening HeavyThe metalnon-pretreated tolerance screening sediments was performedallowed for selected using 276 the highest recovery of cu microorganisms out ofcrobes, 338 isolated colonies. totaling The selection 146 strains (43.2%).wasAmong based on rapid62 these, growth were and suitability isolated from the Sa for spectrophotometric measurements. Consequently, some mycelial fungi mouth, 52 from the Atrato River mouth, and 32 from the Baudó River mouth. T and Actino- mycetota were excluded due to cell aggregate formation that interfered with reproducible were expected as no stressors restricted microbial growth, allowing for a hig data collection. ation rate Screened of vegetative microorganisms cells.21On included the yeast andother hand, (Table 255 bacteria challenging conditions, S2). Tolerance to su imposed by the phenol pretreatment, resulted in lower isolate numbers (50 the metals was observed in microorganisms with different morphologies, consistencies, and textures (creamy and resenting 14.8%sporulated). of totalSome bacteria strains). excreted mucilaginous Phenol-treated sediments substances in the supp selectively presence of 150–350 mg·L−1 Cd2+ (76955) (Figure S2). In addition, the culture of microor- lated slow-growth organisms like Actinomycetota, which are frequently outp ganism 76955 excreted a brown pigment when exposed to Cd2+ and Zn2+ . These could be etative cells (EPS) exopolysaccharides during solid widely culture. reported These microorganisms as molecules excreted by differentwere mainly obtain organisms underAtrato and Baudó stress conditions regions, to chelate metalas microbial ions [48]. Mostgrowth evaluatedwas not detected organisms (<1 CFU·g− showed some level of tolerance tosamples pretreated at least one fromheavy twometal. SanOnly Juan five bacteria River (catalog mouth numbers sites. 76851, merc Regarding 76852, 76975, 76997, and 77085) exhibited complete inhibition at all concentration levels pretreated samples, 142 cultivable microorganisms (42%) were obtained fro of the three individually assessed heavy metal ions (Cd2+ , Pb2+ , and Zn2+ ) (Figure 3). It pling sites: 63, 58, and 21 from the Atrato, San Juan, and Baudó river mouths, It is possible that the mercury pretreatment represented a milder pressure co may more closely resemble the natural environment from which these mic were isolated, given that the sampling areas were characterized by the pres cury resulting from illegal mining processes [22,30]. the presence of 150–350 mg·L−1 Cd2+ (76955) (Figure S2). In addition, the culture of micro- organism 76955 excreted a brown pigment when exposed to Cd2+ and Zn2+. These could be exopolysaccharides (EPS) widely reported as molecules excreted by different organ- isms under stress conditions to chelate metal ions [48]. Most evaluated organisms showed Microorganisms 2023, 11, 2708 some level of tolerance to at least one heavy metal. Only five bacteria (catalog numbers 10 of 24 76851, 76852, 76975, 76997, and 77085) exhibited complete inhibition at all concentration levels of the three individually assessed heavy metal ions (Cd2+, Pb2+, and Zn2+) (Figure 3). It was was observedthat observed thatthe thesamples samplesisolated isolatedfrom fromthe theAtrato AtratoRiver River mouth mouth reported reported mostmost of of the the tolerant strains (n = 111), followed by those from the San Juan River mouth (n == 100) tolerant strains (n = 111), followed by those from the San Juan River mouth (n 100) and and the the Baudó BaudóRiver Rivermouth mouth(n(n= =61) (Figure 61) (Figure 3).3). A total of 121, A total 251,251, of 121, andand214 microorganisms 214 microorgan- displayed isms displayed colony development at some level of cadmium, lead, or respectively colony development at some level of cadmium, lead, or zinc, (Fig- zinc, respectively ure S3).S3). (Figure AmongAmongmicrobial groups, microbial groups,bacteria bacteria exhibited exhibitedhigher highertolerance toleranceto tothe the maximum maximum concentrations concentrations ofof Cd, Cd, Pb, Pb, and and Zn, Zn, with with 106 106 bacteria bacteriagrowing growingat at350 350mg mg·L ·L−−11 of of each each heavy heavy metal, metal, while only 4 yeasts (fungal) isolates developed colonies at this concentration for while only 4 yeasts (fungal) isolates developed colonies at this concentration for all all 33 metals. metals. Isolated Isolated yeasts yeastswere wereparticularly particularlysusceptible susceptible to cadmium, with only eight to cadmium, with only eight cultures cul- tures (38% (38% of fungal of fungal species) species) growing growing at concentrations at concentrations ≥ 150≥ 150 ·L−1−1 mgmg·L . .However, However,all all of of them (except yeast 77149) were tolerant to some level of lead and zinc. Figure 3. Number of tolerant isolates by sampling site. Tolerance screening was performed at vary- Figure 3. Number of tolerant isolates by sampling site. Tolerance screening was performed at varying ing concentrations (50, 150, 250, and 350 mg·L −1) of cadmium, lead, and zinc. A microorganism was concentrations (50, 150, 250, and 350 mg·L−1 ) of cadmium, lead, and zinc. A microorganism was considered tolerant if it exhibited growth at any concentration level of a given metal. considered tolerant if it exhibited growth at any concentration level of a given metal. Among these 110 microorganisms, eleven strains were selected based on growth rate Among these 110 microorganisms, eleven strains were selected based on growth and ease of handling (creamy colonies) (Table S3). Additional screening experiments al- rate and ease of handling (creamy colonies) (Table S3). Additional screening experi- lowed the identification of six microorganisms tolerant to concentrations of up to 750 ments allowed the identification of six microorganisms tolerant to concentrations of up to 750 mg·L−1 of Cd2+ , Pb2+ y Zn2+ ions. Five of these were isolated via direct swabbing (INV PRT212, INV PRT213, 77050, INV PRT215, and INV PRT216), and one (INV PRT211) resulted from the mercury chloride pretreatment. The six promising bacteria were subjected to molecular identification procedures (see Section 3.4). 3.4. Molecular Identification and Phylogenetic Analysis Six bacteria tolerant to concentrations of up to 750 mg·L−1 of Cd2+ , Pb2+ and Zn2+ ions were selected for molecular analyses and taxonomic classification. The sequencing data from one of the samples (bacteria 77050) did not meet the minimal quality requirements, making it impossible to assign this microorganism to a taxonomic group. The resulting phylogenetic tree comprised three clades, each representing species closely related to the evaluated microorganisms. These bacteria were classified within the phylum Pseudomonadota, previously denoted as Proteobacteria [49] (Figure 4). Based on the analysis of 16S rRNA sequences, we identified the genera Stenotrophomonas (INV PRT211 and INV PRT216), Enterobacter (INV PRT212 and INV PRT213), and Pseudomonas sp. INV PRT215. Identity percentages for these bacteria ranged between 97 and 99% when compared to the strains reported in the NCBI’s Reference RNA Sequences database (Table S4). Further experiments were conducted using three out of five molecularly identified microorganisms: INV PRT213, INV PRT215, and INV PRT216. The selection was based on rapid growth after 24 h of culture and isolation region: INV PRT213, INV PRT215, and INV PRT216 were isolated from the Atrato, San Juan, and Baudó regions, respectively. phylum Pseudomonadota, previously denoted as Proteobacteria [49] (Figure 4). Based on the analysis of 16S rRNA sequences, we identified the genera Stenotrophomonas (INV PRT211 and INV PRT216), Enterobacter (INV PRT212 and INV PRT213), and Pseudomonas sp. INV PRT215. Identity percentages for these bacteria ranged between 97 and 99% when compared to the strains reported in the NCBI’s Reference RNA Sequences database (Table Microorganisms 2023, 11, 2708 11 of 24 S4). Figure 4. Phylogenetic tree of five heavy-metal-tolerant bacteria isolated from marine sediments. The tree was constructed using the MEGA-X software, version 10.0.5. Based on the maximum likelihood analysis of the 16S rRNA sequences, the evaluated microorganisms were identified as Stenotrophomonas sp. INV PRT211, Enterobacter sp. INV PRT212, Enterobacter sp. INV PRT213, Pseudomonas sp. INV PRT215, and Stenotrophomonas sp. INV PRT216. 3.5. Viability Test of Enterobacter sp. INV PRT213, Pseudomonas sp. INV PRT215, and Stenotrophomonas sp. INV PRT216 Figure 5 presents the results of viability trials conducted on bacteria INV PRT213, INV PRT215, and INV PRT216 at increasing concentrations of Cd2+ , Pb2+ , and Zn2+ (ranging from 10 to 500 mg·L−1 ). Average viability percentages greater than 100% indicated that microorganisms exposed to the heavy metals showed a higher cell metabolic activity than their respective controls (cultures without heavy metal). This is a phenomenon widely observed in bacteria particularly in heavy-metal tolerant strains, which have shown to increase their division rate as a response to environmental stress [15]. The results indicated that the viability of INV PRT216, INV PRT213 and INV PRT215 remained unaffected by lead exposure at the concentrations assessed in this study. Zinc exposure did not impact cell viability for INV PRT216, but the other two bacteria showed decreased viability after exposure to 250 mg·L−1 Zn2+ . Cadmium had different effects on evaluated microorganisms: INV PRT213 displayed greater susceptibility to this metal, with viability being affected at approximately 35 mg·L−1 , while INV PRT215 exhibited effects at around 125 mg·L−1 and INV PRT216 at 250 mg·L−1 . increase their division rate as a response to environmental stress [15]. The results indicated that the viability of INV PRT216, INV PRT213 and INV PRT215 remained unaffected by lead exposure at the concentrations assessed in this study. Zinc exposure did not impact cell viability for INV PRT216, but the other two bacteria showed decreased viability after exposure to 250 mg·L−1 Zn2+. Cadmium had different effects on evaluated microorganisms: Microorganisms 2023, 11, 2708 INV PRT213 displayed greater susceptibility to this metal, with viability being affected at 12 of 24 approximately 35 mg·L−1, while INV PRT215 exhibited effects at around 125 mg·L−1 and INV PRT216 at 250 mg·L−1. Microorganisms 2023, 11, x FOR PEER REVIEW 13 of 25 Figure 5. Figure 5.Viability Viabilitypercentages percentages of bacteria INVINV of bacteria PRT213, INV PRT215, PRT213, and INV INV PRT215, andPRT216 at increasing INV PRT216 at increasing concentrations of cadmium (blue), lead (yellow), and zinc (black). Concentrations ranged from 10 concentrations of cadmium (blue), lead (yellow), and zinc (black). Concentrations ranged from 10 to to 500 mg·L−1 and curves were adjusted using a nonlinear regression model of four parameters. Dots 500 mg·L− represent 1 and curves were adjusted using a nonlinear regression model of four parameters. Dots average values of triplicate samples and bars represent standard deviation 3.6. Growth represent average values curves of promising isolatesof triplicate sp. Enterobacter samples and bars INV PRT213, representsp. Pseudomonas standard deviation INV PRT215, 3.6. Growth and Steno- trophomonas curves sp. INV PRT216 of promising at different isolates Pb+2 concentrations. Enterobacter sp. INV PRT213, Pseudomonas sp. INV PRT215, and Stenotrophomonas sp. INV PRT216 at different Pb2+ concentrations. Among heavy metal pollutants, lead presence in several environments is of particular concern in Colombia and other countries due to its detrimental effects on human health and ecosystems, even at minimal concentrations exceeding 0.30 µg m−3 [50–53]. Conse- quently, Pb2+ was selected for further evaluation during growth curve construction and removal experiments. 3.6. Bacterial Growth Curves of Promising Isolates at Different Pb2+ Concentrations concentrations up to 300 mg·L−1 Pb2+. It showed an exponential phase until ≈48 h of incu- bation, followed by a stationary phase, and a sharp decline at approximately 72 h (i.e., Microorganisms 2023, 11, 2708 13 of 24 death phase) (Figure 6). A similar trend was observed in the control culture without lead (0 mg Pb2+·L−1). Enterobacter sp. INV PRT213 Among demonstrated heavy metal pollutants, leadrobust presencegrowth in severalat all evaluated environments concentra- is of particular tions, reaching its maximum OD600nm concern in Colombia andafter 24 h. A due other countries slight to itsdecrease detrimentalineffects growth was health on human observed and ecosystems, even at minimal concentrations exceeding 0.30 µg m−3 [50–53]. Conse- between 48 and 72quently, h, afterPbwhich it remained 2+ was selected constant until 120 h of incubation (Figure 6). for further evaluation during growth curve construction and On the other hand, removalitexperiments. was evident that Stenotrophomonas sp. INV PRT216 was signifi- cantly affected by the presence of the metal, recording a maximum OD of 0.05 ± 0.09 at all 3.6. Bacterial Growth Curves of Promising Isolates at Different Pb2+ Concentrations Pb2+ concentrations. This represented an 18-fold reduction in OD compared to the control Figure 6 shows the growth curves of promising isolates at increasing Pb2+ concen- without metal (Figure 6). No trations. Thelagintolerance phase was detectedto lead, as evaluated for the suggested by these results, microorganisms, indicatingseemed their to contradict the findings adaptation to the culture obtained during media. Enterobacter tolerance sp. INV trials andPRT213 demonstrated viability robust growth tests. However, it was at all evaluated concentrations, reaching its maximum OD600nm after 24 h. A slight decrease hypothesized that cells present in the initial inoculum may have high affinity for the metal, in growth was observed between 48 and 72 h, after which it remained constant until 120 h effectively removing it from the of incubation growth (Figure media.sp. 6). Pseudomonas AsINV thePRT215 stressor concentration exhibited adequate growthdecreased, at lead it concentrations up to 300 mg · L −1 Pb2+ . It showed an exponential phase until ≈48 h of allowed the microorganism to divide, explaining the strong growth observed in agar incubation, followed by a stationary phase, and a sharp decline at approximately 72 h plates and the positive viability (i.e., death results. phase) (Figure 6). A similar trend was observed in the control culture without lead (0 mg Pb2+ ·L−1 ). Enterobacter sp. INV PRT213 1.4 1.2 Optical density (OD600nm) 1.0 0.8 0.6 0.4 0.2 0.0 0 20 40 60 80 100 120 Time (h) Pseudomonas sp. INV PRT215 1.4 1.2 Optical density (OD600nm) 1.0 0.8 0.6 0.4 0.2 0.0 0 20 40 60 80 100 120 Time (h) Figure 6. Cont. x FOR PEER REVIEW 15 of 25 Microorganisms 2023, 11, 2708 14 of 24 Stenotrophomonas sp. INV PRT216 1.4 1.2 Optical density (OD600nm) 1.0 0.8 0.6 0.4 0.2 0.0 0 20 40 60 80 100 120 Time (h) 2+ −1 Pb concentration (mg∙L ) 0 10 100 200 300 400 Figure 6. Growth curves of promising bacteria Enterobacter sp. INV PRT213, Pseudomonas sp. INV PRT215, and Stenotrophomonas sp. INV PRT216 at increasing lead concentrations. Dots represent Figure 6. Growth curves of promising bacteria Enterobacter sp. INV PRT213, Pseudomonas sp. INV average values of triplicate samples and bars represent standard deviation. PRT215, and Stenotrophomonas sp. INV PRT216 at increasing lead concentrations. Dots represent On the other hand, it was evident that Stenotrophomonas sp. INV PRT216 was signifi- average values of triplicate samples and bars represent standard deviation. cantly affected by the presence of the metal, recording a maximum OD of 0.05 ± 0.09 at all Pb2+ concentrations. This represented an 18-fold reduction in OD compared to the control 3.7. Evaluation of Pbwithout 2+ Removal Capacity metal (Figure of intolerance 6). The Pr omising Isolates to lead, Enterobacter as suggested by these sp. INV results, PRT213, seemed to contradict the findings obtained during tolerance trials and viability tests. However, it was Pseudomonas sp. INV PRT215, and Stenotrophomonas sp. INV PRT216 hypothesized that cells present in the initial inoculum may have high affinity for the metal, Heavy-metal effectively removalremoving experiment it fromresults the growthindicated that media. As the the wet stressor biomass concentration of Enterobac- decreased, it allowed the microorganism to divide, explaining the strong growth observed in agar plates ter sp. INV PRT213, Pseudomonas sp. INV PRT215, and Stenotrophomonas sp. INV PRT216 and the positive viability results. have different potential for treating lead -containing solutions (400 mg·L−1 Pb2+). Quantifi- 3.7. via Evaluation 2+ Removal Capacity of Promising Isolates Enterobacter sp. INV PRT213, of Pbabsorption cation of the total Pb atomic of the cell-free supernatants showed that Pseu- Pseudomonas sp. INV PRT215, and Stenotrophomonas sp. INV PRT216 domonas sp. INV PRT215 had the highest removal capacity among evaluated bacteria. (Fig- Heavy-metal removal experiment results indicated that the wet biomass of Enterobacter sp. ure 7). Average removal percentages INV PRT213, Pseudomonasranged from 37 sp. INV PRT215, andto 49%, reaching Stenotrophomonas thePRT216 sp. INV maximal values have dif- ferent potential for treating lead -containing solutions (400 mg · L −1 Pb2+ ). Quantification of at biomass concentrations ≥ 16.5 g·L and a contact time of 240 min. Shorter contact times −1 the total Pb via atomic absorption of the cell-free supernatants showed that Pseudomonas sp. with this microorganism INV PRT215 resulted in lower had the highest removal removal capacitypercentages among evaluated ofbacteria. around 36%.7).Statistical (Figure Aver- analysis confirmed that removal efficiency was mediated by the biomass concentration age removal percentages ranged from 37 to 49%, reaching the maximal values at biomass concentrations ≥ 16.5 g·L−1 and a contact time of 240 min. Shorter contact times with this (Tukey test, p < 0.05). A change in culture media coloration was observed when the bio- microorganism resulted in lower removal percentages of around 36%. Statistical analysis mass was exposedconfirmed to the that metal, i.e.,efficiency removal from pale yellowbyto was mediated theabiomass darker color. An concentration increase (Tukey test, in culture pH was also p < detected, 0.05). A change in culture reaching media final coloration values closewastoobserved 7.0 [54]. when the biomass was Enterobacter sp. INV exposed to the metal, i.e., from pale yellow to a darker color. An increase in culture pH PRT213 and Stenotrophomonas was also detected, sp.reaching INV PRT216final valuesexhibited close to 7.0lower average sp. [54]. Enterobacter removal percent- INV PRT213 ages than Pseudomonas sp. INV PRT215. and Stenotrophomonas sp. INVStenotrophomonas PRT216 exhibited lower sp. INV removal average PRT216 removalthan percentages results Pseudomonas sp. INV PRT215. Stenotrophomonas sp. INV varied from 0 to 15%, with the maximum value achieved at a wet biomass dosage −of1 16.5PRT216 removal results varied from 0 to 15%, with the maximum value achieved at a wet biomass dosage of 16.5 g·L g·L−1 after 240 min after of culture. 240 min As previously of culture. notednoted As previously for the Pseudomonas for the strain, Pseudomonas strain, pHpH increased increased during culture to values around 7.0, but no changes in the LB medium’s coloration were observed. Viability results (Section 3.5) indicated that this microorganisms’ growth was largely affected by lead; therefore, we hypothesized that observed removal percentages Microorganisms 2023, 11, 2708 15 of 24 during culture to values around 7.0, but no changes in the LB medium’s coloration were x FOR PEER REVIEW observed. Viability results (Section 3.5) indicated that this microorganisms’ 16 of 25was growth largely affected by lead; therefore, we hypothesized that observed removal percentages can be due to Pb attachment to dead cells. Figure 7. Average lead Figure removal percentages 7. Average + for bacteria Enterobacter sp. INV PRT213, Pseudomonas lead removal percentages + for bacteria Enterobacter sp. INV PRT213, Pseudomonas sp. sp. INV PRT215, and Stenotrophomonas sp. INV PRT216. INV PRT215, and Stenotrophomonas sp. INV Removal experiments PRT216. Removal werewere experiments performed performedatat an an initial Pb2+ concentration of2+400 initial Pb mg·L−1, 30 concentration °C,mg of 400 ·L−RPM, 140 1 , 30 ◦ C, 140 RPM, and an initial pH value of 5.0. Values and an initial pH value of 5.0. Values representexperimental represent differences between differences between runs experimental runs and no and no inoculum inoculum Statistical controls. controls. Statistical comparisons comparisons were made within columns. Means sharing a letter were not statistically different (Tukey test, were made within columns. Means sharing a letter were not statistically different (Tukey test, p < p < 0.05). 0.05). As for Enterobacter sp. INV PRT213, average removal percentages varied from 10 to 26%. Thesp. 3.8. Analysis of Pseudomonas highest INVremoval PRT215 percentages Biomasswere Viaachieved InfraredatSpectroscopy (FT-IR)of 25 g·L−1 biomass concentrations regardless of contact time. No changes in the culture medium coloration were observed, The IR spectra for andthe the metal-free bacterial final pH showed a slight biomass (Pseudomonas increase reaching a value of sp. INV around 6.0.PRT215) as well as those obtained after Pb2+ removal trials are shown in Figure 8. The absorbance 3.8. Analysis of Pseudomonas sp. INV PRT215 Biomass via Infrared Spectroscopy (FT-IR) widebands for the extension vibrations of the hydroxyl and amine groups were located The IR spectra for the metal-free bacterial biomass (Pseudomonas sp. INV PRT215) as between 3433 and 3294 cm−1. The bands located between 2927 and 2850 cm−1 are symmet- well as those obtained after Pb2+ removal trials are shown in Figure 8. The absorbance rical and asymmetrical extensions widebands for theof the C-H extension bond attributed vibrations to alkyl of the hydroxyl chains and amine [54].were groups There located −1 . The bands located between 2927 and 2850 cm−1 are symmet- were also intense bands attributed to the stretching vibrations of carbonyl (C=O) of the between 3433 and 3294 cm rical and asymmetrical amide functional groups at 1654 cm−1, asextensions well as at of the C-H bond 1543 cm−1 forattributed to alkyl chains [54]. There carboxylates (COO-) [54]. were also intense bands attributed to the stretching vibrations of carbonyl (C=O) of the On the other hand, the peaks amide located functional at 1238 groups at 1654cm cm−indicated −1 vibrations 1 , as well as at associated 1543 cm−1 for with carboxylates the[54]. (COO-) possible presence of On -SO 3 groups. the other hand,Finally, the peaks there were located signs at 1238 cm ofindicated − 1 the tension vibrations vibrations associatedofwith the possible presence of -SO groups. Finally, there were phosphate groups at 1080 cm , as well as 3of−1those corresponding to the sulfur–oxygen −1 signs of the tension vibrations of phosphate groups at 1080 cm , as−1well as of those corresponding to the sulfur–oxygen bond (S-O) of organicbond sulphate groups at 709 cm (Table 2) [54]. (S-O) of organic sulphate groups at 709 cm−1 (Table 2) [54]. Table 2. Changes in the functional groups present in the cell membranes of strains treated with and without Pb2+. Wavenumber Group Wavenumber (cm−1) cated in the 3433–3294 cm−1 range, corresponding to the hydroxyl and amine groups, ex- perienced a change in intensity after the addition of the metal ion, possibly due to com- plexing between the metal and these functional groups [58]. It was also evidenced that, regardless of the time (Figure 8a) and biomass concentration (Figure 8b), the changes al- ways Microorganisms took 2023, 11, 2708place within the same functional groups. 16 of 24 (a) (b) Figure 8. FT-IR analysis of Pseudomonas sp. INV PRT215 biomass in heavy-metal-free cultures and Figure 8. FT-IR analysis exposedoftoPseudomonas lead. Changessp. INV PRT215 associated with thebiomass inbetween interaction heavy-metal-free cultures bacterial biomass and of Pseudomonas exposed to lead. Changes associated with the interaction between bacterial biomass of Pseudomonas sp. INV PRT215 and lead ions: (a) same amount of biomass, different times; (b) same contact time, sp. INV PRT215 and lead ions: different (a)concentration. biomass same amount of biomass, different times; (b) same contact time, different biomass concentration. Table 2. Changes in the functional groups present in the cell membranes of strains treated with and without Pb2+ . Wavenumber Group Wavenumber (cm−1 ) References (cm−1 ) Functional after Lead Removal 3433 and 3294 Hydroxyl groups 3263–3140 1080 Phosphate 1118 1780–1640 (C=O) of amides 1658–1539 [54–57] 1543 Carboxylate (COO-) 1519 1238 SO3 1045 Microorganisms 2023, 11, 2708 17 of 24 Close inspection of Figure 8 showed some differences in biomass spectra of Pseudomonas sp. INV PRT215 exposed to a Pb2+ solution. These changes were attributed to interactions of cellular components with the metal ion, which could be described as follows. (1) The carbonyl groups (C=O) of amides likely present in the cell membrane decreased consider- ably [54]. (2) The band located at 1543 cm−1 , which corresponds to the carboxylates (COO-), exhibited a decrease in intensity, possibly due to the biosorption of the metal ion [54]. (3) The decrease in the transmittance percentage at 1080 cm−1 could be attributed to the interaction between the metal ion with phosphate groups [54]. (4) The wideband located in the 3433–3294 cm−1 range, corresponding to the hydroxyl and amine groups, experienced a change in intensity after the addition of the metal ion, possibly due to complexing between the metal and these functional groups [58]. It was also evidenced that, regardless of the time (Figure 8a) and biomass concentration (Figure 8b), the changes always took place within the same functional groups. 4. Discussion The Chocó region in Colombia is considered a biodiversity hotspot located between the Caribbean and the Pacific Ocean. This area is geographically isolated from the Ama- zon basin by the Andes Mountain range, and it harbors one of the largest numbers of endemic species and abundant water and mineral resources. However, reports evaluating its microbial diversity are scarce. Within this region there are three important national water bodies: the Atrato, San Juan, and Baudó Rivers [22]. Unfortunately, several reports have alerted the increasing presence of various serious contaminants in these rivers, including heavy metals, hydro- carbons, pesticides, and others. Heavy metal pollution is of particular concern as even small concentrations of these substances have shown to impact human health and disrupt metabolic processes throughout the food chain [22,59]. The introduction of heavy metals such as cadmium (Cd) and lead (Pb) has been primarily associated with the disposal of tailings from gold mining [60], while the presence of zinc (Zn) has been linked to runoff of superphosphate and other phosphorate fertilizers [61]. In this study, sediment samples were collected from the Atrato, San Juan, and Baudó rivers mouths and cultivable microor- ganisms were isolated from these remote and underexplored ecosystems. The isolated yeast and bacteria were assessed for their ability to tolerate heavy metals of interest (Cd, Pb and Zn), and promising organisms were further evaluated for their potential to remove lead from liquid solutions. Based on the physicochemical parameters of the sampling sites, the salinity values correspond to brackish waters, indicating oligohaline zones due to the influence of the sea on the rivers [47]. The lower salinity values at some sampling points can be attributed to the influx of freshwater from the rivers, which was greatly influenced by the rainy season. The pH values of the surface water samples suggest a zone of freshwater influence, which tends to be more neutral compared to the expected ocean pH (around 8.1). However, in line with the quality criteria established by Colombian government agencies for the preservation flora and fauna [62], the pH levels at the sampling sites align with those found in marine and estuarine waters. Classical microbiology approaches allowed for the isolation and characterization of microorganisms from the different sediment samples. The cultivable microorganisms included fungi and bacteria, while microalgae and other microorganisms were not isolated in this study. Despite using typical media for fungal isolation, such as B&K, malt extract agar, and RBC agar, the majority of the isolates were bacteria. Moreover, these media were supplemented with antibacterial molecules; therefore, bacterial growth indicated that there were antibiotic-resistant microorganisms in the sampled environments. These findings are consistent with numerous marine biodiversity studies that have reported a higher prevalence of cultivable bacteria compared to other microorganisms [63,64]. This is not unexpected, as there are still significant knowledge gaps regarding the appropriate culture of marine microorganisms, particularly of marine fungi development in laboratory Microorganisms 2023, 11, 2708 18 of 24 settings [65–67]. Therefore, alternative approaches, like metagenomic analysis of the environmental DNA, could prove invaluable in elucidating the microbial diversity within the collected samples. A greater number of cultivable strains, exhibiting different morphological characteris- tics were obtained from sampling points near mangrove zones, with isolate counts ranging from 39 to 55. This was in contrast to sampling points closer to the open sea (OBR. n = 32) or a beach (BSJM2, n = 34). Mangrove ecosystems, typically found along the coastal margins and brackish waters of coastal estuaries, provide complex nutrients (organic matter) and more favorable conditions for microbial colonization [68]. This favorable environment could explain the higher number of isolates from these areas. Tolerance screening revealed that the majority of isolated microorganisms were capable of growing in at least one heavy metal at the levels assessed in this study (50–750 mg·L−1 ). Only five microorganisms did not exhibit colony development in any tolerance trials. It has been reported that gold mining occurs upstream of the Atrato, San Juan, and Baudó rivers, with wastewater influence downstream [22,31,32]. Therefore, it is possible that the presence of inorganic pollutants, such as heavy metals, in the collected sediments may have alter microbial metabolism and community structures [69], thus fostering the development of heavy metal-resistant strains in the selected sampling points. The isolated microorganisms demonstrated a higher susceptibility to cadmium than to lead and zinc. Specifically, 64 microorganisms were completely inhibited by cadmium, while only nine were affected by all evaluated levels of Pb and Zn. Higher toxicity of cadmium compared to other heavy metals has been previously reported for several marine- derived microorganisms [70–72]. Authors argued that the ability of bacteria to tolerate different concentrations of heavy metals depends on many factors, including their taxo- nomic genus, metabolic abilities, growth conditions, and growth phase [73]. For instance, some studies have indicated that culture conditions, such as pH and salinity levels, play crucial roles in expressing cadmium resistance genes [74]. Metal-tolerant microorganisms can induce different genomic responses to avoid disrupting metabolic activity and prevent irreversible cell damage caused by heavy metals [75]. One of these defense mechanisms involves the production of protective mucilaginous substances in response to the metal presence, as widely described in the literature [48,76]. For instance, studies conducted in [76] reported the production of exopolysaccharides (EPS) by some creamy bacteria, which could chelate different metal ions (Pb, Cd, Cu, Zn, Ni, Co). Similarly, [77] showed that the bacterium Paenibacillus jamilae produced EPS with a strong affinity for complexing lead (228 mg g−1 ), effectively removing the pollutant from the aqueous solution. While the production of pigments and mucilaginous substances was observed in this study during screening trials, their exact role in heavy metal detoxification remains to be fully elucidated. Molecular identification of selected promising isolates placed these microorganisms within the phylum Pseudomonadota (previously denoted as Proteobacteria), and they were assigned to three genera: Stenotrophomonas, Enterobacter, and Pseudomonas. Pseu- domonadota has been described as the most common phylum in several sediment samples from marine environments [63,64]. Moreover, metagenomic analysis of sediment samples collected in the Colombian Caribbean region had also found Proteobacteria along with Bacteriodetes (Bacteroidota), Actinobacteria (Actinomycetota), and Firmicutes (Bacillota) as the dominant phyla, while Pseudomonas were the most common genera [78,79]. The genera Enterobacter and Stenotrophomonas have been documented in marine samples from around the world [63,80]. For instance, the genus Enterobacter has been isolated from marine water and sediment samples obtained from Cove Rock and Bonza Bay beach of the Eastern Cape Province, South Africa [80]. Similarly, the genus Stenotrophomonas has been reported in samples from the northeastern South China Sea [63]. Therefore, the findings of this study align with the global distribution of these microorganisms in marine environments. The three microorganisms selected for further studies showed different growth trends when exposed to lead-containing solutions. These differences may be attributed to their distinct taxonomic classifications, which are directly associated to differences in metabolic Microorganisms 2023, 11, 2708 19 of 24 capabilities [81]. Consequently, these bacteria showed different lead removal potentials, which could indicate different detoxification mechanisms for each bacterium. For exam- ple, the lead removal results obtained with the Stenotrophomonas strain (0–15%) and its corresponding growth curve data suggested that lead removal from the solution was a surface process, probably an adsorption process independent of metabolism: in this process, the metal was retained in some component of the dead biomass. These results contrast with those obtained by [82], who evaluated the tolerance of Stenotrophomonas maltophilia to different concentrations of Cd, Pb, Ag, Cu, and Ni ions, finding that, within 16 h, this microorganism could tolerate Pb(NO3 )2 concentrations of up to 5 mM. Additionally, strains within this genus have been found to employ at least two mechanisms to over- come metal toxicity. These mechanisms involve the reduction of ions in their elemental state, rendering them non-toxic, and active detoxification processes that require an active metabolism [70,72,82]. Unlike the Stenotrophomonas sp. INV PRT216, Enterobacter sp. INV PRT213 and Pseudomonas sp. INV PRT215 showed higher removal capacities and active biomass divi- sion during the construction of growth curves. It was observed that lead removal was influenced by the interaction between biomass concentration and exposure time: as these two parameters increased, an increased removal percentage was observed. Therefore, these results were associated with a possible biosorption process. Biosorption mechanisms using living cells have been described as a two-stage process: an initial absorption independent of the metabolism that takes place after exposure, followed by a slower active absorption that does depend on active metabolism, generating an intracellular accumulation of metal ions [41]. Considering that short contact times were employed in this study, it is neces- sary to conduct longer exposure experiments that allow for a potentially higher removal percentage and active metal capture [83]. The bacterium Pseudomonas sp. INV PRT215 achieved the highest lead removal per- centage (49%) in this study after 240 min of exposure, representing a removal efficiency of 0.29 mg Pb h−1 mgbiomass−1 . These lead removal results are lower compared to those obtained with Bacillus thioparans strain U3 (1.88 mg Pb h−1 mgbiomass−1 ). However, they are comparable to those obtained with Pseudomonas chengduensis PPSS-4 (0.11 mg Pb h−1 mgbiomass−1 ) isolated from marine sediment samples from India [12], and they are at least 4-fold higher than those reported for gram-positive species such as Lactobacillus plantarum MF042018 (0.07 mg Pb h−1 mgbiomass−1 ) [43], and Bacillus xiamenensis PbRPSD202 (0.06 mg Pb h−1 mgbiomass−1 ) [41]. Gram-positive bacteria possess a thick peptidoglycan layer, which confers a negative charge on the cell wall and, therefore, a greater capacity to attract positively charged molecules such as Pb ions [84]. Even though Pseudomonas are Gram-negative bacteria and, consequently, they lack a heavily negative charge in their cell surface, other authors have reported several Pseudomonas species as an excellent heavy-metal biosorbents [54]. The FTIR analysis conducted on the biomass of the strain Pseudomonas sp. INV PRT215 evidenced possible interactions between Pb ions and functional groups in the cell, such as carboxylates, phosphates, and amines. It was observed on resulting spectra that transmittance decreased in biomass exposed to the metal compared to biomass from controls, indicating that the stretching of the bonds took place to a lesser extent when there was interaction with metals, thus reducing the intensity of the signals. Studies conducted by [54] suggested that the biomass of Pseudomonas stutzeri reduced the intensity of the functional groups after treatment with 300 mg·L−1 of Zn2+ and Pb2+ . It has been reported that the ligands (functional groups) of the cell membrane with a negative load are useful for eliminating metals and other toxic compounds via electrostatic interactions [81]. Thus, the bonding sites with the greatest potential in the microorganisms cells are the carboxyl, amine, phosphate, sulphate, and hydroxyl groups [73], i.e., those recorded in this study during biomass FTIR analysis. Microorganisms 2023, 11, 2708 20 of 24 5. Conclusions Classical microbiology culture methods allowed the isolation of several cultivable bacteria and fungi from sediment samples collected at underexplored tropical marine environments. Isolates exhibited various morphological and phenotypic characteristics, including pigmented, sporulated, filamentous, creamy, and mucilaginous microorgan- isms. Tolerance screening trials, focusing on three heavy metals—cadmium, lead, and zinc—revealed that the majority of the marine organisms exhibited some degree of toler- ance to one or more of these metals at the evaluated concentration levels. Furthermore, the results indicated a more widespread sensitivity to cadmium than to lead and zinc among the evaluated marine organisms. Promising bacteria with high heavy-metal tolerance were identified through 16S rRNA gene sequencing, and they were found to belong to the phylum Pseudomonadota, specifically within the genera Stenotrophomonas, Enterobacter, and Pseudomonas. These assessed microorganisms displayed varying growth patterns when exposed to lead-containing solutions and demonstrated different potential for removing the metal from the aqueous phase. In particular, Gram-negative bacterium Pseudomonas sp. INV PRT215, isolated from San Juan River mouth, exhibited the highest lead removal efficiency surpassing the removal capacities of other marine bacteria, including some Gram- positive organisms. This study aimed to assess microbial diversity from underexplored marine environments and evaluate the heavy-metal tolerance of isolated microorganisms. The demonstrated growth and viability of multiple isolates in highly toxic metal solutions provided valuable insights for further studies aimed at enhancing the performance of marine microorganisms in heavy-metal remediation processes. Supplementary Materials: The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/microorganisms11112708/s1. Table S1. Culture media for microbial isolation. Table S2. Cadmium (Cd2+ ), Lead (Pb2+ ), and Zinc (Zn2+ ) tolerance screening of 276 microorganisms. A microorganism was catalogued as tolerant (+) if growth was observed after 24–48 h of culture; not tolerant (−) if growth was inhibited entirely around wells, or sensitive (±) if some colonies were formed (36). Sampling sites: Atrato river mouth (MPAR, MARM, MRAR), Baudó river mouth (OBR, MVUBR), and San Juan river mouth (MCHSJ2, BSJM2, MSJ2). Table S3. Cadmium (Cd2+ ), Lead (Pb2+ ), and Zinc (Zn2+ ) tolerance screening trials at increased concentrations of Pb2+ , Cd2+ and Zn2+ ions of eleven microorganisms. A microorganism was catalogued as tolerant (+) if growth was observed after 24–48 h of culture; not tolerant (−) if growth was inhibited entirely around wells, or sensitive (±) if some colonies were formed (36). Table S4. Results obtained by comparing the 16S rRNA sequences to those in the Reference RNA Sequences database of the NCBI. Figure S1. Phenotypic characteristics of some isolated colonies. Figure S2. Photographic record of pigments and substances excreted by bacterium 76955 under Cd2+ and Zn2+ stress conditions. Figure S3. Number of strains tolerant to Cd2+ , Pb2+ , and Zn2+ ions by sampling site. Sampling sites: Atrato river mouth (MPAR, MARM, MRAR), Baudó river mouth (OBR, MVUBR), and San Juan river mouth (MCHSJ2, MSJ2, BSJM2). Author Contributions: K.L.A.-C. and M.Q.: Data curation, Formal analysis, Investigation, Methodol- ogy, writing—original draft. B.C.-C. and M.M.-G.: Methodology. E.L.O.-T.: Methodology, Formal anal- ysis. L.B.: Methodology. O.S.: Writing—review and editing. A.Z.-C.: Data curation, Formal analysis, Writing—review and editing. J.G.-L.: Funding acquisition, Project administration, Writing—review and editing. All authors have read and agreed to the published version of the manuscript. Funding: This research and APC was funded by the Ministry of Science, Technology, and Innovation (Minciencias)-Colombia BIO and “Fondo Francisco José de Caldas (FFJC)” Grant code 75973/Contract 80740-568-2020; the Marine and Coastal Research Institute “José Benito Vives de Andréis” (INVE- MAR); and the Ministry of the Environment and Sustainable Development (Minambiente) through the tokens of the National Investment Projects Bank (BPIN) code 20170110000113. Data Availability Statement: The data presented in this study are available in supplementary material. Microorganisms 2023, 11, 2708 21 of 24 Acknowledgments: The authors would like to thank the Ministry of Science, Technology, and Innovation (MinCiencias)-Colombia BIO and FFJC for funding the project “Bio+Biop: plataforma integral BIODIVERSIDAD MICROBIANA + BIOPROSPECCIÓN del Pacífico Norte y Zona Sur del Caribe colombiano, un potencial recurso en la biorremediación de ambientes contaminados por metales pesados”, grant code 75973/Contract #80740-568-2020. Authors thank the Marine and Coastal Research Institute “José Benito Vives de Andréis” (INVEMAR) and its Evaluation and Use of Marine and Coastal Resources Program/Marine Bioprospecting Line (contribution # 1367). The authors also wish to thank the Ministry of the Environment and Sustainable Development (Minambiente) for the funding received through the National Investment Projects Bank (BPIN). 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In Biosorption; Derco, J., Vrana, B., Eds.; IntechOpen: Rijeka, Croacia, 2018; Volume 2, pp. 21–49, ISBN 978-1-78923-473-2. Disclaimer/Publisher’s Note: The statements, opinions and data contained in all publications are solely those of the individual author(s) and contributor(s) and not of MDPI and/or the editor(s). MDPI and/or the editor(s) disclaim responsibility for any injury to people or property resulting from any ideas, methods, instructions or products referred to in the content.