Batch studies on the biodegradation of paracetamol and 1,4-hydroquinone by novel bacterial strains isolated from extreme environmental samples and the identification of candidate catabolic genes

Vol.:(0123456789) Applied Water Science (2024) 14:198 https://doi.org/10.1007/s13201-024-02264-6 ORIGINAL ARTICLE Batch studies onthebiodegradation ofparacetamol and1,4‑hydroquinone bynovel bacterial strains isolated fromextreme environmental samples andtheidentification ofcandidate catabolic genes AlbaLara‑Moreno1,2 · FatmaEl‑Sayed3· CymonJ.Cox1· MariaC.Costa1,3· JorgeD.Carlier1 Received: 23 August 2023 / Accepted: 4 August 2024 / Published online: 13 August 2024 © The Author(s) 2024 Abstract The emerging pollutant paracetamol (APAP) is one of the most prescribed drugs worldwide. In addition, APAP and its main metabolites, namely, 4-aminophenol (4-AP), hydroquinone (H2Q), benzoquinone (BQ), and 2,5-dihydroxy-1,4-benzoquinone (2,5-OH-BQ), among others, are frequently detected in wastewater treatment plants (WWTPs) influents, effluents, and the environment. Thus, continuous release into the environment, especially aquatic environments, is a source of general concern. Six APAP-degrading bacterial strains were isolated from two mine samples from the Iberian Pyrite Belt (Lousal and Poderosa mines). Mycolicibacterium aubagnense HPB1.1, which was isolated using enrichment cultures from the Poderosa mine sample in the presence of H2Q as the sole carbon source, also showed APAP biodegrading capabilities. Pure cultures of this strain degraded 34.3mg L−1 of APAP in 5days and 9.4mg L−1 of H2Q in 4days. Interestingly, BQ and 2,5-OH-BQ were detected as metabolites resulting from H2Q abiotic degradation, but these compounds were removed in the strain’s cultures. Furthermore, M. aubagnense HPB1.1 whole-genome was sequenced, and its encoded proteins were aligned with enzymes of APAP-degrading bacteria recovered from databases and literature aiming to identify candidate catabolic genes. Putative amidases, deaminases, hydroxylases, and dioxygenases, responsible for the degradation of APAP by the HPB1.1 strain, were identified by similarity, corroborating its ability to transform APAP and its intermediate metabolite H2Q into less toxic metabolic compounds due to their capacity to break the aromatic ring of these molecules. Keywords Bioremediation· Aerobic biodegradation· Wastewater treatment plants· Mycolicibacterium aubagnense HPB1.1· Emerging contaminant· Biodegrading genes Introduction Over the last two decades, environmental concerns have increased beyond the awareness of traditional pollutants such as polychlorinated biphenyls, dioxins, organochlorine, and organophosphorus pesticides. These concerns have extended to emerging pollutants introduced into the environment mainly through anthropogenic activities (Ternes etal. 2004; Kock etal. 2023). The true threat behind these emerging pollutants is that their environmental and human toxicological effects have yet to be fully evaluated (Rosenfeld and Feng 2011). Pharmaceuticals such as endocrine-disrupting drugs, analgesics, antibiotics, hormones, anti-inflammatory, antidiabetic, and antiepileptic drugs have been considered emerging pollutants because many studies have detected their presence in different matrices (surface water, groundwater, or soil) (Mohapatra etal. 2021). Pharmaceuticals are primarily introduced into the environment through continuous effluent release from pharmaceutical industries, human and veterinary health facilities, and particularly, wastewater * Alba Lara-Moreno [email protected] Jorge D. Carlier [email protected] 1 Centre ofMarine Sciences (CCMAR) , University oftheAlgarve, Gambelas Campus, 8005-139Faro, Portugal 2 Department ofMicrobiology andParasitology, Faculty ofPharmacy, University ofSeville, 41012Seville, Spain 3 Faculty ofSciences andTechnologies , University oftheAlgarve, Gambelas Campus, 8005-139Faro, Portugal Applied Water Science (2024) 14:198198 Page 2 of 18 treatment plants (WWTPs). This is considered unavoidable due to population growth and aging, which means that these compounds will be increasingly present in urban wastewater (Silva etal. 2021). Paracetamol, also known as N-acetyl-p-aminophenol and frequently shortened as APAP, is one of the most widely used non-prescribed analgesic, and antipyretic drugs (Żur etal. 2018; Shabani etal. 2021). However, APAP and its degradation products 1,4-hydroquinone (H2Q) and 4-ami- nophenol (4-AP) are considered not only emerging pollutants but also micropollutants detected in the environment at concentrations ranging from below ng L−1 to μg L−1 (Kim and Zoh 2016). Despite that the level of APAP in sewage effluents and natural waters is considered low (ng L−1 to μg L−1), varied negative effects have been reported (Heberer 2002). These include reproductive or DNA damage, accumulation in tissues, oxidative stress, lipid peroxidation, and behavioral changes observed in algae, microcrustaceans, mollusks, or teleost fish (Gómez-Oliván etal. 2014; Minguez etal. 2016; Islas-Flores etal. 2017). Based on Regulation (EC) No 1907/2006, APAP was categorized as harmful to aquatic organisms based on an EC50 concentration between 11 and 100mg L−1 and as very toxic to aquatic organisms based on an EC50 concentration < 1mg L−1 after long-term exposure. The intermediate product of APAP biodegradation, 4-AP, is considered a dead-end metabolite (Mackie etal. 2013). 4-AP was reported for its considerable nephrotoxicity, mutagenicity, teratogenicity, and DNA cleavage induction in mouse and human lymphoma cells (Wang and Feng 2023). In addition, H2Q, the main metabolite of APAP, is widespread in the environment since it is usually produced from the advanced oxidation of several aromatic compounds (as APAP) and also due to its extensive use in various activities such as photography, dye intermediates, stabilizers in paints, varnish oils, and motor fuels. However, H2Q has proven to be more toxic and less degradable than phenol (Santos etal. 2004). Indeed, H2Q is known as a hematotoxic and carcinogenic agent associated with harmful effects in occupational environments. Moreover, in the environment, H2Q has shown increased toxicity for aquatic organisms (Enguita and Leitão, 2013). Levels of pollutants, including APAP and its metabolites, are rising in WWTPs, which accordingly accumulate in the environment. The conventional decontamination method of chlorination, as well as advanced methods including membrane bioreactors, nanofiltration, reverse osmosis, and carbon nanocomposites with magnetic properties, are not sufficiently tailored for pharmaceutical removal (Kim etal. 2018; Tiwari etal. 2017). Both current and conventional methods mostly convert APAP to its metabolites which are even more toxic (Chopra and Kumar 2020). Therefore, there is still an urgent need to develop strategies aimed at improving the removal of these chemicals. Bioremediation is a comprehensive strategy that utilizes biological systems, primarily bacteria, fungi, and plants, to clean contaminated environments by oxidizing, immobilizing, or transforming pollutants. Its main objective is to decrease pollution levels to those that are undetectable, nontoxic, or acceptable (i.e., within limits set by regulatory agencies) (Vishwakarma 2020). Microbial biodegradation is mostly considered an effective, sustainable, eco-friendly, and cost-effective approach for the removal of APAP (Vargas-Ordóñez etal. 2023). To boost the effectiveness of degradation, three strategies have been suggested: natural attenuation (using the ability of the intrinsic microbial community to degrade the contaminant), bioaugmentation (the addition of living cells capable of degradation), and biostimulation (the addition of limiting nutrients to support microbial growth) (Lara-Moreno etal. 2022a). Pollutantdegrading microorganisms are obtained using enrichment cultures in the presence of high concentrations of contaminants (Palma etal. 2021; Lara-Moreno etal. 2022b; Vargas- Ordóñez etal. 2023). Several studies have shown improved bioremediation using bioaugmentation in contaminated water by APAP. A varied panel of bacterial strains has been identified during the last two decades for their ability to biodegrade high APAP concentrations (some of them above 50–3000mg L−1) using it as a sole carbon source, including members of Pseudomonas (Poddar etal. 2022; Vargas- Ordóñez etal. 2023; Żur etal. 2018; Hu etal. 2013), Bacillus (Chopra and Kumar 2023), Stenotrophomonas (Zhang etal. 2013), Ensifer (Park and Oh 2020), or Shinella (Chen etal. 2022), among others. Compared with the number of APAP biodegrading strains, fewer microbial strains have been proven for their ability to biodegrade 4-AP and H2Q. For example, Khan etal. (2009) reported that 4-AP biodegradation was induced by Pseudomonas sp. ST-4 at different conditions (pH, temperature, and glucose concentration). In another study, Moraxella, Pseudomonas, Sphingomonas, Burkholderia, Variovorax, Azospirillum, Brachymonas, and Cupriavidus have been described for their ability to utilize H2Q which may be a product of not only APAP but also other phenolic compounds such as 4-chlorophenol, 4-fluoro-, 4-bromo-, 4-iodo-, and 4-nitrophenol degradation (Enguita and Leitão, 2013). More recently, Zhao (2017) reported that H2Q is removed by the endogenous microbiota of granular sludges. Despite its toxicity, only one biodegradation study (Harbison and Belly 1982) has been reported using H2Q as the initial compound. In this work, bacterial strains with the ability to degrade APAP and its metabolites as the only source of carbon and energy were isolated using enrichment cultures inoculated with samples from extreme environments of mines from The Iberian Pyrite Belt. The isolate, Mycolicibacterium aubagnense HPB1.1 was identified as the most effective degrading bacteria. Furthermore, this species was not previously Applied Water Science (2024) 14:198 Page 3 of 18 198 reported as being able to remove the studied compounds. Thus, whole-genome sequences of the strain were obtained, and their encoded proteins compared to APAP-degrading enzymes retrieved from databases and literature aiming to identify candidate catabolic genes for future works on gene function analysis to provide new information about the mechanism of APAP biodegradation that lately will be useful for genetic bioaugmentation to improve wastewater treatment. Materials andmethods Materials Analytical grade paracetamol (N-(4-hydroxyphenyl) acetamide, purity ≥ 99%) and 1,4-Hydroquinone (Benzene- 1,4-diol, purity ≥ 99%) were purchased from Sigma-Aldrich, USA. Five mine samples (LA, LC, PB, PDE, and PF) from the Iberian Pyrite Belt (considered one of the most important metallogenic regions in the world) were used to obtain APAP and H2Q degrading bacteria. LA and LC samples were collected from white and yellow wall sediments of the Lousal mine, respectively (38°02′01.8"N 8°25′32.2"W) located in Grândola (Portugal). The Lousal mine was mostly active from 1900 to 1988, and it was known as an important massive sulfide deposit for fertilizer production. The deposit was primarily mined for pyrite (iron sulfide) with about 50Mt of ore with 1.4% Zn, 0.8% lead, and 0.7% copper (Relvas etal. 2014). PB, PDE, and PF samples were collected in the Poderosa mine (37°44′54.8"N 6°39′19.9"W) based in Campillo (Huelva, Spain), from wall sediment, wall moonmilk, and sand sediment, respectively. The Poderosa mine was working between 1864 and 1924, and it presents high levels of copper as chalcopyrite, chalcocite, and covellite. Poderosa has been an important source of copper, exploiting more than 600 000 tonnes of copper during its activity period (Grande etal. 2016). X-ray diffraction (XRD) characterization by Palma and Costa (2024) revealed for the Lousal mine’s samples a predominant composition of silicates (silicon dioxide, quartz, olivine), phyllosilicates (kaolinite, muscovite), oxides (hematite), carbonates (calcite), sulfide (arsenopyrite, octosulfur), and aluminum phosphate, while for the Poderosa mine’s samples it revealed mainly a composition of silicates (quartz), phyllosilicates (muscovite), sulfates (pickeringite, gypsum, natrojarosite, jarosite), and phosphate (brushite). It is generally accepted that there is a great biotechnological potential in microbial communities from extreme environments, since they have microorganisms that evolved with mechanisms allowing their adaptation to hostile conditions. For example, strains with biotechnological potential have already been isolated from moonmilk, an extreme environment found in caves and mines composed mainly of fine CaCO3 crystals where extremophiles and non-extremophiles are found (Hui etal. 2021; Kosznik-Kwaśnicka etal. 2022). Methods Collecting ofmicrobial consortia frommine samples The samples collected from Lousal (LA and LC) and Poderosa (PB, PDE, and PF) mines were washed with Ringer’s solution (Sigma-Aldrich, USA). This washing procedure was performed in 50mL flasks with 9mL of Ringer’s solution and 1g of sample with 150rpm of orbital agitation for 4h. After that, 500µL of the washing solution was transferred to a 50mL flask with 5mL of Nutrient broth (NB, g L−1: 10.0 peptones; 1.0 beef extract; 2.0 yeast extract; 5.0 NaCl) and incubated at 25°C for 48h. After incubation, cultures were preserved in glycerol (20% v/v) stored at − 80°C. Enrichment culture ofparacetamol and1,4‑hydroquinone degrading consortia 220 μL of microbial suspension from a glycerol stock kept at − 80ºC was inoculated into 20mL of LB broth in a 100mL Erlenmeyer flask and incubated at 30°C under shaking conditions of 160rpm for 24–48h. The grown seed cultures of Poderosa mine consortia (PB, PF, and PDE) and Lousal mine consortia (LA and LC) were centrifuged at 4000rpm for 20min. After discarding the supernatant, pellets were washed twice by resuspension in 10mL of Mineral salt medium ((MSM, g L−1): 0.5 K2HPO4; 0.5 KH2PO4; 1.0 NH4HPO4; 0.5 MgSO4; 0.01 NaCl; 0.2 MgCl2; 0.03 CaCl2; 0.00034 MnSO4; 0.00043 ZnSO4; 0.00035 NH4(Mo‧7O2) 4H2O; 0.0004 CoCl2; 0.0001 EDTA) followed by centrifugation (4000rpm for 20min). The obtained pellet was resuspended in MSM. Enrichment cultures were performed in a 100mL sterilized Erlenmeyer flask. Each flask contained 45mL of MSM supplemented with 500mg L−1 of APAP and a volume of microbial inoculum to reach a final OD600 = 1. The flasks were kept at 30°C under shaking conditions of 160rpm, and every 7d, 10mL of the culture was transferred to a new flask containing the new sterile MSM with 500mg L−1 of APAP and incubated again. This process was repeated three times. The same proceeding was conducted with H2Q (500mg L−1) using PB, PF, and PDE. In the case of APAP, at the end of the incubation period planned for each enrichment culture, 1mL of each culture was centrifuged at 4000rpm for 20min, then tenfold dilution was conducted using sterile MSM and the diluted samples were taken for drug monitorization using UV–vis spectrum scans at 245nm. Applied Water Science (2024) 14:198198 Page 4 of 18 Isolation ofpotential paracetamol and1,4‑hydroquinone degrading bacteria 100µL of each tenfold dilution from final enrichment cultures were spread on MSM agar plates supplemented with APAP and H2Q separately at a concentration of 500mg L−1 as the sole carbon source added; hence, bacteria that grow on the plates are considered potentially drug biodegrading. After confirming the microbial growth, biomass was spread on LB-drug plates to obtain morphologically distinct colonies. During one week of incubation at 30°C, the plates were checked daily, and morphologically distinct colonies were picked separately and streaked on MSM-drug (500mg L−1) plates to confirm the isolate’s ability to utilize the drug as a sole carbon source. To prevent bacterial growth in the solid medium due to impurities from the agar that could serve as carbon sources, we have used Ultrapure Noble Agar (Thermo Scientific, Ref. J10907, LOT: 213,191). Pure colonies of each isolate were suspended into the cryopreservation medium in a cryovial for long-term storage at − 80°C. 16S rRNA gene sequencing andtaxonomic classification Genomic DNA was extracted from a pure culture of each of the 15 potential isolates that were able to utilize APAP or H2Q as the sole carbon source, using NZY microbial gDNA isolation kit (Nzytech, Portugal). 1mL of each culture was centrifuged (4000rpm for 20min) and DNA was extracted from cell pellets as described in the kit. The 16S rRNA gene was amplified using a Supreme NZYTaq 2 × Green Master Mix (Nzytech, Portugal) with universal primers for prokaryotes: the universal primers 8F (also known as fD1) (5′- AGA GTT TGA TCC TGG CTC AG -3′) (Weisburg etal. 1991) and 1492R (5′-GGT TAC CTT GTT ACG ACT T-3 ′) (Lane 1991). The PCR amplification was confirmed by gel electrophoresis using ENDUROTM electrophoresis system (Labnet, USA), which was carried out in 1% (w/v) agarose gel in 1 × TAE buffer. The amplified products were sequenced by the Sanger method in both directions (forward and reverse), with the same primers that were used for PCR amplification. A capillary electrophoresis sequencing system (Genetic Analyzer, Model 3130xl, Applied Biosystems, Foster City, USA) was used for Sanger sequencing. The taxonomic classification was based on the results obtained by nucleotide alignment with the “16S ribosomal RNA sequences (bacteria and archaea)” database, using the BLASTn (Basic Local Alignment Search Tool nucleotide) tool (Altschul etal. 1990) at the NCBI Web BLAST site (https:// blas t. ncbi. nlm. nih. gov/ Blast. cgi). Inoculum preparation An aliquot of bacterial isolate culture was transferred to 5mL of LB broth supplemented with 500mg L−1 of APAP or H2Q. Incubation proceeded for 24 or 48h at 25°C under shaking conditions of 160rpm. 5mL of each grown culture was inoculated into two separate 100mL aliquots of LB- drug (500mg L−1) medium dispended in 200mL bottles and incubated under the same conditions. Microorganisms were harvested and centrifuged after 24–48h and subsequently washed twice using MSM, to remove any possible LB residues, before initiation of the experiments. Paracetamol biodegradation assay insolution APAP biodegradation experiments were performed in duplicates in 200mL sterilized glass bottles containing 60mL of MSM supplemented with APAP or H2Q at a concentration of 50mg L−1. Bacterial inoculum was added in the volume necessary to adjust initial OD600 ~ 1 (108CFU mL−1 approximately). Glass bottles were kept shaking (160rpm) at 30°C in the dark. Samples were taken at the initial time and at different incubation times (0, 5, 10, 15, 19, and 28days). Negative controls of uninoculated media were also tested. At each time interval, 1mL of each test was centrifuged at 4000rpm for 20min and the supernatant was filtered using polyether sulfone syringe filters (0.22μm pore size) for the subsequent HPLC analysis. HPLC conditions were applied as described by Palma etal. (2018) to quantify APAP. A reversed-phase Surf Extreme C18 column (250mm × 4.6mm, 5μm, functional group: a 100Å pore-sized silica), purchased from ImChem, was used for separation. An Xbridge-C18 guard column (4.6 × 20mm, 5μm) purchased from Waters was used at the entry of the separation column. Smartline HPLC set (KNAUER, Germany), with UV-detector 2600 was used and peaks were analyzed using Chromatography-Clarity SW software. The mobile phase consisted of phosphate buffer/ methanol (80/20, v/v). The phosphate buffer solution (pH 4.6) was prepared by dissolving 4.5g KH2PO4 and 0.0314g K2HPO4·3H2O in 500mL of ultrapure water, using phosphoric acid (85%) to adjust pH if necessary. The isocratic mode was applied with 1mL min−1 flow rate. The column was maintained at room temperature. The total run time was 10min for APAP and the injection volume was 20μL. The detection was at 245nm. The samples were quantified by external linear calibration, using six concentrations from 50 to 2.5mg L−1. The obtained calibration curve had an R2 of 0.998 and an estimated Limit of Detection (LOD) of 1.6mg L−1. Applied Water Science (2024) 14:198 Page 5 of 18 198 1,4‑hydroquinone biodegradation assay insolution H2Q biodegradation in solution was studied in three replicates, conducted in 200mL sterilized Erlenmeyers flask with 60mL of MSM, spiked with 10mg L−1 of H2Q as the sole carbon source. The flasks were inoculated with the volume of bacterial inoculum (HPB1.1 strain) necessary to adjust the initial OD600 = 0.8–1. In the case of negative control, the flasks were not inoculated with the bacterium. Then, the Erlenmeyers flasks were kept at 30°C and shaken (125rpm) in the dark for 7days. During this period samples were taken at the initial time and after 2, 4, 6, and 7days. Before measuring the samples in the HPLC, they were processed in the same way as mentioned in the sect Inoculum preparation. The concentration of H2Q in the samples was quantified using an Ultra High Performance Liquid Chromatography Nexera system (SHIMADZU), equipped with an XBridgeTMC18 5µm, 4.6 × 250mm column (Waters, USA). For the H2Q samples, the mobile phase composition was Methanol:Water (10:90v/v), adjusted to a pH ~ 3 with orthophosphoric acid 85%, using an isocratic method. The flow rate was set at 1.3mL min−1, with an injection volume of 200 μL. The total run time was 8min. The detection wavelength was at 190nm for H2Q and 240nm for its metabolites (2,5-OH-1,4-BQ and 1,4-benzoquinone). The retention time for the samples was approximately 4min for H2Q, 4.9 and 5.4min for 2,5-OH-1,4-BQ, and 6.8min for 1,4-benzoqui- none. The samples were quantified by external linear calibration, using five concentrations from 25 to 1mg L−1. In this case, the calibration curve had an R2 of 0.9992 and an estimated LOD of 0.5mg/L−1. 1,4‑hydroquinone adsorption study onbacterial biomass After the H2Q biodegradation test in solution, an extraction assay in triplicates was conducted to recover the concentration of H2Q accumulated in bacterial cells. The supernatant (42mL) and the bacterial biomass were centrifugated (20min, 4000rpm). After centrifugation, the supernatant was separated from the bacterial pellet. The solvents used for the solid–liquid extraction were selected based on Li etal. (2006). For this, 4.2mL of Methanol + 0.1% of acetic acid (pH = 4) were added to the Falcon tubes containing the bacterial cells. To disrupt the cells, the extraction procedure involved 20s vortex mixing, followed by 5min of sonication and 10min of centrifugation at 4000rpm. The quantification of the recovered H2Q from the cells was performed using HPLC, as described in Sect."Methods". Genome sequencing data andgene annotation ofMycolicibacterium aubagnense HPB1.1. M. aubagnense HPB1.1 was inoculated in 5mL of LB liquid medium and incubated at 30°C at 150rpm for 24h. Afterward, an aliquot was centrifugated at 10,000rpm for 3min. The genomic DNA was extracted from the collected cells using NZY microbial gDNA isolation kit (Nzytech, Portugal). The DNA concentration was quantified by an ultra-trace UV spectrophotometer (Nanodrop 1000, Thermo Scientific). The genome of M. aubagnense HPB1.1 was sequenced to obtain useful genetic information about the genes involved in the APAP biodegradation pathway. The HPB1.1 genome was sequenced by Integrated Microbioma Resource (IMR, Dalhousie University, Canada) using PacBio Sequel at coverage from 33 × to 203x (https:// imr. bio). The generated sequencing reads were assembled using PacBio's SMRTlink genome assembler version 10. The bacterial genome has been annotated using the RAST tool (Rapid Annotation Subsystem Technology) (Aziz etal. 2018) and NCBI Prokaryotic Genome Annotation Pipeline. The sequencing data generated in this study were deposited in the National Center for Biotechnology Information (NCBI), under the BioProject ID PRJNA955654 and BioSample ID SAMN34178714. The whole genome of M. aubagnense HPB1.1 has been deposited in DDBJ/ENA/GenBank under accession numbers CP122994-CP12299. In silico analysis: nucleotide andprotein sequence analysis First, a set of reference protein sequences involved in the APAP biodegradation pathway was recovered from databases and literature as explained in more detail in the Results and Discussion section, namely in sub Sect."In silico prediction of genes involved in the paracetamol degradation pathway". Afterward, each reference protein was used as a Query in alignment searches to the genome of M. aubagnense HPB1.1 using the NCBI online search tool tblastn, a variant of the BLAST algorithm that compares a protein sequence against a nucleotide sequence database, where the nucleotide sequences are dynamically translated into protein sequences in all six possible reading frames during the search (https:// blas t. ncbi. nlm. nih. gov/ Blast. cgi). The algorithm parameters used were those set by default (Max target sequences: 100, Expect threshold: 0.05, Word size: 5, Max matches in a query range: 0, Matrix: BLOSUM62, Gap costs: Existence: 11 Extension: 1, Compositional adjustments: conditional compositional score matrix adjustment, Filter: low complexity regions). Finally, the genomic annotation data of the hits obtained by this tblastn search were used to get the complete sequences of the found proteins (hits) from the genome annotation created with RAST. Applied Water Science (2024) 14:198198 Page 6 of 18 The decision on the identification of a candidate catabolic gene was first based on evaluating the alignment percentage of coverage and the percentage of identity. Then, in the case of various sub-unites of the same enzyme, the consecutive genomic position was considered. Moreover, a study of the catalytic domains of the selected proteins was carried out using Conserved Domain Database (CDD) (https:// www. ncbi. nlm. nih. gov/ cdd) and InterProScan (Jones etal. 2014; Paysan-Lafosse etal. 2022). Results anddiscussion Screening/Enrichment ofthemicrobial consortia forparacetamol and1,4‑Hydroquinone removal Two microbial consortia (LA and LC) were obtained from the Lousal mine, while three microbial consortia (PB, PDE, and PF) were obtained from the Poderosa mine. The obtained consortia were enriched in MSM medium with APAP as the sole carbon source. During the incubation period of each culture, a deep brown coloration of MSMAPAP was observed for all enrichment cultures of all microbial consortia except for the LC consortium and the negative control (Fig.1S). Such medium coloration is associated with APAP biodegradation according to De Gusseme etal. (2011) who reported a brown coloration of the synthetic mineral medium during the microbial degradation of APAP by two described isolates, due to the formation, accumulation and polymerization of degradation intermediates, such as catechol and its derivatives (H2Q and 1,4-benzoquinone). In another work, Korotaev etal. (2016) studied the brown precipitate formed in the process of APAP biotransformation in the presence of Rhodococcus Ruber IEGM 77, concluding that 2,5-diaminobenzene-1,4-diol, 4,6-diaminobenzene- 1,3-diol, 2,5-dihydroxy-1,4-benzoquinonediimine and 2-amino-5-hydroxy -1,4-benzoquinoneimine were identified as the structural units of the copolymer responsible of coloration. In summary, the brown precipitate is a consequence of oxidative polycondensation of APAP and 4-aminophenol. Figure1 shows a preliminary study to quantify the APAP removal values according to UV–vis molecular spectroscopy analysis to have an idea about the capacity of the initial consortia to degrade APAP, before proceeding to strains isolation and biodegradation studies with pure cultures. The first enrichment culture of consortium LA from Lousal mine after 7days, showed only 6% APAP removal. On the other hand, the second and third enrichment cultures of consortium LA, which were 14 and 21days old respectively, showed maximal APAP removal capacity as inferred by APAP removal of 99.7% and 84.1%, respectively. The higher extent of APAP removal reported for the second (14days) and third (21days) enrichment cultures compared with the first enrichment culture can be explained based on the enhanced growth of APAP-utilizing strains, favoring the development of bacteria that can use APAP as a source of carbon and energy (Parajuli etal. 2017). The enrichment cultures of Poderosa mine consortia PDE and PB showed values of APAP removal of almost 22%, and in the case of LC and PF consortia removals of 15% were achieved. Furthermore, the PB, PDE, and PF consortia were used aiming to obtain enriched consortia able to degrade H2Q. In this case, deep brown coloration was observed for Poderosa mine enrichment cultures, but also in the negative control medium (Fig.2S). This indicates that H2Q was changing due to abiotic factors. It is known that oxygen can act as 0 10 20 30 40 50 60 70 80 90 100 LA 1LA 2LA 3 LC 1 LC 2LC 3PDE 1PDE 2PDE 3PF 1PF 2PF 3PB 1PB 2PB 3 APAP removal (%) Fig. 1 Paracetamol (APAP) removal in MSM-APAP (500 mg L−1) enrichment cultures of PDE, PB, and PF microbial consortia from Poderosa mine and LA and LC microbial consortia from Lousal mine using UV–vis absorbance (245 nm). 1, 2, and 3 means: 7-day 1st enrichment culture, 14-day-old 2nd enrichment culture, and 21-day- old 3rd enrichment cultures, respectively Applied Water Science (2024) 14:198 Page 7 of 18 198 an oxidant in the oxidative polymerization of H2Q, leading to the formation of brown polymers. Zhang etal. (2012a) associate brown precipitate with the formation of poly hydroquinone (PH2Q), a redox-active polymer with quinone/ H2Q redox-active units in the main chain. Dang etal. (2014) studied something similar and confirmed that H2Q could self-polymerize to form brown polymers based on air oxidation, which is known as PH2Q. Therefore, since there was abiotic transformation of H2Q it was difficult to evaluate if biodegradation occurred or not. Thus, H2Q was not monitored in the enrichment cultures, and all were used to inoculate selective MSM plates aiming to isolate potential H2Q degrading strains. Isolation andidentification ofparacetamol andH2Q degrading bacterial strains Specific potential APAP-degrading strains forming isolated colonies were selected from the solid MSM containing APAP as the only added source of carbon and energy. Fifteen strains were isolated from PB, PDE, PF, LA, and LC consortia. Isolated strains were labeled with the letters P or H (isolated in the presence of APAP or H2Q, respectively) followed by the name of the original consortium (PDE, PB, LC, or LA) plus the enrichment step (2 or 3 for the second or third culture, respectively) and a number from 1 to 4 (PLA2.1, PLA2.2, PLC2.1, PLC2.2, PPDE2.1, PPDE3.1, PPDE3.2, PPDE3.3, PLA3.2, PLA2.3, PLA2.4, PLA3.1, PLC2.3, PLA3.3, PDE3.4). Concerning their taxonomic classification, the NCBI Blast analyses revealed phylogenetic affiliations with identity values of the 16S rRNA sequences with their closest neighbors above 98.99%. However, the sequences presented very high similarities (> 97%) with other close neighbors of the same genus but assigned to different species. Thus, the BLAST alignment was not significant enough to classify the studied isolates at the species level (Yarza etal. 2014). In what concerns H2Q, only one colony was formed and isolated from the plates inoculated with the enrichment cultures, namely from the plate inoculated with the first enrichment of the Poderosa mine microbial consortium PDE; thus, only one strain showed potential ability to use H2Q as a carbon source. The 16S rRNA gene sequence showed a match of 100% to species from Mycolicibacterium aubagnense in the NCBI GenkBank “nt” sequence repository, and our strain was named M. aubagnense HPB1.1 based on the same criteria mentioned above. Members of Mycolicibacterium were previously included in genus Mycobacterium but were separated in a different genus by Gupta etal. in 2018. Table1 shows the classification of potential APAP and H2Q biodegrading bacterial isolates using 16S rRNA gene sequence analysis. Paracetamol biodegradation insolution bypure cultures ofpotentially degrading bacteria For space and time management reasons, it was decided to randomly choose one representative of each genus for subsequent biodegradation tests with pure cultures in a liquid medium. Thus, six of the fifteen isolates with potential APAP degrading capacity were selected. In addition, the Table 1 Phylogenetic affiliations of the bacterial strains isolated from Poderosa and Lousal consortia HPB1.1 is classified to the species level based on its genome sequence (described later in the manuscript) Strain Genus Range of similarity (%) Phylum/Class, family, genus PLA2.1 Bacillus sp. 99.86–100 Bacillota/Bacilli, Bacillaceae, Bacillus PLA2.2 PLC2.1 PLC2.2 PPDE2.1 PPDE3.1 PPDE3.2 PPDE3.3 Rhizobium sp. 99.5–99.8 Pseudomonadota/Alphaproteobacteria, Rhizobiaceae, Rhizobium PLA3.2 Variovorax sp. 99.62–99.85 Pseudomonadota/Betaproteobacteria, Comamonadaceae, Variovorax PLA2.3 Aeromonas sp. 99.88 Pseudomonadota/Gammaproteobacteria, Aeromonadaceae, Aeromonas PLA2.4 PLA3.1 PLC2.3 Niallia sp. 97.55–99.19 Bacillota/Bacilli, Bacillaceae, Niallia PLA3.3 Paraburkholderia sp. 98.5–100 Pseudomonadota/Betaproteobacteria, Burkholderiaceae, Paraburkholderia PDE3.4 HPB1.1* Mycolicibacterium aubagnense 100 Actinomycetota/Actinomycetia, Mycobacteriaceae, Mycolicibacterium Applied Water Science (2024) 14:198198 Page 8 of 18 strain isolated in H2Q selective medium was also selected for APAP biodegradation tests. Thus, seven strains isolated using enrichment cultures from mine samples were selected to conduct APAP biodegradation assays in liquid MSM supplemented with 50mg L−1 of APAP: Niallia sp. PLC2.3, Bacillus sp. PPDE3.1, Rhizobium sp. PPDE3.3, Aeromonas sp. PLA3.1, Variovorax sp. PLA3.2, Paraburkholdeira sp. PLA3.3, Mycolicibacterium aubagnense HPB1.1 (Fig.2). After 28days of the biodegradation assay, 1.1 ± 0.1mg L−1 (degraded concentration ± Mean Deviation) of APAP was degraded in the control test, indicating minimum abiotic degradation as expected (Zhang etal. 2012a; Dang etal. 2014). Niallia sp. PLC2.3, Aeromonas sp. PLA3.1 and Variovorax sp. PLA3.2 achieved the lowest biodegradation (13.9 ± 0.8, 18.8 ± 3.5 and 18.1 ± 7.9mg L−1, respectively after 28days). For Bacillus sp. PPDE3.1, a high biotransformation was observed, reaching a degradation of about 23.3 ± 5.2mg L−1 of APAP after 28days of the assay. Rhizobium sp. PPDE3.3 and Paraburkholderia sp. PLA3.3 seemed the most promising for APAP removal where APAP removal was 31.9 ± 2.5mg L−1 and 31.4 ± 0.6mg L−1, respectively. Interestingly, the Poderosa mine M. aubagnense strain HPB1.1 selected as potential H2Q degrading also showed APAP removal. As shown in Fig.2, after 5days 34.3 ± 0.8mg L−1 APAP was removed, and after 28days, APAP removal was 46.6 ± 0.1mg L−1, in the MSM cultures initially with 50mg L−1 APAP. The negative control did not indicate abiotic degradation and high removal was observed in all tests, suggesting the biodegrading capacity of all isolates. Several authors have reported the ability of some of the studied genera to degrade organic pollutants. Bako etal. (2021) highlighted polychlorinated biphenyl degradation in bioreactors by Paraburkholderia. Rhizobium sp. has been used for phenol compound degradation (Wei etal. 2008). In the study of Patel etal. (2022), Niallia circulans has been reported to degrade azo dye Reactive Red 152. Also, Mycolicibacterium sp. has been stated as polychlorinated biphenyls degrading (Steliga etal. 2020) and Naloka etal. (2021) indicated Mycolicibacterium sp. as a part of an artificial consortium to degrade fuel oil. However, genera Paraburkholderia, Rhizobium, Niallia, and Mycolicibacterium members have not previously been reported as pharmaceuticals-degrading bacteria, including APAP and H2Q. Therefore, more studies should be directed toward further exploring the diversity of potential biodegrading bacteria from mines as representatives of extreme environments. Nevertheless, species of the genera Aeromonas, Bacillus, and Variovorax have been described as drug degraders. Aeromonas sp. was able to remove gliazide in MSM supplemented with 0.5g L−1 of gliazide (Ouartsi etal. 2019). B. thuringiensis B1 degraded 20mg L−1 of ibuprofen in MSM supplemented with 1mg L−1 of glucose (Marchlewicz etal. 2017). Furthermore, Bacillus subtilis and Bacillus cereus ControlAeromonas sp. PLA 3.1 Variovorax sp. PLA3.2 Paraburkholdeira sp. PLA3.3 Nialliasp. PLC2.3 Rhizobiumsp. PPDE3.3 Bacillus sp. PPDE3.1 Mycolicibacterium sp. HPB1.1 0 5 10 15 20 25 30 35 40 45 50 APAP removed(mg L-1) Fig. 2 Paracetamol (APAP) removal in MSM-APAP (50 mg L−1) (Control) and after inoculation of bacterial isolates (Tests) during the biodegradation experiments. [Removed] = [initial]-[after incubation], based on HPLC analysis (described in materials and methods). The error bars represent the mean deviation for two replicates Applied Water Science (2024) 14:198 Page 9 of 18 198 removed 1200mg L−1 and 200mg L−1 of APAP in MSM, respectively (Chopra and Kumar 2020; Palma etal. 2021). Murdoch and Hay (2015) studied the ability of Variovorax sp. Ibu-1 to use 200mg L−1 of ibuprofen as a carbon source. It is important to highlight that previously studied genera, except Bacillus, have not been identified as having APAP degradation capacities. 4‑hydroquinone biodegradation insolution byM. aubagnense HPB1.1 The Poderosa mine isolate M. aubagnense HPB1.1 was the only one selected as a potential H2Q-removing bacterium for the biodegradation tests in liquid MSM monitored by HPLC analysis in the current study. As shown in Fig.3S, H2Q removal in the culture with HPB1.1 strain was not associated with a brownish/pinkish coloration, while in the negative control this coloration, which appeared very quickly after H2Q solubilization and before incubation in the dark, stayed during all the incubation time. This coloration indicates H2Q oxidation in contact with air and light (Enguita and Leitão, 2013) and/or that the medium’s physical–chemical characteristics potentiated H2Q oxidative polymerization (Zhang etal. 2012a). Therefore, it was important to try to understand and monitor as much as possible the abiotic H2Q transformation. It is known that H2Q autoxidation can occur in water at room temperature, resulting in the production of 1,4-benzo- quinone (BQ); even if at a small rate without the addition of a catalyst (Owsik and Kolarz 2002; Chunmei etal. 2015). In detail, it has been suggested that the autoxidation of H2Q to quinones (Q) occurs in free-radical chain (cyclic) processes (Roginsky and Barsukova 2000). On the other hand, it is known that the simplest quinone, the 1,4-benzoquinone (BQ), is light sensitive, and studies on its photolysis have shown that upon excitation by visible light it can go through two pathways of reactions that both result in 2-hydroxy- 1,4-benzoquinone (2-OH-BQ) and H2Q (Ononye etal. 1986; Von Sonntag etal. 2004). Moreover, it has also been shown that the 2-hydroxy-1,4-benzoquinone (2- OH -BQ) can be further oxidized to 2,5-dihydroxy-1,4-benzoquinone (2,5-OH-BQ) (Kurien and Robins 1970; Fónagy etal. 2021). Figure4S illustrates the reactions occurring in the two possible pathways. Thus, when H2Q is solubilized in water and exposed to light, four main compounds can be present at a certain equilibrium: hydroquinone (H2Q), 1,4-benzoqui- none (BQ), 2-hydroxy-1,4-benzoquinone (2- OH-BQ) and 2,5-dihydroxy-1,4-benzoquinone (2,5-OH-BQ). Interestingly, these four compounds have different absorption spectra (Fónagy etal. 2021), which can be used for identification. H2Q has a characteristic absorption peak at ~ 288nm, BQ has a major absorption peak at ~ 246nm, 2-OH-BQ has a major characteristic peak at ~ 256nm and another wider but lower peak at ~ 485nm, 2,5-OH-BQ has a major characteristic peak at ~ 300nm. For example, Rouco etal. (2018) evaluated the reduction of p-benzoquinone (peak at 246nm) into H2Q (peak at 290nm) by spectrophotometric monitoring of the reaction using the spectra. In our study, when H2Q was dissolved in liquid MSM medium, abiotic transformation started to occur as indicated by the brownish/pinkish coloration developed and by the observation of more than one peak by HPLC analysis (details in methods). Thus, a reference material (Hydroquinone TraceCERT®, 74,347—Supelco) was used to confirm that the main peak (retention times (RT):—RT ~ 4.1min) was in fact H2Q. The initial H2Q concentration was ~ 9.4mg L−1 and it dropped to non-detected values in four days in the test with M. aubagnense HPB1.1, while just a small decay from ~ 9,4 to 7.9mg L−1 occurred in the negative (abiotic) control (Fig.3), indicating that most of the H2Q removal in the culture was due to biotic factors. In what concerns H2Q transformation products, three peaks (apart from the H2Q peak) were detected: one with a RT of ~ 4.9min, with the typical absorption spectra of 2,5-OH-BQ; the second with a RT of ~ 5.4min, also with the typical absorption spectra of 2,5-OH-BQ; and the third with a RT ~ 6.8min, with the typical absorption spectra of BQ (Fig.4). According to the absorption spectra, one of the two peaks at ~ 4.9min and ~ 5.4min should correspond to 2,5-OH-BQ, while the other is probably another hydroxybenzoquinone isomer also with two hydroxyls. During the H2Q biodegradation test the peak at RT ~ 5.4min was not detected. Yet, interestingly, the peaks at RT ~ 4.9min and RT ~ 6.8min (2,5-OH-BQ and BQ, respectively) increased in the negative (abiotic) control and decreased to non-detec- tion in the pure culture of M. aubagnense HPB1.1 (Fig.5). 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