Open AccessArticle Biodegradation of the Non-Steroidal Anti-Inflammatory Drug Diclofenac in a Packed-Bed Biofilm Reactor and Its Ecotoxicity Evaluation 1 Centro de Investigaciòn en Ciencias de la Salud, Facultad de Ciencias de la Salud, Universidad Anáhuac México, Av. Universidad Anáhuac 46, Col. Lomas Anáhuac, Huixquilucan 52786, Estado de México, Mexico 2 Instituto Politécnico Nacional, Escuela Nacional de Ciencias Biológicas, Departamento de Ingeniería Bioquímica, Wilfrido MAssieu 399, Nueva Industrial Vallejo, Zacatenco, Alcaldía Gustavo A. Madero, Ciudad de México 07738, Mexico 3 Facultad de Ingenierìa, Universidad Anàhuac Mèxico, Av. Universidad Anàhuac 46, Col. Lomas Anàhuac, Huixquilucan 52786, Estado de Mèxico, Mexico * Authors to whom correspondence should be addressed. Processes 2026, 14(12), 1847; https://doi.org/10.3390/pr14121847 (registering DOI) Submission received: 25 April 2026 / Revised: 4 June 2026 / Accepted: 4 June 2026 / Published: 7 June 2026 The presence of xenobiotics in wastewater, particularly emerging contaminants such as pharmaceuticals, poses an ecotoxicological risk to the environment and human health. One of the main pharmaceutical products detected in water is diclofenac, which can be sold without a prescription. The lack of health regulations indicates the necessity of finding environmentally friendly treatment alternatives to remove this type of contaminant. Among these alternatives, biotechnology, specifically biological processes, offers a sustainable option compared to conventional treatments. Current treatment methods used in wastewater treatment plants are ineffective at removing diclofenac, a chlorinated aromatic compound highly resistant to degradation processes. In recent years, new treatment methods have gained prominence due to the favorable results they have yielded, including physicochemical, biological, and advanced processes. Biological treatments are notable for their low cost and the high level of effectiveness and efficiency with which they can remove toxic compounds. For this reason, the aim of this research project was to evaluate the degradation efficiency of a biological treatment in a bioreactor using a microbial community consisting of five bacterial strains, which was isolated from a pharmaceutical effluent and cultivated in a continuous culture system. Removal efficiencies ranging from 99.38 to 99.98% were achieved at various volumetric loading rates (from 0.087 to 1.043 g L −1d −1). Influents and effluents from the biological reactor were analyzed using bioassays to determine any potential toxic effects. The results showed that the effluents did not elicit a negative response in the bioindicators, indicating high toxicity in the influents. Keywords: bioassays; biological process; diclofenac; emerging pollutants; microbial community 1. Introduction Emerging pollutants, which result from human and industrial activities, are new chemical substances that are not currently regulated and may have adverse effects on ecosystems. These include compounds from the chemical and pharmaceutical industries, whose impact on health and the environment is not yet well understood [ 1, 2]. Therefore, this research offers an opportunity to implement an effective and efficient corrective measure through a specific biological treatment for the removal of diclofenac. This treatment was carried out using a microbial community isolated from a pharmaceutical effluent, which was immobilized in a packed-bed reactor operating under continuous culture conditions. At the same time, the toxicity of the influents and effluents from the biological reactor was evaluated using bioassays to assess the impact on the quality of the bioprocess. This research offers the possibility of integrating different fields of knowledge to understand the bioprocesses involved in the biological degradation of diclofenac. These findings will open up new possibilities to scale up the bioprocess and remediate environments contaminated by this drug, which causes serious environmental and health problems. 2. Material and Methods 2.1. Chemicals For the biodegradation process, pharmaceutical-grade diclofenac [diclofenac sodium (C 14H 10Cl 2NNaO 2)] was purchased from Pharmalife (Pharmalife, Ramos Arizpe, Coahuila, Mexico). For the analytical methods, reagent-grade diclofenac sodium (sodium;2-[2-(2,6-dichloroanilino)phenyl]acetate) was acquired from Sigma-Aldrich (Sigma-Aldrich, St. Louis, MO, USA), with purity higher than 98%. The solvents used for high-performance liquid chromatography (HPLC) were purchased from J.T. Baker (J.T. Baker, Phillipsburg, NJ, USA). 2.2. Culture Medium The mineral salts medium (MSM) containing 100 mg L −1 of diclofenac, which was used to feed the reactor, was a modification of that described by Schmidt et al. [ 48] and Suleiman et al. [ 40]. The composition of MSM was (g L −1): K 2HPO 4, 0.2; KH 2PO 4, 0.05; MgSO 4⋅7H 2O, 0.05; CaHPO 4, 0.01. One milliliter of a solution containing micro-elements was added to obtain a final concentration (mg L −1) of FeSO 4⋅7H 2O, 2.0; ZnSO 4⋅7H 2O, 1.0; MnSO 4⋅7H 2O, 0.2; Na 2MoO 4⋅2H 2O, 0.1; CuSO 4⋅5H 20, 0.2); H 3BO 3, (0.02). All were analytical-grade chemicals (Merck & Co., Kenilworth, NJ, USA). Agar (BD Difco, Franklin Lakes, NJ, USA) was added (18 g L −1) when solid medium was required. For toxicity assay, the algal assay procedure (AAP) medium was used to propagate the freshwater unicellular green microalga Pseudokirchneriella subcapitata [ 49]. 2.3. Selection of the Microbial Community The effluent from pharmaceutical wastewater treatment plant was collected from the industrial area of Naucalpan de Juárez, MX, MEX. This location was chosen because it is the most industrialized municipality in the Estado de México, where diverse industries and residential areas coexist [ 50]. The sample was collected in an amber bottle, stored at 4 °C and immediately transported to the laboratory. The sample was divided into two parts; the first was processed to obtain the physicochemical characteristics and the second was used for the isolation of the microbial community. The isolation of the microbial community was carried out through spread plate method [ 51] and streak plate method [ 51]. In the two methods, solid selective medium was used (MSM plus 100 mg L −1 of diclofenac). 2.4. Isolation of Bacterial Strains, DNA Extraction, Amplification, Purification, and Molecular Identification Bacterial colonies showing morphologic differences were isolated in MSM agar plates, containing 100 mg L −1 of diclofenac (MSM-D). DNA from pure strains was extracted with a silica gel-based membrane method [DNeasy Blood & Tissue Kit, Qiagen (Qiagen, Hilden, Germany)]. DNA was diluted in 50 µL of sterile distilled water and quantified by measuring the absorbance at 260 nm. Bacterial isolates were identified by the analysis of the 16S rDNA sequence using the primers reported by Brandt et al. [ 52] and the conditions described by Relman et al. [ 53] and White et al. [ 54]. For 16S rDNA amplifications, the primers Fd1 (5′-CAG AGT TTG ATC CTG GCT CAG-3′) and R6 (5′-TAC GGT TAC CTT GTT ACG AC-3′) were used [ 52]. The PCR conditions [ 53] were pre-denaturation at 94 °C for 5 min, denaturation at 94 °C for 1 min, annealing at 55 °C for 1 min, extension at 72 °C for 2.5 min and 30 cycles, and a final extension at 72 °C for 10 min, with termination temperature at 10 °C. The size of the fragment amplified from 16S rDNA gene was approximately 1500 bp. PCR products (≈1.5 Kb) were purified using the QIAquick Kit, Qiagen (Qiagen, Hilden, NW, DEU) and sequenced through a DNA sequencing service (the Sanger method). The sequence data were compared using the BLAST tool ( http://www.ncbi.nlm.nih.gov/) [ 55] and aligned with the most similar sequences and with sequences from related species collected from the non-redundant GenBank database of the 16S rDNA from the NCBI. Clustal X v2.0.1 ( http://www.ebi.ac.uk/tools/clustalw2/) [ 56] and SEAVIEW 3.2 ( http://doua.prabi.fr/software/seaview/) [ 57] software packages were used to align and edit the sequences and the MEGA 6 ( http://www.megasoftware.net/) [ 58] program to construct the phylogenetic tree with the Neighbor-joining method [ 59], with 1000 bootstrap replications to assess nodal support in the tree. 2.5. Packed-Bed Biofilm Reactor (PBBR) To study the biodegradation kinetics of diclofenac, a packed-bed biofilm reactor was used [ 60, 61, 62, 63], adapted for continuous culture [ 61, 64, 65]. This biological reactor consisted of a bubble column filled with fragments of volcanic stone. It is made of Pyrex-type borosilicate glass, with internal dimensions of 14.5 cm in height and 10.5 cm in diameter. Filtered air was supplied at a volumetric gas flow rate of 0.2 L min −1 through a sintered glass plate diffuser (with a pore diameter 40–100 µm), located at the bottom of the column. The total volume of the bioreactor was 1200 mL, with an operational volume of 690 mL and a support volume of 510 mL. The weight of support material was 1 ± 0.037 Kg of fragments of porous volcanic stone, with an equivalent diameter of 19.9 ± 1.45 mm, which acted as a support for the biofilm. All experiments were carried out at room temperature (about 23 ± 2 °C) and pH (6.9 to 7.1 ± 0.005) in a laboratory-scale packed-bed biofilm reactor. A schematic diagram of the PBBR is shown in Figure 1. 2.6. PBBR Operation To saturate the porous support with the diclofenac, the PBBR was initially operated in abiotic conditions by feeding it with MSM-D (containing 100 mg L −1 of diclofenac) at a flow rate of 4.3 L d −1, maintaining an airflow rate of 0.2 L min −1. The diclofenac concentration in the outflowing liquid was analyzed twice a day. The support material was saturated when the diclofenac concentrations were equal in the inflowing and outflowing liquid. The PBBR was inoculated with a cell suspension of the microbial community. After inoculation and colonization of the PBBR with the microbial community, the reactor was operated in continuous mode [ 65, 66, 67, 68]. The continuous culture system was carried out at room temperature (about 23 ± 2 °C) and the pH was monitored during the operation (6.9 to 7.1 ± 0.005). 2.7. Analytical Methods 2.7.1. Physicochemical Characteristics of the Pharmaceutical Effluent The physicochemical tests [ 69] performed on the pharmaceutical effluent were pH (Method 8156 Hach), chemical oxygen demand (Method 8000 Hach), chloride (Method 8113 Hach), nitrogen [total (Method 10208 Hach)], phosphorus [reactive (Method 10209 Hach) and total (Method 10210 Hach)] and sulfide (Method 10254 Hach). 2.7.2. Spectrophotometric Determination of Diclofenac For rapid estimation, diclofenac concentration was determined by its UV absorption at 276 nm in a DR 6000 Hach spectrophotometer (Hach, Loveland, CO, USA). 2.7.3. Chemical Oxygen Demand (COD) Chemical oxygen demand was used as an indirect measure of the degradation of diclofenac and the excipients (inactive ingredients) present in pharmaceutical-grade diclofenac tablets. A close reflux method 8000 was used for COD determination, using a DRB 200 Hach reactor (Hach, Loveland, CO, USA). The reactive kit used could determine COD levels from 0.7 to 40 mg L −1 [ 69]. 2.7.4. Chloride Released by Dehalogenation of Diclofenac The mercuric thiocyanate method was used to measure chloride concentrations in the PBBR effluents. A spectrophotometric method 8113 was applied to determine the chloride released by dehalogenation. The reagent kit allowed Cl − to be determined in a range of 0.1 to 25 mg L −1 [ 69]. 2.7.5. Determination of Diclofenac by HPLC A high-performance liquid chromatograph was used to analyze the diclofenac content in the samples. All samples were injected three times. The HPLC analysis was performed on a Knauer Azura Assistant ASM 2.1 L System (Knauer, Steglitz-Zehlendorf, Berlin, Germany). The system was equipped with an analytical column [Nova-Pak C18 (4 µm, 150 × 3.9 mm)] purchased from Waters (Waters, Milford, MA, USA), and a UV detector. The mobile phase contained 0.05 M phosphate buffer (pH 2.7) and acetonitrile (1:1, vv−1) with the flow rate of 1.0 mL min −1. The wavelength of the UV detector was 276 nm. The column temperature was 25 °C and injection volume of samples was 10 µL. 2.7.6. Determination of Cell Concentration 2.7.7. Ecotoxicity Bioassays in the PBBR Effluents Effect of PBBR Effluents Toxicity on Germination of Lactuca sativa Seeds Seeds of Lactuca sativa were used for seed germination test. The conventional environmental bioassay was done according to the ASTM standard germination protocol [ 70]. The seeds were surface-sterilized in sodium hypochlorite solution (0.05%) for half an hour and rinsed with deionized water afterwards, then the L. sativa seeds were placed into a plastic bag and stored at 4 °C for 1 month before germination. Twelve seeds were placed onto filter paper (diameter 90 mm) in a glass Petri dish (diameter 100 mm) and wet with 5 mL of the solution to be tested (PBBR influents or PBBR effluents or deionized water for the controls). The Petri dish was covered with lid, was sealed with laboratory film and was put in an incubator at 25 °C for 120 h in the dark and humidity (80%) conditions. The bioassay was replicated six times per treatment. Toxicity Assessment of the PBBR Effluents to Microalgae Pseudokirchneriella subcapitata Microalgae toxicity testing with Pseudokirchneriella subcapitata was conducted following the OECD Guideline 201 [ 49]. P. subcapitata was axenically cultured in AAP medium [ 49] with PBBR influents or PBBR effluents and AAP medium as control to evaluate the effect of the treatment on diclofenac toxicity. Independent bioassays were started, and each assay had three replicates. AAP medium was used as control. The cell density was determined daily with a Neubauer chamber for four days. 3. Results 3.1. Pharmaceutical Effluent The analytical parameters of the pharmaceutical effluent are shown in Table 1. According to the European Commission [ 71] and Mexican Regulations [ 72, 73, 74], the values of pH, chemical oxygen demand, chloride, nitrogen (total), phosphorus (reactive and total), and sulfide did not exceed the detection limit of the analytical method employed. These values suggest that some pharmaceutical compounds are removed inside the pharmaceutical wastewater treatment plant; for this reason, the pharmaceutical effluent was used only for the isolation of microbial community. It is important to mention that no residues of diclofenac were detected by spectrophotometry or HPLC in the pharmaceutical effluent. 3.2. Identification of the Predominant Bacterial Strains in the Microbial Community A microbial community was obtained from pharmaceutical effluent collected from the industrial area of Estado de México, MEX. Through spread plate method and streak plate method, using 100 mg L −1 of diclofenac, five bacterial strains were isolated. All five strains are capable of growing using diclofenac as their sole source of carbon and nitrogen. The five strains, labeled as A-UA-2019, C-UA-2019, C2-UA-2019, D-UA-2019, and E-UA-2019, were identified by sequence similarity of their 16S rDNA amplicons with known sequences from the NCBI GenBank. Table 2 presents the bacterial identification using 16S rDNA sequencing, and the phylogenic relationships of the bacterial strains identified in the microbial community are represented in Figure 2. The microorganisms present in the microbial community have demonstrated an impressive capacity for adapting to diclofenac. This makes them highly efficient biological agents for environmental remediation and opens opportunities for their use in various treatment strategies. 3.3. Removal of Diclofenac in the Continuously Operated PBBR Before beginning studies on the biodegradation of diclofenac by the microbial community, abiotic control was established in the packed-bed reactor, under the same conditions but without inoculation, thereby avoiding possible microbial contamination. The PBBR was operated in a continuous regime for 200 days. Twelve flow rates (F) were probed throughout PBBR operation. They varied from 0.6 to 7.2 L d −1, corresponding to dilution rates (D from 0.870 to 10.435 d −1 and hydraulic retention times (HRT) from 1.150 to 0.096 d. Figure 3 shows the volumetric removal rates of diclofenac (R VD) as a function of the volumetric loading rates of diclofenac (B VD) probed in the PBBR. The removal rates of diclofenac and the excipients (inactive ingredients) present in the tablets of the pharmaceutical grade diclofenac were also measured in terms of COD ( Figure 4). In both cases, almost complete removal efficiencies were obtained ( Figure 5). For this reason, it was not necessary to observe the inhibitory or synergistic (co substrate) effect of the excipients present in the commercial formulation. It is important to note that the estimated COD equivalence for the diclofenac was 1.509 [g O 2] [g diclofenac] −1, and the carbon and nitrogen contents of diclofenac are 0.528 [g C] [g diclofenac] −1 and 0.044 [mg N] [mg diclofenac] −1, respectively. These values were used to calculate the amount of oxygen required to oxidize the diclofenac into CO 2, ammonia and water. Figure 3Figure 4 show that the volumetric removal rates of diclofenac did not decay, even at the highest volumetric loading rates probed (B VD = 1.043 g L −1d −1), which suggests that the PBBR did not reach its operational limit in 200 days. At all dilution rates ( Figure 5), high removal efficiencies for diclofenac were obtained (from 99.38 to 99.98%). Numerous studies on the biodegradation of diclofenac in batch cultures have been published, and their results are generally reported in terms of the diclofenac removal efficiency [ 24, 32, 36]. However, there is a notable lack of research focused on continuous culture processes. In this context, the results of this study, which used a packed-bed biofilm reactor for diclofenac biodegradation in continuous culture, were more efficient than those of previous studies. Mohamed et al. [ 37] reported removal efficiencies of 97% at a diclofenac concentration of 40 mg L −1 in batch culture, whereas Suleiman et al. [ 40] were unable to biodegrade the diclofenac (100 mg L −1) in continuous culture. Although several studies report efficiencies of over 90%, these usually use additional carbon [ 33, 34, 35] and nitrogen sources [ 22, 34, 36]. 3.4. Diclofenac Dechlorination Based on the chlorine content of the diclofenac molecule, 0.2232 [g Cl −] [g diclofenac] −1, the calculated concentration of chloride ion at the PBBR influent was 0.02232 [g Cl −] L −1. The release of chlorides during the biodegradation of diclofenac provides an idea of the low reactivity of the possible metabolites formed during the biodegradation process ( Figure 6). 3.5. Cell Concentration 3.6. Ecotoxicity Bioassays 3.6.1. The PBBR Effluents’ Toxicity on Germination of Lactuca sativa Seeds The results indicate ( Figure 8) that there are no significant differences between the PBBR effluents and the controls (Dunnett’s test, p > 0.05). However, significant differences are observed between the PBBR influents and the PBBR effluents (Tukey’s pairwise comparisons, p 0.05). 4. Discussion The predominant bacterial genera identified in the microbial community were Bacillus, KosakoniaLysinibacillus ( Table 2). Identification was performed through PCR amplification and bidirectional sequencing of bacterial 16S rDNA amplicons of approximately 1500 base pairs. These were then compared with known 16S rDNA sequences from the NCBI GenBank using the Basic Local Alignment Search Tool (BLAST; http://www.ncbi.nlm.nih.gov/). Additionally, Figure 2 shows the phylogenetic tree, which was constructed using MEGA6 software ( http://www.megasoftware.net/) [ 58] with 1000 bootstrap replications to assess nodal support. The Jukes–Cantor substitution model was used. There are reports on the ability of the genus Bacillus to degrade diclofenac; for example, Bacillus subtilis BMT4i can degrade diclofenac (67%) within 72 h in batch cultures and, when supplied with additional sources of carbon and nitrogen, the percentage of degradation increases by 20% [ 34]. A bacterial consortium consisting of Bacillus thuringiensis B1 and Pseudomonas moorei KB4 in a batch reactor is able to degrade paracetamol, ibuprofen, naproxen, and diclofenac [ 24]. Bacillus paralicheniformis HAS-1 has the ability to remove 38.88% of diclofenac after 15 days in a batch culture [ 89]. Bacillus safensis HS4–2 and Bacillus haynesii TXO1–1SG1 are known to metabolize diclofenac, ibuprofen and ketoprofen [ 90]. Bacillus paranthracis DYD-1 is able to degrade 50 mg L −1 of diclofenac within 45 h in a batch reactor [ 39]. To date, the degradation of diclofenac has not been confirmed in any Kosakonia or Lysinibacillus strain. Consequently, these microorganisms have not been genetically characterized and, as a result, the sequences of the genes encoding the enzymes involved in the biodegradation of diclofenac are unknown. In biodegradation studies, the removal rate and removal efficiency are two parameters that can be used to evaluate the degradation capacity of microorganisms in a bioprocess [ 24, 38, 91]. Figure 3 shows the behavior of the volumetric removal rates of diclofenac, as measured by HPLC, at different volumetric loading rates in the PBBR. Removal efficiencies ranging from 99.38 to 99.98% were obtained at all dilution rates tested ( Figure 5), indicating that the PBBR can support high loadings of diclofenac. The biodegradation of diclofenac was indirectly estimated using COD ( Figure 4), which showed a similar effect to the HPLC measurement and achieved high removal efficiencies ( Figure 5). These results suggest the removal of diclofenac and its possible biodegradation products, as high removal rates were achieved with both methods [ 7, 27, 32]. It is noteworthy that, during the biodegradation process, the excipients or inactive ingredients in pharmaceutical-grade diclofenac tablets, specifically talc (silicates), did not interfere with or alter the process; their role was limited to acting as inert ingredients. Finally, toxicity tests were conducted to evaluate the adverse effects of the PBBR influents and effluents on the test organisms [ 49, 70, 71], with the purpose of verifying if the presence of possible metabolic intermediates formed during the diclofenac biodegradation process could generate toxicity higher than the original compound. The results clearly showed that the PBBR influents had an inhibitory effect on L. sativa germination (50%) and P. subcapitata growth (80%), indicating that they are highly toxic [ 49, 70, 80, 81]. By contrast, the results for the PBBR effluents were very similar to the controls, indicating no significant differences between the two groups (Dunnett’s test, p > 0.05). However, significant differences are observed between the PBBR influents and the PBBR effluents (Tukey’s pairwise comparisons, p < 0.05). The different values obtained in the toxicity bioassays are due to interspecies variability and their high sensitivity [ 42, 43, 44, 45, 46, 47]. This reflects the level of confidence, accuracy, and reproducibility of the toxicity tests [ 42, 43, 44, 45, 46, 47, 80]. Recent research shows that the toxic effects of diclofenac are evaluated using more than one test organism, with the aim of understanding the negative impact it has on the environment, specifically on water bodies [ 42, 43, 44, 45, 46, 47, 49, 70]. Through toxicity bioassays, it is possible to determine water quality or the effectiveness of the treatment used to decontaminate it. Two of the bioindicators used for this purpose are L. sativa [ 45, 46, 47, 70] and P. subcapitata [ 42, 43, 44, 49], as they have short response times and are highly sensitive to toxic substances. Based on these results, it can be concluded that the proposed reaction system for the biodegradation process of diclofenac is functional, operationally stable and therefore effective and efficient. 5. Conclusions A microbial community capable of degrading diclofenac was selected from a pharmaceutical effluent sample and immobilized in a PBBR operating under continuous culture conditions. The removal efficiencies obtained, both by HPLC and COD, along with the release of chlorides, suggest the degradation of diclofenac. It is also important to note that the microbial community did not require a co substrate, as diclofenac was utilized as its sole source of carbon and nitrogen. The proposed reaction system permitted high concentrations of both planktonic and sessile cells, thereby facilitating diclofenac degradation at high volumetric loading rates. At all dilution rates tested, the influent to the PBBR was toxic to both test organisms. Therefore, ecotoxicity bioassays were conducted to evaluate the effectiveness of the treatment and assess the quality of the PBBR effluent. Author Contributions Conceptualization, Y.B.-M., L.C.C.-C. and O.A.R.-M.; methodology, Y.B.-M., L.C.C.-C., O.A.R.-M., D.T.-A. and S.B.-R.; investigation, Y.B.-M., L.C.C.-C., O.A.R.-M., D.T.-A. and S.B.-R.; writing—original draft preparation, Y.B.-M., L.C.C.-C., O.A.R.-M., D.T.-A. and S.B.-R.; writing—review and editing, Y.B.-M., L.C.C.-C., O.A.R.-M., D.T.-A. and S.B.-R.; supervision, L.C.C.-C. and O.A.R.-M.; funding acquisition, L.C.C.-C. and O.A.R.-M. All authors have read and agreed to the published version of the manuscript. Funding This research received no external funding. Data Availability Statement The original contributions presented in this study are included in the article. Further inquiries can be directed to the corresponding authors. Acknowledgments The authors would like to express their sincere thanks to Universidad Anáhuac and Instituto Politécnico Nacional. Conflicts of Interest The authors declare no conflicts of interest. References Wilkinson, J.; Hooda, P.S.; Barker, J.; Barton, S.; Swinden, J. Occurrence, fate and transformation of emerging contaminants in water: An overarching review of the field. Environ. Pollut. 2017, 23, 954–970. [] [ CrossRef] Chen, K.; Zeng, X.; Hao, Q.; Yang, H.; Meng, R.; Sun, Z.; Jin, Z. The multi-faceted impacts of anthropogenic groundwater recharge on groundwater environment: A review. Phys. Chem. Earth 2026, 143, 104396. [] [ CrossRef] Nguyen, M.K.; Lin, C.; Bui, X.T.; Rakib, M.R.J.; Nguyen, H.L.; Truong, Q.M.; Hoang, H.G.; Tran, H.T.; Malafaia, G.; Idris, A.M. 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Air vent port (1), sample port (2), liquid and air output (3), air input (4), sintered glass plate diffuser (5), reactor support (6), liquid medium input (7), packed-bed (8), liquid level (9), inoculation port (10). Figure 1. Laboratory-scale packed-bed reactor. Air vent port (1), sample port (2), liquid and air output (3), air input (4), sintered glass plate diffuser (5), reactor support (6), liquid medium input (7), packed-bed (8), liquid level (9), inoculation port (10). Figure 2. Phylogenetic tree based on 16S rDNA gene sequences showing the relationship of the bacterial strains identified in the microbial community. GenBank accession numbers are indicated in parentheses. The bar indicates the number of substitutions per
