Bioremediation Applications Research Articles

Profiling The Growth Conditions and Persistent Organic Pollutants (POPS) Tolerance of Phenoliferia glacialis USM-PSY62

Antarctica is characterized by extreme cold, isolated, and unique ecosystems. Nevertheless, Antarctica harbors diverse species of microorganisms, particularly in its ice-covered lakes and subglacial environments. These microorganisms have special adaptations to extreme cold and low-nutrient conditions. Some extremophiles, like psychrophiles can thrive in these harsh environments. Phenoliferia glacialis USM-PSY62, previously identified as Rhodotorula sp. USM-PSY62 is a psychrophilic yeast isolated from the ice brine of Antarctica. However, there is very little information on this psychrophilic yeast. This study aims to characterize the P. glacialis USM-PSY62 through the identification of the optimum growth parameters in different media (Yeast Peptone Dextrose, YPD & Yeast Malt, YM), temperature (4°C, 15°C, 20°C) and pH (6, 7, 8, 9) as well as their ability in carbon assimilation and extracellular enzyme production. It has an optimal growth in YPD compared to YM broth media. P. glacialis USM-PSY62 grows optimally at 15°C and pH 7.0. This Antarctic yeast enters the stationary phase on day six of incubation under optimum conditions. It appeared mainly as elongated-shape and oval-shaped with budding formation and was found to produce extracellular enzymes such as protease and amylase in the presence of 2% glucose concentration in YM media. P. glacialis USM-PSY62 also can assimilate various types of carbon sources including raffinose, arabinose, and maltose. Interestingly, the psychrophilic yeast presented growth in media supplemented with Persistent Organic Pollutants (POPs) such as dichlorophenyldichloroethylene (DDE) and polychlorinated biphenyl (PCB). These preliminary findings suggest that P. glacialis USM-PSY62 has tremendous potential for bioremediation application in polluted cold regions, as well as deepening our knowledge of its optimal growth conditions.

Reductive dehalogenase of Dehalococcoides mccartyi strain CBDB1 reduces cobalt- containing metal complexes enabling anodic respiration

Microorganisms capable of direct or mediated extracellular electron transfer (EET) have garnered significant attention for their various biotechnological applications, such as bioremediation, metal recovery, wastewater treatment, energy generation in microbial fuel cells, and microbial or enzymatic electrosynthesis. One microorganism of particular interest is the organohalide-respiring bacterium Dehalococcoides mccartyi strain CBDB1, known for its ability to reductively dehalogenate toxic and persistent halogenated organic compounds through organohalide respiration (OHR), using halogenated organics as terminal electron acceptors. A membrane-bound OHR protein complex couples electron transfer to proton translocation across the membrane, generating a proton motive force, which enables metabolism and proliferation. In this study we show that the halogenated compounds can be replaced with redox mediators that can putatively shuttle electrons between the OHR complex and the anode, coupling D. mccartyi cells to an electrode via mediated EET. We identified cobalt-containing metal complexes, referred to as cobalt chelates, as promising mediators using a photometric high throughput methyl viologen-based enzyme activity assay. Through various biochemical approaches, we show that cobalt chelates are specifically reduced by CBDB1 cells, putatively by the reductive dehalogenase subunit (RdhA) of the OHR complex. Using cyclic voltammetry, we also demonstrate that cobalt chelates exchange electrons with a gold electrode, making them promising candidates for bioelectrochemical cultivation. Furthermore, using the AlphaFold 2-calculated RdhA structure and molecular docking, we found that one of the identified cobalt chelates exhibits favorable binding to RdhA, with a binding energy of approximately −28 kJ mol−1. Taken together, our results indicate that bioelectrochemical cultivation of D. mccartyi with cobalt chelates as anode mediators, instead of toxic halogenated compounds, is feasible, which opens new perspectives for bioremediation and other biotechnological applications of strain CBDB1.

Effects of simulated low-temperature thermal remediation on the microbial community of a tropical creosote contaminated soil.

In the search for more sustainable remediation strategies for PAH-contaminated soils, an integrated application of thermal remediation and bioremediation (TEB) may allow the use of less impacting temperatures by associating heating to biological degradation. However, the influence of heating on soil microbiota remains poorly understood, especially in soils from tropical regions. This work investigated the effects of low-temperature heating on creosote-contaminated soil bacteria. We used culture-dependent and 16S rRNA sequencing methods to compare the microbial community of soil samples heated to 60 and 100 oC for 1h in microcosms. Heating to 60°C reduced the density of cultivable heterotrophic bacteria compared to control soil (p < 0.05), and exposure to 100°C inactivated the viable heterotrophic community. Burkholderia-Caballeronia-Paraburkholderia (BCP) group and Sphingobium were the predominant genera. Temperature and incubation time affected the Bray-Curtis dissimilarity index (p < 0.05). At 60°C and 30 days incubation, the relative abundance of Sphingobium decreased and BCP increased dominance. The network of heated soil after 30 days of incubation showed fewer nodes and edges but maintained its density and complexity. Both main genera are associated with PAH degradation, suggesting functional redundancy and a likely potential of soil microbiota to maintain biodegradation ability after exposure to higher temperatures. We concluded that TEB can be considered as a potential strategy to bioremediate creosote-contaminated soils, allowing biodegradation in temperature ranges where thermal remediation does not completely remove contaminants. However, we recommend further research to determine degradation rates with this technology.

A New Bacterial Strain Producing Both of the Surfactin and Fengycin Lipopeptide Biosurfactant with Strong Emulsifications on Crude Oil.

A new lipopeptide-producing strain Cytobacillus sp. R3-1 was isolated from the production water of the Daqing oilfield in China and identified based on 16S rRNA gene sequence analyses. The strain R3-1 is capable of simultaneously producing both of the surfactin and fengycin, the two major families of the lipopeptide biosurfactant. The chemical structures of the surfactin and fengycin were confirmed by a combination of the ESI-MS, FT-IR, and amino acid analyses, and the impact of various temperatures, pH, and NaCl concentrations on the emulsifying activity (E24) was investigated. The lipopeptide biosurfactant produced by the strain R3-1 exhibited strong emulsifying activity with E24 value over 60% on crude oil and different hydrocarbons, including the cyclohexane, hexadecane, benzene, toluene, kerosene, diesel oil, and liquid paraffin. Meanwhile, it showed excellent emulsifying activity across a broad range of conditions of the temperature up to 60 °C, NaCl tolerance up to 100 g/L, and pH values between 5 and 9, which suggests that the strain R3-1 is a valuable microbial candidate for the simultaneous production of the surfactin and fengycin lipopeptide biosurfactant with strong emulsifying properties and stability under diverse environmental conditions and is a potential application in environmental bioremediation and enhanced oil recovery.

Screening and Optimization of Pathogenic Strains of Pseudomonas sp. for Cholinesterase Enzyme Activity

The identification and optimization of pathogenic strains of Pseudomonas sp. with significant Cholinesterase enzyme activity has promising implications for environmental and medical applications. This study aimed to screen various Pseudomonas strains to identify those exhibiting high cholinesterase activity and to optimize the conditions for maximal enzyme production. Multiple Pseudomonas strains were isolated and characterized from diverse clinical samples, employing selective media and biochemical assays to determine cholinesterase activity. The strains with maximum enzyme activity were further subjected to optimization experiments, including variations in growth conditions such as Temperature, pH and Nutrient availability. The findings revealed sample no.39 among the collected 50 samples had significant enzyme activity of 0.19151 IU is obtained when choline chloride was 0.3999%, Ammonium Chloride was 0.01M, pH was 7.0, Temperature was 27°C and Time of incubation was 24hrs. These results not only advance our understanding of Pseudomonas sp. enzyme capabilities but also offer potential applications in bioremediation, biosensor development and pharmaceutical industries. Future research will focus on the molecular characterization of the cholinesterase enzyme and its applications in various industrial processes.

Investigating the role of extracellular polymeric substances produced by Parachlorella kessleri in Zn(II) bioremediation using atomic force microscopy

Microalgae, such as Parachlorella kessleri, have significant potential for environmental remediation, especially in removing heavy metals like zinc from water. This study investigates how P. kessleri, isolated from a polluted river in Argentina, can remediate zinc. Using atomic force microscopy (AFM), the research examined the interactions between Zn particles and cells grown with different nitrogen sources—nitrate or ammonium. The results showed that cells grown with nitrate produced extracellular polymeric substances (EPS), while those grown with ammonium did not. Raman spectroscopy revealed distinct metabolic responses based on the nitrogen source, with nitrate-grown cells showing altered profiles after zinc exposure. Zinc exposure also changed the surface roughness and nanomechanical properties of the cells, particularly in those producing EPS. AFM force spectroscopy experiments then confirmed strong Zn binding to EPS in nitrate-grown cells, while interactions were weaker in ammonium-grown cells that lacked EPS. Overall, our results elucidate the critical role of EPS in Zn removal by P. kessleri cells and show that Zn remediation is mediated by EPS adsorption. This study underscores the significance of regulating nitrogen sources to stimulate EPS production, offering insights that are essential for subsequent bioremediation applications.

New Insights on the Glyphosate-Degrading Enzymes C-P Lyase and Glyphosate Oxidoreductase Based on Bioinformatics

Bioremediation, the degradation of environmental pollutants by living organisms, has immense potential to lead to a greener planet. Bioinformatics analysis can contribute to the identification of novel microorganisms, which biodegrade contaminants, or of participating proteins and enzymes, and the elucidation of the complex metabolic pathways involved. In this study, we focus on C-P lyase and glyphosate oxidoreductase (Gox), two enzymes which degrade glyphosate, a widely used pesticide. Amino acid sequences of the two enzymes were collected from a broad range of microorganisms using the KEGG database and BLAST. Based on this, we identified additional lineages, with putative glyphosate-degrading activity, for which no glyphosate-degrading species have been reported yet. The conserved residues in each enzyme were identified via multiple alignments and mapped onto the 3D structures of the enzymes, using PyMOL, leading to novel insights into their function. As the experimental structure of Gox is still unknown, we created structural models based on three different programs and compared the results. This approach can be used to yield insights into the characteristics of potential glyphosate-degrading enzymes. Given the limited information available, such a step is important to gain further knowledge about them, which can contribute to their application in bioremediation in the future.

Insights into the community structure and environmental functions of water hyacinth rhizobiome in urban river ecosystem.

Water hyacinth (WH) is a widespread floating invasive aquatic plant with a prolific reproductive and dispersion rate. With the aid of its root-associated microbes, WH significantly modulates the ecosystem's functioning. Despite their irrevocable importance, the WH microbiome remains unexplored in detail. Here, we present a shotgun sequencing analysis of WH rhizobiome predominant in urban rivers and their surrounding water to unveil the diversity drivers and functional relationship. The core microbiome of the WH mainly consisted of the methane-metabolizing archaebacteria and sulfate-reducing bacteria, which are probably driving the methane and sulfur metabolic flux along the vegetative zone in the water. The beta diversity analysis revealed temporal variations (River WH_2020 vs. WH_2022) (R of 0.8 to 1 and R2 of 0.17 to 0.41), which probably could be attributed to the transient taxa as there was a higher sharing of core bacteria (48%). Also, the WH microbiome significantly differed (R = 0.46 to 1.0 and R2 of 0.18 to 0.52) from its surrounding water. Further, the functional analysis predicted 140 pollutant-degrading enzymes (PDEs) well-implicated in various xenobiotic pollutant degradation, including hydrocarbons, plastics, and aromatic dyes. These PDEs were mapped to bacterial genera such as Hydrogenophaga, Ideonella, Rubrivivax, Dechloromonas, and Thauera, which are well-reported for facilitating the metabolism of xenobiotic compounds. The higher prevalence of metal and biocide resistance genes further highlighted the persistence of resistant microbes assisting WH in bioremediation applications.

Complete biodegradation of glyphosate with microbial consortium YS622: Structural analysis, biochemical pathways, and environmental bioremediation

The large-scale use of glyphosate has led to the continuous accumulation of residues in the environment, disrupting ecological balance and posing a threat to human health. The main objective of this study was to investigate the potential role of microbial consortium YS622 for the bioremediation of glyphosate-contaminated water/soil environments. YS622 could degrade 100 % of 50 mg/L glyphosate within 36 h, an efficiency higher than that of any other consortia or pure microorganisms reported so far. The kinetic parameters of maximum specific degradation rate, half-rate constant, and inhibition coefficient were determined to be 0.2 h−1, 139.0 mg/L, and 25.1 mg/L, respectively. YS622 metabolized glyphosate via the aminomethylphosphonic acid pathway via breaking the C–N bond. In addition, the sequencing analysis revealed that Azospirillum, Cloacibacterium, and Ochrobactrum were the dominant genera of YS622 in the degradation process, indicating that these genera might play pivotal roles in glyphosate biodegradation. Furthermore, the inoculation of YS622 in both sterilized and unsterilized water–sediment systems significantly promoted the degradation of glyphosate, with over 97 % degradation of 60 mg/L glyphosate within 36 h. The discovery of YS622 fills a gap in the synergistic metabolism of glyphosate at the community level and provides a potential candidate for bioremediation applications.

Enhanced phosphorus availability and cadmium remediation using phosphate-solubilizing bacteria-loaded biochar in contaminated soils

Phosphate-solubilizing bacteria (PSB) have garnered extensive attention for their ability to improve soil phosphorus (P) availability and their potential applications in bioremediation. However, the efficacy of PSB is contingent upon their colonization in soils, which can be hindered by the toxicity of heavy metals such as cadmium (Cd). Utilizing biochar as a protective carrier for PSB presents a promising strategy to address this challenge. This study investigated the effects of PSB-loaded biochar on soil P pools and wheat (Triticum aestivum L.) growth under Cd-contaminated conditions. Results indicated that biochar loading facilitated the colonization and functioning of the inoculated PSB strain (Bacillus subtilis), as evidenced by increased soil cultivatable PSB abundance and phosphatase activity as well as increased relative abundance of Bacillus in bacterial community. Consequently, improved soil P availability and reduced soil exchangeable Cd content were observed, leading to a 30.3% increase in wheat shoot biomass, a 50.4% increase in P accumulation, and a 24.1% reduction in Cd accumulation in wheat shoots under the PSB-loaded biochar treatment. Significant shifts in bacterial community composition were revealed by 16S rRNA amplicon sequencing and enhanced microbe-mediated metabolic activities were observed, as demonstrated by soil enzyme assays and metabolome analysis. Overall, these findings underscore the potential of PSB-loaded biochar to enhance P availability, mitigate Cd toxicity, and shape the soil microbial community, offering a promising strategy for sustainable nutrient management and bioremediation in Cd-contaminated agricultural soils.

Antibacterial Activity of Zinc Oxide Nanoparticles Synthesized by Green Eichhornia Crassipes Extract Method

Purpose: Zinc Oxide Nanoparticle (ZnO NPs) production has become more common because of the benefits of the green strategy, which include its ease of use, environmental friendliness, and cheap operating costs. In order to accomplish the goal of green chemistry, the green synthesis process also uses safe solvents like water and ethanol. ZnO NPs are among the metal oxide-NPs used in antibacterial and bioremediation applications. Materials and Methods: Energy Dispersive X-ray spectroscopy (EDX), Fourier Transform Infrared spectroscopy (FT-IR), X-Ray Diffractometer (XRD), and Field Emission Scanning Electron Microscopy (FESEM) were used to analyze the green-produced ZnO NPs. Results: ZnO nanoparticles have an average size of 22.89 nm, which is corroborated by FESEM pictures, indicating that the particles are tiny. The excellent purity of ZnO NPs has been confirmed by EDX data. The antibacterial activity of ZnO NPs was assessed against a few dangerous pathogens. Zinc oxide nanoparticles were shown to have an interesting antibacterial action against both gram-positive and gram-negative bacteria at micromolar concentrations because they exhibit the maximum diameter of inhibition zone at concentrations 100 mg/ml of S. aureus, E. coli, K. pneumonia, Acintobacret spp, S. fecalis reaching (27,19,18,17, and 14) mm, respectively while S. pneumonia were resistant.The ZnO NPs recorded at a concentration of 12.5 mg/ml lowest areas of the inhibition zone against the same isolates reaching (16, 11, 11, 12, and 10) mm while S. pneumonia were resistant, respectively, as well. Conclusion: ZnO NPs, since they have excellent antibacterial properties, and are biocompatible, they will open up a new line of inquiry for antibacterial agent research because they are stable, nontoxic, and harmless.

Novel Feruloyl Esterase for the Degradation of Polyethylene Terephthalate (PET) Screened from the Gut Microbiome of Plastic-Degrading Mealworms (Tenebrio Molitor Larvae).

Mealworms (Tenebrio molitor) larvae can degrade both plastics and lignocellulose through synergistic biological activities of their gut microbiota because they share similarities in chemical and physical properties. Here, a total of 428 genes encoding lignocellulose-degrading enzymes were screened from the gut microbiome of T. molitor larvae to identify poly(ethylene terephthalate) (PET)-degrading activities. Five genes were successfully expressed in E. coli, among which a feruloyl esterase-like enzyme named TmFae-PETase demonstrated the highest PET degradation activity, converting PET into MHET (0.7 mgMHETeq ·h-1·mgenzyme-1) and TPA (0.2 mgTPAeq ·h-1·mgenzyme-1) at 50 °C. TmFae-PETase showed a preference for the hydrolysis of ferulic acid methyl ester (MFA) in the presence of both PET and MFA. Site-directed mutagenesis and molecular dynamics simulations of TmFae-PETase revealed similar catalytic mechanisms for both PET and MFA. TmFae-PETase effectively depolymerized commercial PET, making it a promising candidate for application. Additionally, the known PET hydrolases IsPETase, FsC, and LCC also hydrolyzed MFA, indicating a potential origin of PET hydrolytic activity from its lignocellulosic-degrading abilities. This study provides an innovative strategy for screening PET-degrading enzymes identified from lignocellulose degradation-related enzymes within the gut microbiome of plastic-degrading mealworms. This discovery expands the existing pool of plastic-degrading enzymes available for resource recovery and bioremediation applications.

Microalgae-fungal pellets as novel dual-bioadsorbents for dye and their practical applications in bioremediation of palm oil mill effluent

Microalgae-fungal pellets were applied as novel dual-biosorbents for dye removal compared to fungal pellets. Both pellet types effectively removed anionic dyes better than cationic dyes, with the maximum adsorbing efficiency being nearly 100 % at a wide pH range of 3–8. The adsorption isotherms of anionic Congo Red dye and Coomassie brilliant blue R-250 dye using both pellet types and their biosorption kinetics were intensively studied. Noteworthy, the maximum adsorption capacity and affinity of microalgae-fungal pellets were much higher than those of fungal pellets. Both fungal pellets were also applied in the bioremediation of palm oil mill effluent (POME). The repeated treatment of POME by replacing pellets every 12 h enhanced the percent removal of color, phenolic compounds, and COD up to 90.97 ± 0.36 %, 70.71 ± 0.90 % and 56.55 ± 1.98 %, respectively. This study has demonstrated the promising potential for addressing dye removal and bioremediation of colored-industrial effluent in a sustainable and economically viable manner.

Entrapment of Cyanase from Thermomyces lanuginosus Using Biomimetic Silica and Its Application for Cyanate Bioremediation.

Cyanate, a toxic product from the chemical oxidation treatment of highly toxic cyanide, can be converted to harmless ammonia and carbon dioxide by cyanase (EC 4.2.1.104). Cyanase from Thermomyces lanuginosus was entrapped in biomimetic silica to improve stability and reusability. After entrapment, the enzyme's activity increased by two-fold, and the residual activity after 30-min of incubation at 60 °C also increased by two-fold, compared to the free enzyme. After being stored at room temperature for 28 days, the entrapped cyanase retained 79% of the initial activity, while the free form retained 61%. The immobilized cyanase was successfully applied to cyanate detoxification; the co-entrapment of carbonic anhydrase from Sulfurihydrogenibium azorense decreased the amount of bicarbonate necessary for cyanate detoxification by 50%. The cyanate degradation retained 53% of the initial value after the co-entrapped cyanate and carbonic anhydrase were reused five times.

Mitogenomic profiling and gut microbial analysis of the newly identified polystyrene-consuming lesser mealworm in Kenya

Plastic waste has recently become a major global environmental concern and one of the biggest challenges has been seeking for alternative management options. Several studies have revealed the potential of several coleopteran species to degrade plastics, and this is the first research paper on plastic-degradation potential by lesser mealworms from Africa. This study evaluated the whole mitogenomic profile of the lesser mealworm to further identify the insect. The ability of the mealworm to consume Polystyrene (PS) was also evaluated alongside its associated gut microbiota diversity. Our results showed a complete circular mitochondrial genome which clustered closely to the Alphitobius genus but also suggested that our insect might be a new subspecies which require further identification. During the PS feeding trials, overall survival rates of the larvae decreased when fed a sole PS diet while PS intake was observed to increase over a 30-day period. The predominant bacteria observed in larvae fed PS diets were Kluyvera, Lactococcus, Klebsiella, Enterobacter, and Enterococcus, while Stenotrophomonas dominated the control diet. These findings demonstrated that the newly identified lesser mealworm can survive on a PS diet and has a consortium of important bacteria strongly associated with PS degradation. This work provides a better understanding of bioremediation applications, paving the way for further research into the metabolic pathways of plastic-degrading microbes and bringing hope to solving plastic waste pollution while providing high-value insect protein towards a circular economy.

Radiation Resilience in Bacteria: Mechanism and Bioremediation Applications

Radiation Resilience in Bacteria: Mechanism and Bioremediation Applications

The INSIGHT platform: Enhancing NAD(P)-dependent specificity prediction for co-factor specificity engineering

Enzyme specificity towards cofactors like NAD(P)H is crucial for applications in bioremediation and eco-friendly chemical synthesis. Despite their role in converting pollutants and creating sustainable products, predicting enzyme specificity faces challenges due to sparse data and inadequate models. To bridge this gap, we developed the cutting-edge INSIGHT platform to enhance the prediction of coenzyme specificity in NAD(P)-dependent enzymes. INSIGHT integrates extensive data from principal bioinformatics resources, concentrating on both NADH and NADPH specificities, and utilizes advanced protein language models to refine the predictions. This integration not only strengthens computational predictions but also meets the practical demands of high-throughput screening and optimization. Experimental validation confirms INSIGHT's effectiveness, boosting our ability to engineer enzymes for efficient, sustainable industrial and environmental processes. This work advances the practical use of computational tools in enzyme research, addressing industrial needs and offering scalable solutions for environmental challenges.

Fabrication and characterization of polyvinyl alcohol-chitosan composite nanofibers for carboxylesterase immobilization to enhance the stability of the enzyme

Electrospinning stands out as a flexible and viable method, presenting designed nanoscale materials with customized properties. This research demonstrates the immobilization of carboxylesterase protein Ha006a, reported for its adequacy in pesticide bioremediation by utilizing the electrospinning strategy. This strategy was utilized to create nanofibers by incorporating variable mixtures of biodegradable and cost-effective polyvinyl alcohol (PVA)-chitosan (CS) nanofiber solution (PVA100, PVA96, PVA94, PVA92 and PVA90). All the mixtures were electrospun at a reliable voltage of 21 kV, maintaining a gap of 12 cm from the nozzle. The Ha006a, sourced from Helicoverpa armigera, was consolidated into the optimized PVA90 polymer mixture. The electrospun nanofibers experienced comprehensive characterization utilizing distinctive microscopy and spectroscopy procedures counting FESEM, TGA, XRD and FTIR. The comparative investigation of the esterase property, ideal parameters and stability of the unbound and bound/immobilized Ha006a was scrutinized. The results uncovered an essential elevation in the ideal conditions of enzyme activity post-immobilization. The PVA-CS control nanofiber and Ha006a-PVA-CS showed a smooth structure, including an average breadth of around 170.5 ± 44.2 and 222.5 ± 66.5 nm, respectively. The enzyme-immobilized nanofibers displayed upgraded stability and comprehensive characterization of the nanofiber, which guaranteed genuineness and reproducibility, contributing to its potential as a potent device for bioremediation applications. This investigation opens the way for the manufacture of pesticide-resistant insect enzyme-based nanofibers, unlocking their potential for assorted applications, counting pesticide remediation and ensuring environmental sustainability.

Discovery of the 1-naphthylamine biodegradation pathway reveals a broad-substrate-spectrum enzyme catalyzing 1-naphthylamine glutamylation.

1-Naphthylamine (1NA), which is harmful to human and aquatic animals, has been used widely in the manufacturing of dyes, pesticides, and rubber antioxidants. Nevertheless, little is known about its environmental behavior and no bacteria have been reported to use it as the growth substrate. Herein, we describe a pathway for 1NA degradation in the isolate Pseudomonas sp. strain JS3066, determine the structure and mechanism of the enzyme NpaA1 that catalyzes the initial reaction, and reveal how the pathway evolved. From genetic and enzymatic analysis, a five gene-cluster encoding a dioxygenase system was determined to be responsible for the initial steps in 1NA degradation through glutamylation of 1NA. The γ-glutamylated 1NA was subsequently oxidized to 1,2-dihydroxynaphthalene which was further degraded by the well-established pathway of naphthalene degradation via catechol. A glutamine synthetase-like (GS-like) enzyme (NpaA1) initiates 1NA glutamylation, and this enzyme exhibits a broad substrate selectivity toward a variety of anilines and naphthylamine derivatives. Structural analysis revealed that the aromatic residues in the 1NA entry tunnel and the V201 site in the large substrate-binding pocket significantly influence NpaA1's substrate preferences. The findings enhance understanding of degrading polycyclic aromatic amines, and will also enable the application of bioremediation at naphthylamine contaminated sites.

Different Roles of Dioxin-Catabolic Plasmids in Growth, Biofilm Formation, and Metabolism of Rhodococcus sp. Strain p52.

Microorganisms harbor catabolic plasmids to tackle refractory organic pollutants, which is crucial for bioremediation and ecosystem health. Understanding the impacts of plasmids on hosts provides insights into the behavior and adaptation of degrading bacteria in the environment. Here, we examined alterations in the physiological properties and gene expression profiles of Rhodococcus sp. strain p52 after losing two conjugative dioxin-catabolic megaplasmids (pDF01 and pDF02). The growth of strain p52 accelerated after pDF01 loss, while it decelerated after pDF02 loss. During dibenzofuran degradation, the expression levels of dibenzofuran catabolic genes on pDF01 were higher compared to those on pDF02; accordingly, pDF01 loss markedly slowed dibenzofuran degradation. It was suggested that pDF01 is more beneficial to strain p52 under dibenzofuran exposure. Moreover, plasmid loss decreased biofilm formation, especially after pDF02 loss. Transcriptome profiling revealed different pathways enriched in upregulated and downregulated genes after pDF01 and pDF02 loss, indicating different adaptation mechanisms. Based on the transcriptional activity variation, pDF01 played roles in transcription and anabolic processes, while pDF02 profoundly influenced energy production and cellular defense. This study enhances our knowledge of the impacts of degradative plasmids on native hosts and the adaptation mechanisms of hosts, contributing to the application of plasmid-mediated bioremediation in contaminated environments.