Structural Elucidation of a Metagenomic Urethanase and Its Engineering Towards Enhanced Hydrolysis Profiles.

While plastics like polyethylene terephthalate can already be degraded efficiently by the activity of hydrolases, other synthetic polymers like polyurethanes (PUs) and polyamides (PAs) largely resist biodegradation. In this study, we solved the first crystal structure of the metagenomic urethanase UMG-SP-1, identified highly flexible loop regions to comprise active site residues, and targeted a total of 20 potential hot spots by site-saturation mutagenesis. Engineering campaigns yielded variants with single mutations, exhibiting almost 3- and 8-fold improved activity against highly stable N-aryl urethane and amide bonds, respectively. Furthermore, we demonstrated the release of the corresponding monomers from a thermoplastic polyester-PU and a PA (nylon 6) by the activity of a single, metagenome-derived urethanase after short incubation times. Thereby, we expanded the hydrolysis profile of UMG-SP-1 beyond the reported low-molecular weight carbamates. Together, these findings promise advanced strategies for the bio-based degradation and recycling of plastic materials and waste, aiding efforts to establish a circular economy for synthetic polymers.

Removal of artificial sweeteners in wastewater treatment plants and their degradation during sewage sludge composting with micro- and nano-sized kaolin.

This study surveyed the fates of artificial sweeteners in influent, effluent, and sewage sludge (SS) in wastewater treatment plant, and investigated the effects of Micro-Kaolin (Micro-KL) and Nano-Kaolin (Nano-KL) on nitrogen transformation and sucralose (SUC) and acesulfame (ACE) degradation during SS composting. Results showed the cumulative rate of ACE and SUC in SS was ∼76 %. During SS composting, kaolin reduced NH3 emissions by 30.2-45.38 %, and N2O emissions by 38.4-38.9 %, while the Micro-KL and Nano-KL reduced nitrogen losses by 14.8 % and 12.5 %, respectively. Meanwhile, Micro-KL and Nano-KL increased ACE degradation by 76.8 % and 84.2 %, and SUC degradation by 75.3 % and 77.7 %, and significantly shifted microbial community structure. Furthermore, kaolin caused a positive association between Actinobacteria and sweetener degradation. Taken together, kaolin effectively inhibited nitrogen loss and promoted the degradation of ACE and SUC during the SS composting, which is of great significance for the removal of emerging organic pollutants in SS.

Enhanced biodegradation of benzo[a]pyrene with Trametes versicolor stimulated by citric acid.

Polycyclic aromatic hydrocarbons (PAHs) are a class of persistent organic pollutants with carcinogenic, mutagenic and teratogenic effects. The white-rot fungi in the fungal group have significant degradation ability for high molecular weight organic pollutants. However, exogenous fungi are easily antagonized by indigenous microorganisms. Low molecular weight organic acids, a small molecular organic matter secreted by plants, can provide carbon sources for soil microorganisms. Combining organic acids with white rot fungi may improve the nutritional environment of fungi. In this study, immobilized Trametes versicolor was used to degrade benzo[a]pyrene in soil, and its effect on removing benzo[a]pyrene in soil mediated by different low molecular weight organic acids was investigated. The results showed that when the degradation was 35 days, the removal effect of the experimental group with citric acid was the best, reaching 43.7%. The degradation effect of Trametes versicolor on benzo[a]pyrene was further investigated in the liquid medium when citric acid was added, and the effects of citric acid on the biomass, extracellular protein concentration and laccase activity of Trametes versicolor were investigated by controlling different concentrations of citric acid. In general, citric acid can act as a carbon source for Trametes versicolor and promote its extracellular protein secretion and laccase activity, thereby accelerating the mineralization of benzo[a]pyrene by Trametes versicolor. Therefore, citric acid can be used as a biostimulant in the remediation of PAHs contaminated soil with Trametes versicolor.

Microbial diversity and oil biodegradation potential of northern Barents Sea sediments.

The Arctic, an essential ecosystem on Earth, is subject to pronounced anthropogenic pressures, most notable being the climate change and risks of crude oil pollution. As crucial elements of Arctic environments, benthic microbiomes are involved in climate-relevant biogeochemical cycles and hold the potential to remediate upcoming contamination. Yet, the Arctic benthic microbiomes are among the least explored biomes on the planet. Here we combined geochemical analyses, incubation experiments, and microbial community profiling to detail the biogeography and biodegradation potential of Arctic sedimentary microbiomes in the northern Barents Sea. The results revealed a predominance of bacterial and archaea phyla typically found in the deep marine biosphere, such as Chloroflexi, Atribacteria, and Bathyarcheaota. The topmost benthic communities were spatially structured by sedimentary organic carbon, lacking a clear distinction among geographic regions. With increasing sediment depth, the community structure exhibited stratigraphic variability that could be correlated to redox geochemistry of sediments. The benthic microbiomes harbored multiple taxa capable of oxidizing hydrocarbons using aerobic and anaerobic pathways. Incubation of surface sediments with crude oil led to proliferation of several genera from the so-called rare biosphere. These include Alkalimarinus and Halioglobus, previously unrecognized as hydrocarbon-degrading genera, both harboring the full genetic potential for aerobic alkane oxidation. These findings increase our understanding of the taxonomic inventory and functional potential of unstudied benthic microbiomes in the Arctic.

Unraveling the mechanisms of benzo[a]pyrene degradation by Pigmentiphaga kullae strain KIT-003 using a multi-omics approach.

Polycyclic aromatic hydrocarbons (PAHs), notably benzo[a]pyrene (BaP), are environmental contaminants with multiple adverse ecological implications. Numerous studies have suggested the use of BaP biodegradation using various bacterial strains to remove BaP from the environment. This study investigates the BaP biodegradation capability of Pigmentiphaga kullae strain KIT-003, isolated from the Nak-dong River (South Korea) under specific environmental conditions. The optimum conditions of biodegradation were found to be pH 7.0, 35°C, and a salinity of 0 %. GC-MS analysis suggested alternative pathways by which KIT-003 produced catechol from BaP through several intermediate metabolites, including 4-formylchrysene-5-carboxylic acid, 5,6-dihydro-5,6-dihydroxychrysene-5-carboxylic acid (isomer: 3,4-dihydro-3,4-dihydroxychrysene-4-carboxylic acid), naphthalene-1,2-dicarboxylic acid, and 2-hydroxy-1-naphthoic acid. Proteomic profiles indicated upregulation of enzymes associated with aromatic compound degradation, such as nahAc and nahB, and of those integral to the tricarboxylic acid cycle, reflecting the strain's adaptability to and degradation of BaP. Lipidomic analysis of KIT-003 demonstrated that BaP exposure induced an accumulation of glycerolipids such as diacylglycerol and triacylglycerol, indicating their crucial role in bacterial adaptation mechanisms under BaP stress. This study provides significant scientific knowledge regarding the intricate mechanisms involved in BaP degradation by microorganisms.

Biodegradation of humic acids by Streptomyces rochei to promote the growth and yield of corn.

Humic acids (HAs) are organic macromolecules that play an important role in improving soil properties, plant growth and agronomic parameters. However, the feature of relatively complex aromatic structure makes it difficult to be degraded, which restricts the promotion to the crop growth. Thus, exploring microorganisms capable of degrading HAs may be a potential solution. Here, a HAs-degrading strain, Streptomyces rochei L1, and its potential for biodegradation was studied by genomics, transcriptomics, and targeted metabolomics analytical approaches. The results showed that the high molecular weight HAs were cleaved to low molecular aliphatic and aromatic compounds and their derivatives. This cleavage may be associated with the laccase (KatE). In addition, the polysaccharide deacetylase (PdgA) catalyzes the removal of acetyl groups from specific sites on the HAs molecule, resulting in structural changes. The field experiment showed that the degraded HAs significantly promote the growth of corn seedlings and increase the corn yield by 3.6 %. The HAs-degrading products, including aromatic and low molecular weight aliphatic substances as well as secondary metabolites from S. rochei L1, might be the key components responsible for the corn promotion. Our findings will advance the application of HAs as soil nutrients for the green and sustainable agriculture.

Draft genome sequence of the Tremellomycetes yeast Papiliotrema laurentii 5307AH, isolated from aircraft.

Papiliotrema laurentii 5307AH was isolated from an aircraft polymer-coated surface. The genome size is 19,510,785 bp with a G + C content of 56%. The genome harbors genes encoding oxygenases, cutinases, lipases, and enzymes for styrene degradation, all of which could play a critical role in survival on xenobiotic surfaces.

Pseudomonas putida KT2440: the long journey of a soil-dweller to become a synthetic biology chassis.

Although members of the genus Pseudomonas share specific morphological, metabolic, and genomic traits, the diversity of niches and lifestyles adopted by the family members is vast. One species of the group, Pseudomonas putida, thrives as a colonizer of plant roots and frequently inhabits soils polluted with various types of chemical waste. Owing to a combination of historical contingencies and inherent qualities, a particular strain, P. putida KT2440, emerged time ago as an archetype of an environmental microorganism amenable to recombinant DNA technologies, which was also capable of catabolizing chemical pollutants. Later, the same bacterium progressed as a reliable platform for programming traits and activities in various biotechnological applications. This article summarizes the stepwise upgrading of P. putida KT2440 from being a system for fundamental studies on the biodegradation of aromatic compounds (especially when harboring the TOL plasmid pWW0) to its adoption as a chassis of choice in metabolic engineering and synthetic biology. Although there are remaining uncertainties about the taxonomic classification of KT2440, advanced genome editing capabilities allow us to tailor its genetic makeup to meet specific needs. This makes its traditional categorization somewhat less important, while also increasing the strain's overall value for contemporary industrial and environmental uses.

Study on the biodegradation characteristics and mechanism of tetracycline by Serratia entomophila TC-1.

Microbial degradation is an important solution for antibiotic pollution in livestock and poultry farming wastes. This study reports the isolation and identification of the novel bacterial strain Serratia entomophila TC-1, which can degrade 87.8 % of 200 mg/L tetracycline (TC) at 35 °C, pH 6.0, and an inoculation amount of 1 % (v/v). Based on the intermediate products, a possible biological transformation pathway was proposed, including dehydration, oxidation ring opening, decarbonylation, and deamination. Using Escherichia coli and Bacillus subtilis as biological indicators, TC degraded metabolites have shown low toxicity. Whole-genome sequencing showed that the TC-1 strain contained tet (d) and tet (34), which resist TC through multiple mechanisms. In addition, upon TC exposure, TC-1 participated in catalytic and energy supply activities by regulating gene expression, thereby playing a role in TC detoxification. We found that TC-1 showed less interference with changes in the bacterial community in swine wastewater. Thus, TC-1 provided new insights into the mechanisms responsible for TC biodegradation and can be used for TC pollution treatment.

Synergistic preparation of a straw fiber/polylactic acid composite with high toughness and strength through interfacial compatibility enhancement and elastomer toughening.

Plant fiber-reinforced polylactic acid (PLA) composites are extensively utilized in eco-friendly packaging, sports equipment, and various other applications due to their environmental benefits and cost-effectiveness. However, PLA suffers from brittleness and poor toughness, which restricts its use in scenarios demanding high toughness. To expand the application range of plant fiber-reinforced PLA-based composites and enhance their poor toughness, this study employed a two-step process involving wheat straw fiber (WF) to improve the interfacial compatibility between WF and PLA. Additionally, four elastomeric materials-poly (butylene adipate-co-terephthalate) (PBAT), poly (butylene succinate) (PBS), polycaprolactone (PCL), and polyhydroxyalkanoate (PHA)-were incorporated to achieve a mutual reactive interface enhancement and elastomeric toughening. The results demonstrated that Fe3+/TsWF/PLA/PBS exhibited a tensile strength, elongation at break, and impact strength of 34.01 MPa, 14.23 %, and 16.2 kJ/m2, respectively. These values represented a 2.4 %, 86.7 %, and 119 % increase compared to the unmodified composites. Scanning electron microscopy analysis revealed no fiber exposure in the cross-section, indicating excellent interfacial compatibility. Furthermore, X-ray diffraction and differential scanning calorimetry tests confirmed improvements in the crystalline properties of the composites. This work introduces a novel approach for preparing fiber-reinforced PLA-based composites with exceptional toughness and strength.

Environmental stressor assessment of hydrocarbonoclastic bacteria biofilms from a marine oil spill.

The environmental and economic impact of an oil spill can be significant. Biotechnologies applied during a marine oil spill involve bioaugmentation with immobilised or encapsulated indigenous hydrocarbonoclastic species selected under laboratory conditions to improve degradation rates. The environmental factors that act as stressors and impact the effectiveness of hydrocarbon removal are one of the challenges associated with these applications. Understanding how native microbes react to environmental stresses is necessary for effective bioaugmentation. Herein, Micrococcus luteus and M. yunnanensis isolated from a marine oil spill mooring system showed hydrocarbonoclastic activity on Maya crude oil in a short time by means of total petroleum hydrocarbons (TPH) at 144 h: M. luteus up to 98.79 % and M. yunnanensis 97.77 % removal. The assessment of Micrococcus biofilms at different temperature (30 °C and 50 °C), pH (5, 6, 7, 8, 9), salinity (30, 50, 60, 70, 80 g/L), and crude oil concentration (1, 5, 15, 25, 35 %) showed different response to the stressors depending on the strain. According to response surface analysis, the main effect was temperature > salinity > hydrocarbon concentration. The hydrocarbonoclastic biofilm architecture was characterised using scanning electron microscopy (SEM) and atomic force microscopy (AFM). Subtle but significant differences were observed: pili in M. luteus by SEM and the topographical differences measured by AFM Power Spectral Density (PSD) analysis, roughness was higher in M. luteus than in M. yunnanensis. In all three domains of life, the Universal Stress Protein (Usp) is crucial for stress adaptation. Herein, the uspA gene expression was analysed in Micrococcus biofilm under environmental stressors. The uspA expression increased up to 2.5-fold in M. luteus biofilms at 30 °C, and 1.3-fold at 50 °C. The highest uspA expression was recorded in M. yunnanensis biofilms at 50 °C with 2.5 and 3-fold with salinities of 50, 60, and 80 g/L at hydrocarbon concentrations of 15, 25, and 35 %. M. yunnanensis biofilms showed greater resilience than M. luteus biofilms when exposed to harsh environmental stressors. M. yunnanensis biofilms were thicker than M. luteus biofilms. Both biofilm responses to environmental stressors through uspA gene expression were consistent with the behaviours observed in the response surface analyses. The uspA gene is a suitable biomarker for assessing environmental stressors of potential microorganisms for bioremediation of marine oil spills and for biosensing the ecophysiological status of native microbiota in a marine petroleum environment.

Evaluating the low-rank coal degradation efficiency bioaugmented with activated sludge.

Microbial bioaugmentation of coal is considered as a viable and ecologically sustainable approach for the utilization of low-rank coals (LRC). The search for novel techniques to derive high-value products from LRC is currently of great importance. In response to this demand, endeavors have been undertaken to develop microbially based coal solubilization and degradation techniques. The impact of supplementing activated sludge (AS) as a microbial augmentation to enhance LRC biodegradation was investigated in this study. The LRC and their biodegradation products were characterized using the following methods: excitation-emission Matrices detected fluorophores at specific wavelength positions (O, E, and K peaks), revealing the presence of organic complexes with humic properties. FTIR indicated the increased amount of carboxyl groups in the bioaugmented coals, likely due to aerobic oxidation of peripheral non-aromatic structural components of coal. The bacterial communities of LRC samples are primarily composed of Actinobacteria (up to 36.2%) and Proteobacteria (up to 25.8%), whereas the Firmicutes (63.04%) was the most abundant phylum for AS. The community-level physiological profile analysis showed that the microbial community AS had high metabolic activity of compared to those of coal. Overall, the results demonstrated successful stimulation of LRC transformation through supplementation of exogenous microflora in the form of AS.

Biodegradation by Cancer Cells of Magnetite Nanoflowers with and without Encapsulation in PS-b-PAA Block Copolymer Micelles.

Magnetomicelles were produced by the self-assembly of magnetite iron oxide nanoflowers and the amphiphilic poly(styrene)-b-poly(acrylic acid) block copolymer to deliver a multifunctional theranostic agent. Their bioprocessing by cancer cells was investigated in a three-dimensional spheroid model over a 13-day period and compared with nonencapsulated magnetic nanoflowers. A degradation process was identified and monitored at various scales, exploiting different physicochemical fingerprints. At a collective level, measurements were conducted using magnetic, photothermal, and magnetic resonance imaging techniques. At the nanoscale, transmission electron microscopy was employed to identify the morphological integrity of the structures, and X-ray absorption spectroscopy was used to analyze the degradation at the crystalline phase and chemical levels. All of these measurements converge to demonstrate that the encapsulation of magnetic nanoparticles in micelles effectively mitigates their degradation compared to individual nonencapsulated magnetic nanoflowers. This protective effect consequently resulted in better maintenance of their therapeutic photothermal potential. The structural degradation of magnetomicelles occurred through the formation of an oxidized iron phase in ferritin from the magnetic nanoparticles, leaving behind empty spherical polymeric ghost shells. These results underscore the significance of encapsulation of iron oxides in micelles in preserving nanomaterial integrity and regulating degradation, even under challenging physicochemical conditions within cancer cells.

Integrating genomics, molecular docking, and protein expression to explore new perspectives on polystyrene biodegradation.

Faced with the escalating challenge of global plastic pollution, this study specifically addresses the research gap in the biodegradation of polystyrene (PS). A PS-degrading bacterial strain was isolated from the gut of Tenebrio molitor, and genomics, molecular docking, and proteomics were employed to thoroughly investigate the biodegradation mechanisms of Pseudomonas putida H-01 against PS. Using scanning electron microscopy (SEM), Fourier-transform infrared spectroscopy (ATR-FTIR), and contact angle analysis, significant morphological and structural changes in the PS films under the influence of the H-01 strain were observed. The study revealed several potential degradation genes and ten enzymes that were specifically upregulated in the PS degradation environment. Additionally, a novel protein with laccase-like activity, LacQ1, was purified from this strain for the first time, and its crucial role in the PS degradation process was confirmed. Through molecular docking and molecular dynamics (MD) simulations, the interactions between the enzymes and PS were detailed, elucidating the binding and catalytic mechanisms of the degradative enzymes with the substrate. These findings have deepened our understanding of PS degradation.

Enhancing electron transfer in anaerobic process by supercapacitor materials: Polyaniline functionated activated carbon.

Strengthening the direct interspecies electron transfer (DIET) is an effective strategy to improve the performance of anaerobic digestion (AD) process. In this study, the polyaniline functionated activated carbon (AC-PANi) was prepared by chemical oxidative polymerization. This material possessed pseudo-capacitance properties as well as excellent charge transfer capability. The experimental results demonstrated that the incorporation of AC-PANi in AD process could efficiently increase the chemical oxygen demand (COD) removal (18.6 %) and daily methane production rate (35.3 %). The AC-PANi can also act as an extracellular acceptor to promote the synthesis of adenosine triphosphate (ATP) and secretion of extracellular enzymes as well as cytochrome C (Cyt-C). The content of coenzyme F420 on methanogens was also shown to be increased by 60.9 % with the addition of AC-PANi in AD reactor. Overall, this work provides an easy but feasible way to enhance AD performance by promoting DIET between acetate-producing bacteria and methanogens.

Optimizing microbial strain selection for pyrethroid biodegradation in contaminated environments through a TOPSIS-based decision-making system.

Improved and contemporary agriculture relies heavily on pesticides, yet some can be quite persistent and have a stable chemical composition, posing a significant threat to the ecology. Removing harmful effects is upon their degradability. Biodegradation must be emphasized to lower pesticide degradation costs, especially in the soil. Here, a decision-making system was used to determine the best microbial strain for the biodegradation of the pyrethroid-contaminated soil. In this system, the criteria chosen as: pH (C1), Temp (C2), RPM (C3), Conc. (C4), Degradation (%) (C5) and Time required for degradation(hrs) (C6); and five alternatives were Bacillus (A1), Acinetobacter (A2), Escherichia (A3), Pseudomonas (A4), and Fusarium (A5). The best alternative was selected by applying the TOPSIS (technique for order performance by similarity to ideal solution) method, which evaluates based on their closeness to the ideal solution and how well they meet specific requirements. Among all the specified criteria, Acinetobacter (A2) was the best and optimal based on the relative closeness value (( R i ∗ ) = 0.740 (A2) > 0.544 (A5) > 0.480 (A1) > 0.403 (A4) > 0.296 (A3)). However, the ranking of the other alternatives is also obtained in the order Fusarium (A5), Bacillus (A1), Pseudomonas (A4), Escherichia (A3). Hence this study suggests Acinetobacter is the best microbial strain for biodegradation of pyrethroids; while least preference should be given to Escherichia. Acinetobacter, versatile metabolic nature with various xenobiotic compounds' degradation ability, is gram-negative, aerobic, coccobacilli, nonmotile, and nonspore forming bacteria. Due to less study about Acinetobacter it is not in that much frame as the other microorganisms. Hence, considering the Acinetobacter strain for the biodegradation study will give more optimal results than the other microbial strains. Novelty of this study, the TOPSIS method is applied first time in selecting the best microbial strain for the biodegradation of pyrethroid-contaminated soil, considering this selection process as multi-criteria decision-making (MCDM) problem.

Microplastics Biodegradation by Estuarine and Landfill Microbiomes.

Plastic pollution poses a worldwide environmental challenge, affecting wildlife and human health. Assessing the biodegradation capabilities of natural microbiomes in environments contaminated with microplastics is crucial for mitigating the effects of plastic pollution. In this work, we evaluated the potential of landfill leachate (LL) and estuarine sediments (ES) to biodegrade polyethylene (PE), polyethylene terephthalate (PET), and polycaprolactone (PCL), under aerobic, anaerobic, thermophilic, and mesophilic conditions. PCL underwent extensive aerobic biodegradation with LL (99 ± 7%) and ES (78 ± 3%) within 50-60 days. Under anaerobic conditions, LL degraded 87 ± 19% of PCL in 60 days, whereas ES showed minimal biodegradation (3 ± 0.3%). PE and PET showed no notable degradation. Metataxonomics results (16S rRNA sequencing) revealed the presence of highly abundant thermophilic microorganisms assigned to Coprothermobacter sp. (6.8% and 28% relative abundance in anaerobic and aerobic incubations, respectively). Coprothermobacter spp. contain genes encoding two enzymes, an esterase and a thermostable monoacylglycerol lipase, that can potentially catalyze PCL hydrolysis. These results suggest that Coprothermobacter sp. may be pivotal in landfill leachate microbiomes for thermophilic PCL biodegradation across varying conditions. The anaerobic microbial community was dominated by hydrogenotrophic methanogens assigned to Methanothermobacter sp. (21%), pointing at possible syntrophic interactions with Coprothermobacter sp. (a H2-producer) during PCL biodegradation. In the aerobic experiments, fungi dominated the eukaryotic microbial community (e.g., Exophiala (41%), Penicillium (17%), and Mucor (18%)), suggesting that aerobic PCL biodegradation by LL involves collaboration between fungi and bacteria. Our findings bring insights on the microbial communities and microbial interactions mediating plastic biodegradation, offering valuable perspectives for plastic pollution mitigation.

The novel hydrolase IpcH initiates the degradation of isoprocarb in a newly isolated strain Rhodococcus sp. D-6.

Isoprocarb (IPC), a representative monocyclic carbamate insecticide, poses risks of environmental contamination and harm to non-target organisms. However, its degradation mechanism has not been reported. In this study, a newly IPC-degrading strain D-6 was isolated from the genus Rhodococcus, and its degradation characteristics and pathway of IPC were analyzed. A novel hydrolase IpcH, responsible for hydrolyzing IPC to 2-isopropylphenol (IPP), was identified. IpcH exhibited low similarity (< 27 %) with other reported hydrolases, including previously characterized carbamate insecticides hydrolases, indicating its novelty. The Km and kcat values of IpcH towards IPC were 69.99 ± 8.33 µM and 95.96 ± 4.02 s-1, respectively. Also, IpcH exhibited catalytic activity towards various types of carbamate insecticides, including monocyclic carbamates (IPC, fenobucarb and propoxur), bicyclic carbamates (carbaryl and carbofuran), and linear carbamates (oxamyl and aldicarb). The molecular docking and site-directed mutagenesis revealed that His254, His256, His329 and His376 were essential for IpcH activity. Strain D-6 can effectively reduce the toxicity of IPC and IPP towards sensitive organisms through its degradation ability. This study presents the initial report on IPC degradation pathway and molecular mechanism of IPC degradation, and provides a good potential strain for bioremediating IPC and IPP-contaminated environments.

Ancestral sequence reconstruction of the prokaryotic three-domain laccases for efficiently degrading polyethylene.

Biodegradation of polyethylene (PE) plastics is environmentally friendly. To obtain the laccases that can efficiently degrade PE plastics, we generated 9 ancestral laccases from 23 bacterial three-domain laccases through ancestral sequence reconstruction. The optimal temperatures of the ancestral laccases were between 60 °C-80 °C, while their optimal pHs were at 3.0 or 4.0. Without substrate pretreatment and mediator addition, all the ancestral laccases can degrade low-density polyethylene (LDPE) films at pH 7.0 and 60 °C. Among them, Anc52, which shared low sequence identity (18 %-41.7 %) with the reported PE-degrading laccases, was the most effective for LDPE degradation. After the catalytic reactions at 90 °C for 14 h, Anc52 (0.2 mg/mL) induced clear wrinkles and deep pits on the PE film surface detected by scanning electron microscope, and its carbonyl and hydroxyl indices reached 2.08 and 2.42, respectively. Then, we identified the residues 203 and 288 critical for PE degradation through site-directed mutation on Anc52. Moreover, Anc52 be activated by heat treatment (60 °C and 90 °C) at pH 7.0, which gave it a high catalytic efficiency (kcat/Km= 191.73 mM-1·s-1) and thermal stability (half-life at 70 °C = 13.70 h). The ancestral laccases obtained here could be good candidates for PE biodegradation.

Biotransformation of Chlorpyrifos Shewanella oneidensis MR-1 in the Presence of Goethite: Experimental Optimization and Degradation Products.

In this study, the degradation system of Shewanella oneidensis MR-1 and goethite was constructed with chlorpyrifos as the target contaminant. The effects of initial pH, contaminant concentration, and temperature on the removal rate of chlorpyrifos during the degradation process were investigated. The experimental conditions were optimized by response surface methodology with a Box-Behnken design (BBD). The results show that the removal rate of chlorpyrifos is 75.71% at pH = 6.86, an initial concentration of 19.18 mg·L-1, and a temperature of 30.71 °C. LC-MS/MS analyses showed that the degradation products were C4H11O3PS, C7H7Cl3NO4P, C9H11Cl2NO3PS, C7H7Cl3NO3PS, C9H11Cl3NO4P, C4H11O2PS, and C5H2Cl3NO. Presumably, the degradation pathways involved are: enzymatic degradation, hydrolysis, dealkylation, desulfur hydrolysis, and dechlorination. The findings of this study demonstrate the efficacy of the goethite/S. oneidensis MR-1 complex system in the removal of chlorpyrifos from water. Consequently, this research contributes to the establishment of a theoretical framework for the microbial remediation of organophosphorus pesticides in aqueous environments.