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Eco-toxicity investigation of polymer materials was considered extremely necessary for their potential menace, which was widely use as mulching materials in agricultural. In this study, polyethylene (PE), polystyrene (PS) and synthetic biomaterials-Ecoflex and cellulose were applying into soil cultivated with two potential indicator plants species: oat (Avena sativa) and red radish (Raphanus sativum). Variety of chemical, biochemical parameters and enzyme activity in soil were proved as effective approach to evaluate polymers phytotoxicity in plant-soil mesocosm. The F-value of biomass, pH, heavy metal and electoral conductivity of Raphanus behaved significant different from T0. Significant analysis results indicated biodegradation was fast in PE than PS, besides, heavy metals were dramatically decrease in the end implied the plant absorption may help decrease heavy metal toxicity. The increase value at T2 of Dehydrogenase activity (0.84 higher than average value for Avena & 0.91 higher for Raphanus), Metabolic Index (3.12 higher than average value for Avena & 3.81 higher for Raphanus) means during soil enzyme activity was promoted by biodegradation for its heterotrophic organisms' energy transportation was stimulated. Statistics analysis was carried on Biplot PC1 (24.2% of the total variance), PC2 (23.2% of the total variance), versus PC3 (22.8% of the total variance), which indicated phosphatase activity and metabolic index was significant correlated, and high correlation of ammonium and protease activity. Furthermore, the effects were more evident in Raphanus treatments than in Avena, suggesting the higher sensitivity of Raphanus to polymers treatment, which indicate biodegradation of polymers in Raphanus treatment has produced intermediate phytotoxic compounds.
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Raphanus , Poluentes do Solo , Biodegradação Ambiental , Polímeros/toxicidade , Solo , Poluentes do Solo/análise , Poluentes do Solo/toxicidadeRESUMO
Rice fungal pathogens, responsible for severe rice yield loss and biotoxin contamination, cause increasing concerns on environmental safety and public health. In the paddy environment, we observed that the asymptomatic rice phyllosphere microenvironment was dominated by an indigenous fungus, Aspergillus cvjetkovicii, which positively correlated with alleviated incidence of Magnaporthe oryzae, one of the most aggressive plant pathogens. Through the comparative metabolic profiling for the rice phyllosphere microenvironment, two metabolites were assigned as exclusively enriched metabolic markers in the asymptomatic phyllosphere and increased remarkably in a population-dependent manner with A. cvjetkovicii. These two metabolites evidenced to be produced by A. cvjetkovicii in either a phyllosphere microenvironment or artificial media were purified and identified as 2(3H)-benzofuranone and azulene, respectively, by gas chromatography coupled to triple quadrupole mass spectrometry and nuclear magnetic resonance analyses. Combining with bioassay analysis in vivo and in vitro, we found that 2(3H)-benzofuranone and azulene exerted dissimilar actions at the stage of infection-related development of M. oryzae. A. cvjetkovicii produced 2(3H)-benzofuranone at the early stage to suppress MoPer1 gene expression, leading to inhibited mycelial growth, while azulene produced lately was involved in blocking of appressorium formation by downregulation of MgRac1. More profoundly, the microenvironmental interplay dominated by A. cvjetkovicii significantly blocked M. oryzae epidemics in the paddy environment from 54.7 to 68.5% (p < 0.05). Our study first demonstrated implication of the microenvironmental interplay dominated by indigenous and beneficial fungus to ecological balance and safety of the paddy environment.
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Magnaporthe , Oryza , Aspergillus , Proteínas Fúngicas , Cromatografia Gasosa-Espectrometria de Massas , Incidência , Doenças das Plantas , TemefósRESUMO
Cadmium (Cd) and arsenic (As) are two of the most toxic elements. However, the chemical behaviors of these two elements are different, making it challenging to utilize a single adsorbent with high adsorption capacity for both Cd(II) and As(V) removal. To solve this problem, we synthesized HA/Fe-Mn oxides-loaded biochar (HFMB), a novel ternary material, to perform this task, wherein scanning electron microscopy (SEM) combined with EDS (SEM-EDS) was used to characterize its morphological and physicochemical properties. The maximum adsorption capacity of HFMB was 67.11â¯mg/g for Cd(II) and 35.59â¯mg/g for As(V), which is much higher compared to pristine biochar (11.06â¯mg/g, 0â¯mg/g for Cd(II) and As(V), respectively). The adsorption characteristics were investigated by adsorption kinetics and the effects of the ionic strength and pH of solutions. X-ray photoelectron spectroscopy (XPS) and Fourier-transform infrared spectroscopy (FT-IR) revealed that chelation and deposition were the adsorption mechanisms that bound Cd(II) to HFMB, while ligand exchange was the adsorption mechanism that bound As(V).
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Arsênio/química , Cádmio/química , Poluentes Ambientais/química , Carvão Vegetal , Óxidos/químicaRESUMO
Cell cycle re-entry, in which Fra-1 (transcription factor FOS-related antigen 1) plays an important role, is a key process in neuronal apoptosis. However, the expression and function of Fra-1 in retinal ganglion cell (RGC) apoptosis are unknown. To investigate whether Fra-1 was involved in RGC apoptosis, we performed a light-induced retinal damage model in adult rats. Western blot revealed that up-regulation of Fra-1 expression appeared in retina after light exposure (LE). Immunostaining indicated that increased Fra-1 was mainly expressed in RGCs in retinal ganglion cell layer (GCL) after LE. Co-localization of Fra-1 with active caspase-3 or TUNEL-positive cells in GCL after LE was also detected. In addition, Fra-1 expression increased in parallel with cyclin D1 and phosphorylated mitogen-activated protein kinase p38 (p-p38) expression in retina after LE. Furthermore, Fra-1, cyclin D1, and active caspase-3 protein expression decreased by intravitreal injection of SB203580, a highly selective inhibitor of p38 MAP kinase (p38 MAPK). All these results suggested that Fra-1 may be associated with RGC apoptosis after LE regulated by p38 MAPK through cell cycle re-entry mechanism.
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Apoptose/fisiologia , Estimulação Luminosa/efeitos adversos , Proteínas Proto-Oncogênicas c-fos/biossíntese , Células Ganglionares da Retina/metabolismo , Células Ganglionares da Retina/patologia , Animais , Masculino , Ratos , Ratos Wistar , Retina/metabolismo , Retina/patologiaRESUMO
Pyruvate kinase isozyme type M2 (PKM2), a key glycolytic enzyme, which is involved in ATP generation and pyruvate production, participates in tumor metabolism, growth, and other multiple cellular processes. However, one attractive biological function of PKM2 is that it translocates to the nucleus and induces cell apoptosis. Recently, increased PKM2 has been found in age-related macular degeneration (AMD), but little is known regarding its function in the AMD pathophysiology. To investigate whether PKM2 participated in retinal degeneration, we performed a light-induced retinal damage model in adult rats. Western blot and immunohistochemistry analysis showed a significant up-regulation of PKM2 in retinal ganglion cells (RGCs) layer (GCL) after light exposure. Immunofluorescent labeling indicated that PKM2 located mainly in RGCs. Co-localization of PKM2 and active caspase-3 as well as TUNEL in RGCs suggested that PKM2 might participate in RGC apoptosis. In addition, the expression patterns of cyclin D1 and phosphorylated extracellular signal-regulated kinase (p-ERK) were parallel with that of PKM2. Furthermore, PKM2, cyclin D1, and active caspase-3 protein expression decreased by intravitreal injection of U0126, a highly selective inhibitor of MAPK/ERK kinase. Collectively, we hypothesized that PKM2 might participate in RGC apoptosis after light-induced retinal damage medicated by p-ERK through cycle re-entry mechanism.
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Proteínas de Transporte/biossíntese , Luz/efeitos adversos , Proteínas de Membrana/biossíntese , Células Ganglionares da Retina/metabolismo , Células Ganglionares da Retina/patologia , Hormônios Tireóideos/biossíntese , Regulação para Cima/fisiologia , Fatores Etários , Animais , Apoptose , Injeções Intravítreas , Masculino , Ratos , Ratos Sprague-Dawley , Retina/metabolismo , Retina/patologia , Proteínas de Ligação a Hormônio da TireoideRESUMO
The epiphytic bacteria in aquatic ecosystems, inhabiting a unique ecological niche with significant ecological function, have long been the subject of attention. Habitat characteristics and plant species are believed to be important in controlling the assembly of epiphytic bacteria. However, the underlying principle governing the assembly of the epiphytic bacterial community on macrophytes is far from clear. In this study, we systematically compared the diversity and community composition of epiphytic bacteria both in different habitats and on different species of macrophytes where they were attached. Results suggested that neither the plant species nor the habitat had a significant effect on the diversity and community of epiphytic bacteria independently, indicating that the epiphytic bacterial community composition was correlated to both geographical distance and individual species of macrophytes. Furthermore, almost all of the abundant taxa were shared between different lake regions or macrophyte species, and the most abundant bacteria belonged to Proteobacteria and Firmicutes. Our results demonstrated that the competitive lottery model may explain the pattern of epiphytic bacterial colonization of submerged macrophyte surfaces. This research could provide a new perspective for exploring plant-microbe interaction in aquatic systems and new evidence for the lottery model as the mechanism best explaining the assembly of epiphytic bacteria.
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The organic loading rate (OLR) is a critical parameter affecting the stability of dry anaerobic digestion (AD) of kitchen waste (KW), and significantly impacting the variations in physicochemical parameters and microbial communities. However, the evolution of quorum sensing (QS) and their role on anaerobic biochemical metabolism during the increase in OLR in dry AD remain unknown. Therefore, this study systematically elucidated the matter through multi-omics analysis based on a pilot-scale dry AD of KW. The results demonstrated that fluctuations in the OLR significantly influenced the microbial QS in dry AD. When the OLR ≤4.0 g·VS/L·d, the system operated stably, and methane production increased. The enrichment of Proteobacteria was crucial for sustaining high levels of functional genes associated with various types of QS, including acyl-homoserine lactones (AI-1), autoinducer-2 (AI-2), autoinducer-3 (AI-3), and gamma-aminobutyric acid (GABA). This enabled cooperative communication among microbes under low OLR. Furthermore, most genes associated with these QS processes positively affected hydrolysis, acidogenesis, and methanogenesis. When the OLR increased to 6.0 g·VS/L·d, the fatty acids and hydrogen partial pressure increased significantly. The autoinducing peptides (AIP)-type became the predominant QS and was positively correlated with fatty acids abundance. Syntrophaceticus and Syntrophomonas may promote syntrophic oxidation of acetate at high OLR through AIP-type QS. These findings provided new insights into the QS processes of microbes during dry AD of KW and a theoretical foundation for optimizing biochemical metabolic processes in dry AD through QS.
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Percepção de Quorum , Anaerobiose , Reatores Biológicos/microbiologia , Metano/metabolismo , Proteobactérias/metabolismo , Proteobactérias/genéticaRESUMO
Organic loading rate (OLR) is crucial for determining the stability of dry anaerobic digestion (AD). Digestate recirculation contributes to reactor stability and enhances methane production. Nevertheless, the understanding of how OLR and digestate recirculation affect the abundance and diversity of antibiotics and antibiotic resistance genes (ARGs), as well as the mechanisms involved in the dissemination of ARGs, remains limited. This study thoroughly investigated this critical issue through a long-term pilot-scale experiment. The metabolome analyses revealed the enrichment of various antibiotics, such as aminoglycoside, tetracycline, and macrolide, under low OLR conditions (OLR ≤ 4.0 g·VS/L·d) and the reactor instability. Antibiotics abundance decreased by approximately 19.66-31.69 % during high OLR operation (OLR ≥ 6.0 g·VS/L·d) with digestate recirculation. The metagenome analyses demonstrated that although low OLR promoted reactor stability, it facilitated the proliferation of antibiotic-resistant bacteria, such as Pseudomonas, and triggered functional profiles related to ATP generation, oxidative stress response, EPS secretion, and cell membrane permeability, thereby facilitating horizontal gene transfer (HGT) of ARGs. However, under stable operation at an OLR of 6.0 g·VS/L·d, there was a decrease in ARGs abundance but a notable increase in human pathogenic bacteria (HPB) and mobile genetic elements (MGEs). Subsequently, during reactor instability, the abundance of ARGs and HPB increased. Notably, during digestate recirculation at OLR levels of 6.0 and 7.0 g·VS/L·d, the process attenuated the risk of ARGs spread by reducing the diversity of ARGs hosts, minimizing interactions among ARGs hosts, ARGs, and MGEs, and weakening functional profiles associated with HGT of ARGs. Overall, digestate recirculation aids in reducing the abundance of antibiotics and ARGs under high OLR conditions. These findings provide advanced insights into how OLR and digestate recirculation affect the occurrence patterns of antibiotics and ARGs in dry AD.
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Antibacterianos , Reatores Biológicos , Resistência Microbiana a Medicamentos , Antibacterianos/farmacologia , Anaerobiose , Resistência Microbiana a Medicamentos/genética , Eliminação de Resíduos LíquidosRESUMO
Emerging evidence suggests a link between alterations in the gut microbiome and adverse health outcomes in the hosts exposed to environmental pollutants. Yet, the causal relationships and underlying mechanisms remain largely undefined. Here we show that exposure to biotoxins can affect gut pathobiome assembly in amphibians, which in turn triggers the toxicity of exogenous pollutants. We used Xenopus laevis as a model in this study. Tadpoles exposed to tropolone demonstrated notable developmental impairments and increased locomotor activity, with a reduction in total length by 4.37%-22.48% and an increase in swimming speed by 49.96%-84.83%. Fusobacterium and Cetobacterium are predominant taxa in the gut pathobiome of tropolone-exposed tadpoles. The tropolone-induced developmental and behavioral disorders in the host were mediated by assembly of the gut pathobiome, leading to transcriptome reprogramming. This study not only advances our understanding of the intricate interactions between environmental pollutants, the gut pathobiome, and host health but also emphasizes the potential of the gut pathobiome in mediating the toxicological effects of environmental contaminants.
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Resident microbiota produces small molecules that influence the chemical microenvironments on leaves, but its signalling roles in pathogen defence are not yet well understood. Here we show that Aspergillus cvjetkovicii, enriched in rice leaf microbiota, subverts Rhizoctonia solani infections via small-molecule-mediated interspecies signalling. 2,4-Di-tert-butylphenol (2,4-DTBP), identified as a key signalling molecule within the Aspergillus-enriched microbiota, effectively neutralizes reactive oxygen species-dependent pathogenicity by switching off bZIP-activated AMT1 transcription in R. solani. Exogenous application of A. cvjetkovicii and 2,4-DTBP demonstrated varying degrees of protective effects against R. solani infection in diverse crops, including cucumber, maize, soybean and tomato. In rice field experiments, they reduced the R. solani-caused disease index to 19.7-32.2%, compared with 67.2-82.6% in the control group. Moreover, 2,4-DTBP showed activity against other rice phytopathogens, such as Fusarium fujikuroi. These findings reveal a defensive strategy against phytopathogens in the phyllosphere, highlighting the potential of symbiotic microbiota-driven neutralization of pathogenicity.
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In this study, modified converter slag (CS) was characterized in relation to its physicochemical structure, and used for the simultaneous removal of NH4(+) and PO4(3-) at low concentrations from aqueous solutions. The effects of contact time, pH, adsorbent dosage, and temperature on the adsorption process were studied in batch experiments. The results showed that the adsorption capacity of modified converter slag was found to sharply increase as a result of modification. The optimum pH is 5-8. The adsorption process was able to reach equilibrium in 90 minutes. Kinetic data were best described by the pseudo-second-order model. The sorption isotherms were a good fit with the Langmuir model. The maximum adsorption capacities of modified converter slag for NH4(+) and PO4(3-) were 2.59 mg/g and 1.185 mg/g, respectively. Thermodynamic studies indicated that the adsorption was a spontaneous and endothermic process. The calculated values of enthalpy change indicated that ligand exchange, chemical reactions, and precipitation are dominating mechanisms of PO4(3-) removal, while physisorption and ion-exchange are major mechanisms of NH4(+) removal.
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Amônia/isolamento & purificação , Fosfatos/isolamento & purificação , Água/química , Adsorção , Amônia/análise , Cinética , Microscopia Eletrônica de Varredura , Modelos Teóricos , Fosfatos/análise , Espectrometria por Raios X , Termodinâmica , Difração de Raios XRESUMO
The electroplating process of copper pyrophosphate (Cu2P2O7) results in the production of a large volume of wastewater that contains a high concentration of copper (Cu). Currently, conventional lime precipitation creates a substantial amount of secondary pollution, which adds extra economic and environmental burdens. In this study, we suggest a straightforward method for on-site recovery of Cu from Cu2P2O7 electroplating wastewater. By optimizing various parameters, characterizing the resulting product, assessing its electroplating capabilities, and analyzing the speciation during the reaction, we comprehensively investigated the feasibility and mechanism of this technique. The results demonstrated that, under the optimal conditions (Cu/P molar ratio of 0.96, pH of 5.0, and a reaction time of 5.0 min), the concentration of residual Cu remained stable between 22.2 and 27.7 mg/L, even when the initial Cu concentrations varied. The addition of Cu triggered a series of hydrolysis and ionization reactions, primarily leading to the formation of Cu2P2O7·3H2O. The harvested Cu2P2O7·3H2O proved to be suitable for practical electroplating applications, exhibiting comparable performance to commercially available Cu2P2O7·3H2O. This demonstrates the feasibility of recovering high-purity Cu2P2O7·3H2O from copper electroplating wastewater, offering a promising approach for on-site copper reuse and concurrently reducing the demand for natural copper resources. Furthermore, this approach significantly reduces the generation of solid waste, aligning with the principles of sustainable development.
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Águas Residuárias , Poluentes Químicos da Água , Cobre/química , Galvanoplastia/métodos , Difosfatos , Poluentes Químicos da Água/análiseRESUMO
Single pathogen-targeted disease management measure has shown drawbacks in field efficacy under the scenario of global change. An in-depth understanding of plant pathogenesis will provide a promising solution but faces the challenges of the emerging paradigm involving the plant microbiome. While the beneficial impact of the plant microbiome is well characterized, their potential role in facilitating pathological processes has so far remained largely overlooked. To address these unsolved controversies and emerging challenges, we hereby highlight the pathobiome, the disease-assisting portion hidden in the plant microbiome, in the plant pathogenesis paradigm. We review the detrimental actions mediated by the pathobiome at multiple scales and further discuss how natural and human triggers result in the prevalence of the plant pathobiome, which would probably provide a clue to the mitigation of plant disease epidemics. Collectively, the article would advance the current insight into plant pathogenesis and also pave a new way to cope with the upward trends of plant disease by designing the pathobiome-targeted measure.
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Mutualistic interactions between host plants and their microbiota have the potential to provide disease resistance. Most research has focused on the rhizosphere, but it is unclear how the microbiome associated with the aerial surface of plants protects against infection. Here we identify a metabolic defence underlying the mutualistic interaction between the panicle and the resident microbiota in rice to defend against a globally prevalent phytopathogen, Ustilaginoidea virens, which causes false-smut disease. Analysis of the 16S ribosomal RNA gene and internal transcribed spacer sequencing data identified keystone microbial taxa enriched in the disease-suppressive panicle, in particular Lactobacillus spp. and Aspergillus spp. Integration of these data with primary metabolism profiling, host genome editing and microbial isolate transplantation experiments revealed that plants with these taxa could resist U. virens infection in a host branched-chain amino acid (BCAA)-dependent manner. Leucine, a predominant BCAA, suppressed U. virens pathogenicity by inducing apoptosis-like cell death through H2O2 overproduction. Additionally, preliminary field experiments showed that leucine could be used in combination with chemical fungicides with a 50% reduction in dose but similar efficacy to higher fungicide concentrations. These findings may facilitate protection of crops from panicle diseases prevalent at a global scale.
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Oryza , Ustilaginales , Oryza/genética , Peróxido de Hidrogênio , LeucinaRESUMO
An FI-KR non-separated method coupled with FAAS for the determination of Fe(II) and Fe(III) was developed. With 60 s of sampling at a flow rate of 6.0 mL x min(-1), EF of 41 for Fe(III) and 9 for Fe(II) were obtained. The precision (RSD, n = 11) for Fe(III) and Fe(II) was 2.3% and 3.1% at the 0.04 mg x L(-1) level respectively. When 0.1 per thousand phi TEA was used as masking reagent, the recovery rate for Fe(III) and Fe(II) was from 97% to 101% and from 96% to 100% respectively.
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A flow injection two steps elution method on-line sorption and preconcentration system coupled to flame atomic absorption spectrometry (FAAS) was developed for the determination of trace Zn in water samples. The conventional elution procedure was divided into two steps: elution procedure and detection procedure. During the elution procedure, the eluent was pumped into KR by the suction of the peristaltic pump and through PTFE tube instead of peristaltic pump tube. By the new method, the dispersion of the analyte was decreased notably, and high absorbance peak value was achieved. Because the eluent was not through the peristaltic pump tube, the peristaltic pump tube was protected from being eroded. Emptying procedure was added in order to insure the veracity and repeatability of the experiment of every time. With 60 s (sample throughput of 37 x h(-1)) of sampling at a flow rate of 6.0 mL x min(-1), an enhancement factor (EF) of 28 (higher than 9 achieved by conventional elution method) and a detection limit (3sigma) of 0.35 x L(-1) were obtained. The precision (RSD, n=11) was 2.1% at the 20 microg x L(-1) level. When 0.1% phi triethannolamine was used as masking reagent, the recovery rate was from 98.7% to 99.6%.
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The increasing input of fungicides has emerged as a global concern for agroecosystem stability and sustainability. Agroecosystem resilience has been linked to microbiome response, however, is not well understood. Focusing on a widespread triazole-class fungicide triadimefon in the paddy ecosystem, we characterized that the soils and sediments were dominant triadimefon reservoirs with the peak level at 195 µg kg-1 and 31.3 µg kg-1, respectively, but essential for the resilience of paddy ecosystem to triadimefon. In paddy simulation models, the half-life of triadimefon in soil-sediment was 8.4-28.9 days, while it was prolonged to 86.6-115.5 days after elimination of resident microbial community. Phospholipid fatty acid profiling and high-throughput sequencing showed that the distinctive bacterial community responses contributed to variable degradation of triadimefon in paddy soils and sediments. Sphingomonas and Xanthomonas were identified as positive responders of the keystone taxa in the responsive bacteriome, whereas Enterobacter were negative responders that declined over time. Synthetic assemblages combined with quantitative polymerase chain reaction further validated that Sphingomonas and Xanthomonas were involved in sustaining soil-sediment resilience to triadimefon contamination. Collectively, our results revealed that the shaping of soil and sediment bacteriomes was responsible for the resilience of the paddy agroecosystem to fungicide contamination.
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Fungicidas Industriais , Microbiota , Ecossistema , Fungicidas Industriais/análise , Solo , Microbiologia do Solo , TriazóisRESUMO
China has adopted nuclear power generation as one of the strategic energy sources to resolve the dilemma between its ever-growing energy demand and the associated environmental issues. To achieve the latter, a systematic assessment of the state of the ecosystem near nuclear power plants and its restoration via ongoing recovery actions would be highly desirable and much needed. Current assessment methods are mostly based on the individual components of the ecosystem and the methods are therefore not integrated. In this paper, we report a set of system-based assessment indices to study the restoration of Daya Bay in Guangdong, China where a nuclear power plant has been in operation for 15 years. The results show that decades of intensive exploitation by the various coastal activities have pushed Daya Bay's ecosystem away from its baseline and its structure and functions are impaired; ecosystem restoration does not make up for the weakening of the ecological carrying capacity due to anthropogenic sea-use, nonetheless, the potential for recovery still exists. The case study suggests that the system-based indices can provide integrated information for ecosystem restoration assessment and management.
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Ecossistema , Centrais Nucleares , China , Recuperação e Remediação Ambiental , Poluentes Radioativos da ÁguaRESUMO
The increasing production of waste activated sludge (WAS) from wastewater treatment plants presents an inherent environmental burden. In this study, Free nitrous acid combined with potassium ferrate (FNAâ¯+â¯PF) pretreatment was used to enhance solubilization and biochemical methane potential of WAS. Results indicated that the maximum removal rates of total suspended solid by PF, FNA, and PFâ¯+â¯FNA pretreatment were 21.84%, 38.09%, and 56.17%, respectively. The biochemical methane potential of WAS without pretreatment reached 61.22 L CH4/kg VSS added while this value increased to 147.07 L CH4/kg VSS added after FNAâ¯+â¯PF pretreatment (0.06â¯g/g TSS NaNO2 and 0.25â¯g/g TSS K2FeO4). Shotgun metagenomic analysis revealed that FNAâ¯+â¯PF pretreatment could increase the diversity and stability of microbial communities by shifting methanogenic pathways from strictly acetoclastic to acetoclastic/hydrogenotrophic, thereby enhancing methane production. This study suggested that FNAâ¯+â¯PF pretreatment is a promising technology to reduce WAS and enhance methane production by pretreated WAS during anaerobic digestion.