Mechanistic insights into sulfur source-driven physiological responses and metabolic reorganization in the fuel-biodesulfurizing Rhodococcus qingshengii IGTS8.

Comparative proteomics and untargeted metabolomics were combined to study the physiological and metabolic adaptations of Rhodococcus qingshengii IGTS8 under biodesulfurization conditions. After growth in a chemically defined medium with either dibenzothiophene (DBT) or MgSO4 as the sulfur source, many differentially produced proteins and metabolites associated with several metabolic and physiological processes were detected including the metabolism of carbohydrates, amino acids, lipids, nucleotides, vitamins, protein synthesis, transcriptional regulation, cell envelope biogenesis, and cell division. Increased production of the redox cofactor mycofactocin and associated proteins was one of the most striking adaptations under biodesulfurization conditions. While most central metabolic enzymes were less abundant in the presence of DBT, a key enzyme of the glyoxylate shunt, isocitrate lyase, was up to 26-fold more abundant. Several C1 metabolism and oligotrophy-related enzymes were significantly more abundant in the biodesulfurizing culture. R. qingshengii IGTS8 exhibited oligotrophic growth in liquid and solid media under carbon starvation. Moreover, the oligotrophic growth was faster on the solid medium in the presence of DBT compared to MgSO4 cultures. In the DBT culture, the cell envelope and phospholipids were remodeled, with lower levels of phosphatidylethanolamine and unsaturated and short-chain fatty acids being the most prominent changes. Biodesulfurization increased the biosynthesis of osmoprotectants (ectoine and mannosylglycerate) as well as glutamate and induced the stringent response. Our findings reveal highly diverse and overlapping stress responses that could protect the biodesulfurizing culture not only from the associated sulfate limitation but also from chemical, oxidative, and osmotic stress, allowing efficient resource management. IMPORTANCE Despite decades of research, a commercially viable bioprocess for fuel desulfurization has not been developed yet. This is mainly due to lack of knowledge of the physiology and metabolism of fuel-biodesulfurizing bacteria. Being a stressful condition, biodesulfurization could provoke several stress responses that are not understood. This is particularly important because a thorough understanding of the microbial stress response is essential for the development of environmentally friendly and industrially efficient microbial biocatalysts. Our comparative systems biology studies provide a mechanistic understanding of the biology of biodesulfurization, which is crucial for informed developments through the rational design of recombinant biodesulfurizers and optimization of the bioprocess conditions. Our findings enhance the understanding of the physiology, metabolism, and stress response not only in biodesulfurizing bacteria but also in rhodococci, a precious group of biotechnologically important bacteria.

Mass spectrometry imaging for biosolids characterization to assess ecological or health risks before reuse.

Biosolids are byproducts of wastewater treatment. With the increasing global population, the amounts of wastewater to be treated are expanding, along with the amounts of biosolids generated. The reuse of biosolids is now accepted for diversified applications in fields such as agriculture, engineering, agro-forestry. However, biosolids are known to be potential carriers of compounds that can be toxic to living beings or alter the environment. Therefore, biosolid reuse is subject to regulations, mandatory analyses are performed on heavy metals, persistent organic pollutants or pathogens. Conventional methods for the analysis of heavy metals and persistent organic pollutants are demanding, lengthy, and sometimes unsafe. Here, we propose mass spectrometry imaging as a faster and safer method using small amounts of material to monitor heavy metals and persistent organic pollutants in different types of biosolids, allowing for ecological and health risk assessment before reuse. Our methodology can be extended to other soil-like matrices.

Contamination Pattern and Risk Assessment of Polar Compounds in Snow Melt: An Integrative Proxy of Road Runoffs.

To assess the contamination and potential risk of snow melt with polar compounds, road and background snow was sampled during a melting event at 23 sites at the city of Leipzig and screened for 489 chemicals using liquid chromatography high-resolution mass spectrometry with target screening. Additionally, six 24 h composite samples were taken from the influent and effluent of the Leipzig wastewater treatment plant (WWTP) during the snow melt event. 207 compounds were at least detected once (concentrations between 0.80 ng/L and 75 µg/L). Consistent patterns of traffic-related compounds dominated the chemical profile (58 compounds in concentrations from 1.3 ng/L to 75 µg/L) and among them were 2-benzothiazole sulfonic acid and 1-cyclohexyl-3-phenylurea from tire wear and denatonium used as a bittern in vehicle fluids. Besides, the analysis unveiled the presence of the rubber additive 6-PPD and its transformation product N-(1.3-dimethylbutyl)-N'-phenyl-p-phenylenediamine quinone (6-PPDQ) at concentrations known to cause acute toxicity in sensitive fish species. The analysis also detected 149 other compounds such as food additives, pharmaceuticals, and pesticides. Several biocides were identified as major risk contributors, with a more site-specific occurrence, to acute toxic risks to algae (five samples) and invertebrates (six samples). Ametryn, flumioxazin, and 1,2-cyclohexane dicarboxylic acid diisononyl ester are the main compounds contributing to toxic risk for algae, while etofenprox and bendiocarb are found as the main contributors for crustacean risk. Correlations between concentrations in the WWTP influent and flow rate allowed us to discriminate compounds with snow melt and urban runoff as major sources from other compounds with other dominant sources. Removal rates in the WWTP showed that some traffic-related compounds were largely eliminated (removal rate higher than 80%) during wastewater treatment and among them was 6-PPDQ, while others persisted in the WWTP.

Biodesulfurization Induces Reprogramming of Sulfur Metabolism in Rhodococcus qingshengii IGTS8: Proteomics and Untargeted Metabolomics.

Sulfur metabolism in fuel-biodesulfurizing bacteria and the underlying physiological adaptations are not understood, which has impeded the development of a commercially viable bioprocess for fuel desulfurization. To fill these knowledge gaps, we performed comparative proteomics and untargeted metabolomics in cultures of the biodesulfurization reference strain Rhodococcus qingshengii IGTS8 grown on either inorganic sulfate or the diesel-borne organosulfur compound dibenzothiophene as a sole sulfur source. Dibenzothiophene significantly altered the biosynthesis of many sulfur metabolism proteins and metabolites in a growth phase-dependent manner, which enabled us to reconstruct the first experimental model for sulfur metabolism in a fuel-biodesulfurizing bacterium. All key pathways related to assimilatory sulfur metabolism were represented in the sulfur proteome, including uptake of the sulfur sources, sulfur acquisition, and assimilatory sulfate reduction, in addition to biosynthesis of key sulfur-containing metabolites such as S-adenosylmethionine, coenzyme A, biotin, thiamin, molybdenum cofactor, mycothiol, and ergothioneine (low-molecular weight thiols). Fifty-two proteins exhibited significantly different abundance during at least one growth phase. Sixteen proteins were uniquely detected and 47 proteins were significantly more abundant in the dibenzothiophene culture during at least one growth phase. The sulfate-free dibenzothiophene-containing culture reacted to sulfate starvation by restricting sulfur assimilation, enforcing sulfur-sparing, and maintaining redox homeostasis. Biodesulfurization triggered alternative pathways for sulfur assimilation different from those operating in the inorganic sulfate culture. Sulfur metabolism reprogramming and metabolic switches in the dibenzothiophene culture were manifested in limiting sulfite reduction and biosynthesis of cysteine, while boosting the production of methionine via the cobalamin-independent pathway, as well as the biosynthesis of the redox buffers mycothiol and ergothioneine. The omics data underscore the key role of sulfur metabolism in shaping the biodesulfurization phenotype and highlight potential targets for improving the biodesulfurization catalytic activity via metabolic engineering. IMPORTANCE For many decades, research on biodesulfurization of fossil fuels was conducted amid a large gap in knowledge of sulfur metabolism and its regulation in fuel-biodesulfurizing bacteria, which has impeded the development of a commercially viable bioprocess. In addition, lack of understanding of biodesulfurization-associated metabolic and physiological adaptations prohibited the development of efficient biodesulfurizers. Our integrated omics-based findings reveal the assimilatory sulfur metabolism in the biodesulfurization reference strain Rhodococcus qingshengii IGTS8 and show how sulfur metabolism and oxidative stress response were remodeled and orchestrated to shape the biodesulfurization phenotype. Our findings not only explain the frequently encountered low catalytic activity of native fuel-biodesulfurizing bacteria but also uncover unprecedented potential targets in sulfur metabolism that could be exploited via metabolic engineering to boost the biodesulfurization catalytic activity, a prerequisite for commercial application.

Phase Transfer and Biodegradation of Pesticides in Water-Sediment Systems Explored by Compound-Specific Isotope Analysis and Conceptual Modeling.

Current approaches are often limited to evaluating the contribution of pesticide dissipation processes in water-sediment systems as both degradation and phase transfer, that is, sorption-desorption, contribute to the apparent decrease of pesticide concentration. Here, the dissipation of widely used herbicides acetochlor and S-metolachlor was examined in laboratory by water-sediment microcosm experiments under oxic and anoxic conditions. Compound-specific isotope analysis (CSIA) emphasized insignificant carbon isotope fractionation in the sediment, indicating prevailing pesticide degradation in the water phase. Conceptual modeling accounting for phase transfer and biodegradation indicated that biodegradation may be underestimated when phase transfer is not included. Phase transfer does not affect carbon isotope fractionation for a wide spectrum of molecules and environmental conditions, underscoring the potential of pesticide CSIA as a robust approach to evaluate degradation in water-sediment systems. CSIA coupled with the identification of transformation products by high-resolution tandem mass spectrometry suggests the degradation of acetochlor and S-metolachlor to occur via nucleophilic substitution and the predominance of oxalinic acids as transformation products under both anoxic and oxic conditions. Altogether, combining the pesticide CSIA, the identification of transformation products, and the use of conceptual phase-transfer models improves the interpretation of pesticide dissipation in water-sediment systems.

Acute-onset delirium in intensive care COVID patients: association of imperfect brain repair with foodborne micro-pollutants.

BACKGROUND AND PURPOSE: COVID-19 affects the brain in various ways, amongst which delirium is worrying. An assessment was made of whether a specific, long-lasting, COVID-19-related brain injury develops in acute respiratory distress syndrome patients after life-saving re-oxygenation. METHODS: Ten COVID+ patients (COVID+) with unusual delirium associated with neuroimaging suggestive of diffuse brain injury and seven controls with non-COVID encephalopathy were studied. The assessment took place when the intractable delirium started at weaning off ventilation support. Brain magnetic resonance imaging (MRI) was performed followed by standard cerebrospinal fluid (CSF) analyses and assessment of CSF erythropoietin concentrations (as a marker for the assessment of tissue repair), and of non-targeted CSF metabolomics using liquid chromatography high resolution mass spectrometry. RESULTS: Patients were similar as regards severity scores, but COVID+ were hospitalized longer (25 [11.75; 25] vs. 9 [4.5; 12.5] days, p = 0.03). On admission, but not at MRI and lumbar puncture performance, COVID+ were more hypoxic (p = 0.002). On MRI, there were leptomeningeal enhancement and diffuse white matter haemorrhages only in COVID+. In the latter, CSF erythropoietin concentration was lower (1.73 [1.6; 2.06] vs. 3.04 [2.9; 3.91] mIU/ml, p = 0.01), and CSF metabolomics indicated (a) increased compounds such as foodborne molecules (sesquiterpenes), molecules from industrialized beverages and micro-pollutants (diethanolamine); and (b) decreased molecules such as incomplete breakdown products of protein catabolism and foodborne molecules (glabridin). At 3-month discharge, fatigue, anxiety and depression as well as MRI lesions persisted in COVID+. CONCLUSIONS: Some COVID+ are at risk of a specific delirium. Imperfect brain repair after re-oxygenation and lifestyle factors might influence long-lasting brain injuries in a context of foodborne micro-pollutants.

Distribution and degradation trend of micropollutants in a surface flow treatment wetland revealed by 3D numerical modelling combined with LC-MS/MS.

Conventional wastewater treatment plants are not designed to treat micropollutants; thus, for 20 years, several complementary treatment systems, such as surface flow wetlands have been used to address this issue. Previous studies demonstrate that higher residence time and low global velocities promote nutrient removal rates or micropollutant photodegradation. Nevertheless, these studies were restricted to the system limits (inlet/outlet). Therefore, detailed knowledge of water flow is crucial for identifying areas that promote degradation and optimise surface flow wetlands. The present study combines 3D water flow numerical modelling and liquid chromatography coupled with high-resolution mass spectrometry (LC-HRMS/MS). Using this numerical model, validated by tracer experimental data, several velocity areas were distinguished in the wetland. Four areas were selected to investigate the waterflow influence and led to the following results: on the one hand, the number and concentration of micropollutants are independent of the waterflow, which could be due to several assumptions, such as the chronic exposure associated with a low Reynolds number; on the other hand, the potential degradation products (metabolites) were also assessed in the sludge to investigate the micropollutant biodegradation processes occurring in the wetland; micropollutant metabolites or degradation products were detected in higher proportions (both number and concentration) in lower flow rate areas. The relation to higher levels of plant and microorganism metabolites suggests higher biological activity that promotes degradation.

Large scale micropollutants and lipids screening in the sludge layers and the ecosystem of a vertical flow constructed wetland.

Wastewater is one of the major sources of micropollutant release into the environment. In order to reduce the impact of wastewater, wastewater treatment plants (WWTP) have been set up, in the instance of vertical flow constructed wetlands (VFCWs). Besides, micropollutants could represent a vast diversity of compounds and compound's choice could bias studies focused on their fate. To overcome this bias, non-targeted screening approaches can be performed. Therefore, the diffusion of micropollutants from raw wastewater in the VFCW compartments (wastewater, plants and sludge) as well as their fate have been investigated using this non-target approach with liquid chromatography (LC) coupled to high resolution mass spectrometry (HRMS) and gas chromatography (GC) coupled to mass spectrometry. To help the operators in their sludge management, this study will be focused on the following question: Is there a specific distribution of micropollutants according to sludge layers? To eliminate the background contamination found both inside the CW and in the surrounding environment, a control coring was performed in bank. A specific distribution could be observed in the top (191 compounds) and bottom layers (38 compounds). However, a distribution over the whole depth for xenobiotics was observed. Micropollutants classes and the main microbial productivity were preferably found in the top layer. The micropollutants fate could however not be restricted to the sludge compartment. Therefore, the specific micropollutants distribution was analyzed in the outputs of the system in their interactions with wastewater (effluent, sludge, and reed rhizomes) to understand their fate. In our study, the results highlighted a consistent part of compounds found in at least two or three of these compartments, with a similar trend in each compartment. These results underline the interactions between the compartments and the global issues of micropollutants distribution as well as its wide spreading in the whole CW ecosystem.

Investigation of Xenobiotics Metabolism In Salix alba Leaves via Mass Spectrometry Imaging.

The method presented uses mass spectrometry imaging (MSI) to establish the metabolic profile of S. alba leaves when exposed to xenobiotics. Using a non-targeted approach, plant metabolites and xenobiotics of interest are identified and localized in plant tissues to uncover specific distribution patterns. Then, in silico prediction of potential metabolites (i.e., catabolites and conjugates) from the identified xenobiotics is performed. When a xenobiotic metabolite is located in the tissue, the type of enzyme involved in its alteration by the plant is recorded. These results were used to describe different types of biological reactions occurring in S. alba leaves in response to xenobiotic accumulation in the leaves. The metabolites were predicted in two generations, allowing the documentation of successive biological reactions to transform xenobiotics in the leaf tissues.

Xenobiotics metabolization in Salix alba leaves uncovered by mass spectrometry imaging.

INTRODUCTION: Micropollutants are increasingly monitored as their presence in the environment is rising due to human activities, and they are potential threats to living organisms. OBJECTIVES: This study aimed at understanding the role of plants in xenobiotics removal from polluted environments by following xenobiotics metabolism in leaf tissues. METHODS: Different classes of micropollutants were investigated using liquid chromatography (LC) coupled to quadrupole-time of flight (Q-TOF) high resolution mass spectrometry (HRMS). The tissue localization of xenobiotics in the leaves of a spontaneous (not planted by humans) Salix alba growing near the water flux was further investigated using matrix-assisted laser desorption ionization (MALDI) mass spectrometry imaging (MSI). RESULTS: The LC-Q-TOF analysis revealed the distribution of micropollutants in three different compartments of a tertiary treatment wetland. When further investing the metabolic profile of S. alba leaves using MSI, different distribution patterns were observed in specific leaf tissues. Xenobiotic metabolites were predicted and could also be tentatively identified in S. alba leaves, shedding new light on the metabolic processes at play in leaves to manage xenobiotics uptake from a polluted environment. CONCLUSION: Using complementary metabolomics approaches, this study performed a large-scale exploration of micropollutants spreading in the environment at the exit of a tertiary treatment wetland. The use of MSI coupled with the prediction of xenobiotic metabolites yielded novel insights into plant metabolism during chronical exposure to low doses of a mixture of micropollutants.