Microbial Interactions Drive the Complete Catabolism of the Antibiotic Sulfamethoxazole in Activated Sludge Microbiomes.

Microbial communities are believed to outperform monocultures in the complete catabolism of organic pollutants via reduced metabolic burden and increased robustness to environmental challenges; however, the interaction mechanism in functional microbiomes remains poorly understood. Here, three functionally differentiated activated sludge microbiomes (S1: complete catabolism of sulfamethoxazole (SMX); S2: complete catabolism of the phenyl part of SMX ([phenyl]-SMX) with stable accumulation of its heterocyclic product 3-amino-5-methylisoxazole (3A5MI); A: complete catabolism of 3A5MI rather than [phenyl]-SMX) were enriched. Combining time-series cultivation-independent microbial community analysis, DNA-stable isotope probing, molecular ecological network analysis, and cultivation-dependent function verification, we identified key players involved in the SMX degradation process. Paenarthrobacter and Nocardioides were primary degraders for the initial cleavage of the sulfonamide functional group (-C-S-N- bond) and 3A5MI degradation, respectively. Complete catabolism of SMX was achieved by their cross-feeding. The co-culture of Nocardioides, Acidovorax, and Sphingobium demonstrated that the nondegraders Acidovorax and Sphingobium were involved in the enhancement of 3A5MI degradation. Moreover, we unraveled the internal labor division patterns and connections among the active members centered on the two primary degraders. Overall, the proposed methodology is promisingly applicable and would help generate mechanistic, predictive, and operational understanding of the collaborative biodegradation of various contaminants. This study provides useful information for synthetic activated sludge microbiomes with optimized environmental functions.

Subsistence and complexity of antimicrobial resistance on a community-wide level.

There are a multitude of resistance strategies that microbes can apply to avoid inhibition by antimicrobials. One of these strategies is the enzymatic modification of the antibiotic, in a process generally termed inactivation. Furthermore, some microorganisms may not be limited to the mere inactivation of the antimicrobial compounds. They can continue by further enzymatic degradation of the compounds' carbon backbone, taking nutritional and energetic advantage of the former antibiotic. This driving force to harness an additional food source in a complex environment adds another level of complexity to the reasonably well-understood process of antibiotic resistance proliferation on a single cell level: It brings bioprotection into play at the level of microbial community. Despite the possible implications of a resistant community in a host and a lurking antibiotic failure, knowledge of degradation pathways of antibiotics and their connections is scarce. Currently, it is limited to only a few families of antibiotics (e.g. ß-lactams and sulfonamides). In this article, we discuss the fluctuating nature of the relationship between antibiotic resistance and the biodegradation of antibiotics. This distinction mainly depends on the genetic background of the microbe, as general resistance genes can be recruited to function in a biodegradation pathway.

Casimicrobium huifangae gen. nov., sp. nov., a Ubiquitous "Most-Wanted" Core Bacterial Taxon from Municipal Wastewater Treatment Plants.

Microorganisms in wastewater treatment plants (WWTPs) play a key role in the removal of pollutants from municipal and industrial wastewaters. A recent study estimated that activated sludge from global municipal WWTPs harbors 1 × 109 to 2 × 109 microbial species, the majority of which have not yet been cultivated, and 28 core taxa were identified as "most-wanted" ones (L. Wu, D. Ning, B. Zhang, Y. Li, et al., Nat Microbiol 4:1183-1195, 2019, https://doi.org/10.1038/s41564-019-0426-5). Cultivation and characterization of the "most-wanted" core bacteria are critical to understand their genetic, physiological, phylogenetic, and ecological traits, as well as to improve the performance of WWTPs. In this study, we isolated a bacterial strain, designated SJ-1, that represents a novel cluster within Betaproteobacteria and corresponds to OTU_16 within the 28 core taxa in the "most-wanted" list. Strain SJ-1 was identified and nominated as Casimicrobium huifangae gen. nov., sp. nov., of a novel family, Casimicrobiaceae. C. huifangae is ubiquitously distributed and is metabolically versatile. In addition to mineralizing various carbon sources (including carbohydrates, aromatic compounds, and short-chain fatty acids), C. huifangae is capable of nitrate reduction and phosphorus accumulation. The population of C. huifangae accounted for more than 1% of the bacterial population of the activated sludge microbiome from the Qinghe WWTP, which showed seasonal dynamic changes. Cooccurrence analysis suggested that C. huifangae was an important module hub in the bacterial network of Qinghe WWTP.IMPORTANCE The activated sludge process is the most widely applied biotechnology and is one of the best ecosystems to address microbial ecological principles. Yet, the cultivation of core bacteria and the exploration of their physiology and ecology are limited. In this study, the core and novel bacterial taxon C. huifangae was cultivated and characterized. This study revealed that C. huifangae functioned as an important module hub in the activated sludge microbiome, and it potentially plays an important role in municipal wastewater treatment plants.

Biodegradation of Polyethylene and Polystyrene by Greater Wax Moth Larvae (Galleria mellonella L.) and the Effect of Co-diet Supplementation on the Core Gut Microbiome.

Plastics waste and microplastics including polyethylene (PE) and polystyrene (PS) have been an environmental concern for years. Recent research has revealed that larvae of Galleria mellonella are capable of biodegrading low density PE film. In this study, we tested the feasibility of enhancing larval survival and the effect of supplementing the co-diet on plastic degradation by feeding the larvae beeswax or wheat bran as a co-diet. Significant mass loss of plastic was observed over a 21-day period, i.e., with respective consumption of 0.88 and 1.95 g by 150 larvae fed only either PS or PE. The formation of C═O and C-O containing functional groups and long chain fatty acids as the metabolic intermediates of plastics in the residual polymers indicated depolymerization and biodegradation. Supplementing beeswax and bran increased the survival rates but decreased the consumption of plastic. The changes in the gut microbiome revealed that Bacillus and Serratia were significantly associated with the PS and PE diets. Beeswax and bran showed different shaping effects on the core gut microbiome of larvae fed the PE and PS. These results suggest that supplementing the co-diet affected the physiological properties of the larvae and plastic biodegradation and shaped the core gut microbiome.

Living with sulfonamides: a diverse range of mechanisms observed in bacteria.

Sulfonamides are the oldest class of synthetic antibiotics still in use in clinical and veterinary settings. The intensive utilization of sulfonamides has been leading to the widespread contamination of the environment with these xenobiotic compounds. Consequently, in addition to pathogens and commensals, also bacteria inhabiting a wide diversity of environmental compartments have been in contact with sulfonamides for almost 90 years. This review aims at giving an overview of the effect of sulfonamides on bacterial cells, including the strategies used by bacteria to cope with these bacteriostatic agents. These include mechanisms of antibiotic resistance, co-metabolic transformation, and partial or total mineralization of sulfonamides. Possible implications of these mechanisms on the ecosystems and dissemination of antibiotic resistance are also discussed. KEY POINTS: • Sulfonamides are widespread xenobiotic pollutants; • Target alteration is the main sulfonamide resistance mechanism observed in bacteria; • Sulfonamides can be modified, degraded, or used as nutrients by some bacteria.

State-of-the-Art Production Chains for Peas, Beans and Chickpeas-Valorization of Agro-Industrial Residues and Applications of Derived Extracts.

The world is confronted with the depletion of natural resources due to their unsustainable use and the increasing size of populations. In this context, the efficient use of by-products, residues and wastes generated from agro-industrial and food processing opens the perspective for a wide range of benefits. In particular, legume residues are produced yearly in very large amounts and may represent an interesting source of plant proteins that contribute to satisfying the steadily increasing global protein demand. Innovative biorefinery extraction cascades may also enable the recovery of further bioactive molecules and fibers from these insufficiently tapped biomass streams. This review article gives a summary of the potential for the valorization of legume residual streams resulting from agro-industrial processing and more particularly for pea, green bean and chickpea by-products/wastes. Valuable information on the annual production volumes, geographical origin and state-of-the-art technologies for the extraction of proteins, fibers and other bioactive molecules from this source of biomass, is exhaustively listed and discussed. Finally, promising applications, already using the recovered fractions from pea, bean and chickpea residues for the formulation of feed, food, cosmetic and packaging products, are listed and discussed.

Accumulation and Transformation of 2,2',4,4'-Tetrabrominated Diphenyl Ether (BDE47) by the Earthworm Metaphire vulgaris in Soil.

The accumulation and transformation of 2,2',4,4'-tetrabrominated diphenyl ether (BDE47), one congener of the flame retardants polybrominated diphenyl ethers (PBDEs), in soil-feeding fauna are still unknown. Using radioactivity tracer, we incubated 14C-labelled BDE47 in soil for 21 days in the presence and absence of the geophagous earthworm Metaphire vulgaris. BDE47 accumulated in the earthworm predominantly via oral ingestion of soil, giving a biota-soil accumulation factor (BSAF) value of 1.3 for radioactivity at the end of incubation, and was mostly located in intestine, followed by clitellum (organs region) and skin of earthworms. Accumulation was accompanied by significant decrease of BDE47 concentration in soil porewater and BDE47 mineralization in soil. BDE47 was transformed in the earthworm gut into two metabolites with higher polarities than BDE47. The results provide for the first time insights into accumulation and transformation of lower-brominated congeners of PBDEs in geophagous earthworms, being helpful for environmental risk assessment of PBDEs.

Fate of 2,4,6-Tribromophenol in Soil Under Different Redox Conditions.

Fate of 2,4,6-tribromophenol (TBP) in environmental matrices is obscure. We used 14C-tracer to investigated mineralization, transformation, and non-extractable residue (NER)-formation of TBP in a soil under continuously oxic, continuously anoxic, and anoxic-oxic alteration conditions. In all cases, TBP rapidly dissipated, mineralized to CO2, and formed NERs in the soil. Considerable amounts of transformation products (2-12%) were detected during the incubation. Marked mineralization (13-26%) indicated that soil microorganisms used TBP as their energy source. About 62-70% of the initial radioactivity was transformed into NERs, being mainly attributed to binding to humic and fulvic acid fractions. TBP transformation was significantly faster under oxic conditions than under anoxic conditions, and was boosted when the soil redox changed from anoxic to oxic state. The results provide new insights into fate of TBP in soil and suggest the importance to evaluate the stability of NERs for risk assessment of TBP in soil.

Comparative genomics reveals a novel genetic organization of the sad cluster in the sulfonamide-degrader 'Candidatus Leucobacter sulfamidivorax' strain GP.

BACKGROUND: Microbial communities recurrently establish metabolic associations resulting in increased fitness and ability to perform complex tasks, such as xenobiotic degradation. In a previous study, we have described a sulfonamide-degrading consortium consisting of a novel low-abundant actinobacterium, named strain GP, and Achromobacter denitrificans PR1. However, we found that strain GP was unable to grow independently and could not be further purified. RESULTS: Previous studies suggested that strain GP might represent a new putative species within the Leucobacter genus (16S rRNA gene similarity < 97%). In this study, we found that average nucleotide identity (ANI) with other Leucobacter spp. ranged between 76.8 and 82.1%, further corroborating the affiliation of strain GP to a new provisional species. The average amino acid identity (AAI) and percentage of conserved genes (POCP) values were near the lower edge of the genus delimitation thresholds (65 and 55%, respectively). Phylogenetic analysis of core genes between strain GP and Leucobacter spp. corroborated these findings. Comparative genomic analysis indicates that strain GP may have lost genes related to tetrapyrrole biosynthesis and thiol transporters, both crucial for the correct assembly of cytochromes and aerobic growth. However, supplying exogenous heme and catalase was insufficient to abolish the dependent phenotype. The actinobacterium harbors at least two copies of a novel genetic element containing a sulfonamide monooxygenase (sadA) flanked by a single IS1380 family transposase. Additionally, two homologs of sadB (4-aminophenol monooxygenase) were identified in the metagenome-assembled draft genome of strain GP, but these were not located in the vicinity of sadA nor of mobile or integrative elements. CONCLUSIONS: Comparative genomics of the genus Leucobacter suggested the absence of some genes encoding for important metabolic traits in strain GP. Nevertheless, although media and culture conditions were tailored to supply its potential metabolic needs, these conditions were insufficient to isolate the PR1-dependent actinobacterium further. This study gives important insights regarding strain GP metabolism; however, gene expression and functional studies are necessary to characterize and further isolate strain GP. Based on our data, we propose to classify strain GP in a provisional new species within the genus Leucobacter, 'Candidatus Leucobacter sulfamidivorax'.

Biotransformation of Sulfonamide Antibiotics in Activated Sludge: The Formation of Pterin-Conjugates Leads to Sustained Risk.

The presence of antibiotics in treated wastewater and consequently in surface and groundwater resources raises concerns about the formation and spread of antibiotic resistance. Improving the removal of antibiotics during wastewater treatment therefore is a prime objective of environmental engineering. Here we obtained a detailed picture of the fate of sulfonamide antibiotics during activated sludge treatment using a combination of analytical methods. We show that pterin-sulfonamide conjugates, which are formed when sulfonamides interact with their target enzyme to inhibit folic acid synthesis, represent a major biotransformation route for sulfonamides in laboratory batch experiments with activated sludge. The same major conjugates were also present in the effluents of nine Swiss wastewater treatment plants. The demonstration of this biotransformation route, which is related to bacterial growth, helps explain seemingly contradictory views on optimal conditions for sulfonamide removal. More importantly, since pterin-sulfonamide conjugates show retained antibiotic activity, our findings suggest that risk from exposure to sulfonamide antibiotics may be less reduced during wastewater treatment than previously assumed. Our results thus further emphasize the inadequacy of focusing on parent compound removal and the importance of investigating biotransformation pathways and removal of bioactivity to properly assess contaminant removal in both engineered and natural systems.

Biodegradation of sulfamethoxazole by a bacterial consortium of Achromobacter denitrificans PR1 and Leucobacter sp. GP.

In the last decade, biological degradation and mineralization of antibiotics have been increasingly reported feats of environmental bacteria. The most extensively described example is that of sulfonamides that can be degraded by several members of Actinobacteria and Proteobacteria. Previously, we reported sulfamethoxazole (SMX) degradation and partial mineralization by Achromobacter denitrificans strain PR1, isolated from activated sludge. However, further studies revealed an apparent instability of this metabolic trait in this strain. Here, we investigated this instability and describe the finding of a low-abundance and slow-growing actinobacterium, thriving only in co-culture with strain PR1. This organism, named GP, shared highest 16S rRNA gene sequence similarity (94.6-96.9%) with the type strains of validly described species of the genus Leucobacter. This microbial consortium was found to harbor a homolog to the sulfonamide monooxygenase gene (sadA) also found in other sulfonamide-degrading bacteria. This gene is overexpressed in the presence of the antibiotic, and evidence suggests that it codes for a group D flavin monooxygenase responsible for the ipso-hydroxylation of SMX. Additional side reactions were also detected comprising an NIH shift and a Baeyer-Villiger rearrangement, which indicate an inefficient biological transformation of these antibiotics in the environment. This work contributes to further our knowledge in the degradation of this ubiquitous micropollutant by environmental bacteria.

A Biocatalytic Nanomaterial for the Label-Free Detection of Virus-Like Particles.

The design of nanomaterials that are capable of specific and sensitive biomolecular recognition is an on-going challenge in the chemical and biochemical sciences. A number of sophisticated artificial systems have been designed to specifically recognize a variety of targets. However, methods based on natural biomolecular detection systems using antibodies are often superior. Besides greater affinity and selectivity, antibodies can be easily coupled to enzymatic systems that act as signal amplifiers, thus permitting impressively low detection limits. The possibility to translate this concept to artificial recognition systems remains limited due to design incompatibilities. Here we describe the synthesis of a synthetic nanomaterial capable of specific biomolecular detection by using an internal biocatalytic colorimetric detection and amplification system. The design of this nanomaterial relies on the ability to accurately grow hybrid protein-organosilica layers at the surface of silica nanoparticles. The method allows for label-free detection and quantification of targets at picomolar concentrations.

The crystal structures of native hydroquinone 1,2-dioxygenase from Sphingomonas sp. TTNP3 and of substrate and inhibitor complexes.

The crystal structure of hydroquinone 1,2-dioxygenase, a Fe(II) ring cleaving dioxygenase from Sphingomonas sp. strain TTNP3, which oxidizes a wide range of hydroquinones to the corresponding 4-hydroxymuconic semialdehydes, has been solved by Molecular Replacement, using the coordinates of PnpCD from Pseudomonas sp. strain WBC-3. The enzyme is a heterotetramer, constituted of two subunits α and two ß of 19 and 38kDa, respectively. Both the two subunits fold as a cupin, but that of the small α subunit lacks a competent metal binding pocket. Two tetramers are present in the asymmetric unit. Each of the four ß subunits in the asymmetric unit binds one Fe(II) ion. The iron ion in each ß subunit is coordinated to three protein residues, His258, Glu264, and His305 and a water molecule. The crystal structures of the complexes with the substrate methylhydroquinone, obtained under anaerobic conditions, and with the inhibitors 4-hydroxybenzoate and 4-nitrophenol were also solved. The structures of the native enzyme and of the complexes present significant differences in the active site region compared to PnpCD, the other hydroquinone 1,2-dioxygenase of known structure, and in particular they show a different coordination at the metal center.

Sequential lignin depolymerization by combination of biocatalytic and formic acid/formate treatment steps.

Lignin, a complex three-dimensional amorphous polymer, is considered to be a potential natural renewable resource for the production of low-molecular-weight aromatic compounds. In the present study, a novel sequential lignin treatment method consisting of a biocatalytic oxidation step followed by a formic acid-induced lignin depolymerization step was developed and optimized using response surface methodology. The biocatalytic step employed a laccase mediator system using the redox mediator 1-hydroxybenzotriazole. Laccases were immobilized on superparamagnetic nanoparticles using a sorption-assisted surface conjugation method allowing easy separation and reuse of the biocatalysts after treatment. Under optimized conditions, as much as 45 wt% of lignin could be solubilized either in aqueous solution after the first treatment or in ethyl acetate after the second (chemical) treatment. The solubilized products were found to be mainly low-molecular-weight aromatic monomers and oligomers. The process might be used for the production of low-molecular-weight soluble aromatic products that can be purified and/or upgraded applying further downstream processes.

Engineering Nanosized Organosilica for Molecular Recognition and Biocatalysis Applications.

A series of synthetic nanomaterials capable of molecular recognition and/or biocatalysis have been produced by exploiting the self-sorting, self-assembly and polycondensation of organosilane building blocks around protein templates. The established methodology allows for the production of thin organosilica layers of controlled thickness, down to nanometer precision. Fully synthetic virus recognition materials have been shown to specifically bind their target virus down to picomolar concentrations. The shielding of natural enzymes allowed producing nanobiocatalysts functioning under harsh operational conditions.

Production of superparamagnetic nanobiocatalysts for green chemistry applications.

Immobilization of enzymes on solid supports is a convenient method for increasing enzymatic stability and enabling enzyme reuse. In the present work, a sorption-assisted surface conjugation method was developed and optimized to immobilize enzymes on the surface of superparamagnetic nanoparticles. An oxidative enzyme, i.e., laccase from Trametes versicolor was used as model enzyme. The immobilization method consists of the production of superparamagnetic nanoparticles by co-precipitation of FeCl2 and FeCl3. Subsequently, the particle surface is modified with an organosilane containing an amino group. Next, the enzymes are adsorbed on the particle surface before a cross-linking agent, i.e., glutaraldehyde is added which links the amino groups on the particle surface with the amino groups of the enzymes and leads to internal cross-linking of the enzymes as well. The method was optimized using response surface methodology regarding optimal enzyme and glutaraldehyde amounts, pH, and reaction times. Results allowed formulation of biocatalysts having high specific enzymatic activity and improved stability. The biocatalysts showed considerably higher stability compared with the dissolved enzymes over a pH range from 3 to 9 and in the presence of several chemical denaturants. To demonstrate the reusability of the immobilized enzymes, they were applied as catalysts for the production of a phenoxazinone dye. Virtually, 100 % of the precursor was transformed to the dye in each of the ten conducted reaction cycles while on average 84.5 % of the enzymatic activity present at the beginning of a reaction cycle was retained after each cycle highlighting the considerable potential of superparamagnetic biocatalysts for application in industrial processes.

Enzyme Shielding in an Enzyme-thin and Soft Organosilica Layer.

The fragile nature of most enzymes is a major hindrance to their use in industrial processes. Herein, we describe a synthetic chemical strategy to produce hybrid organic/inorganic nanobiocatalysts; it exploits the self-assembly of silane building blocks at the surface of enzymes to grow an organosilica layer, of controlled thickness, that fully shields the enzyme. Remarkably, the enzyme triggers a rearrangement of this organosilica layer into a significantly soft structure. We demonstrate that this change in stiffness correlates with the biocatalytic turnover rate, and that the organosilica layer shields the enzyme in a soft environment with a markedly enhanced resistance to denaturing stresses.

Carbon Stable Isotope Fractionation of Sulfamethoxazole during Biodegradation by Microbacterium sp. Strain BR1 and upon Direct Photolysis.

Carbon isotope fractionation of sulfamethoxazole (SMX) during biodegradation by Microbacterium sp. strain BR1 (ipso-hydroxylation) and upon direct photolysis was investigated. Carbon isotope signatures (δ(13)C) of SMX were measured by LC-IRMS (liquid chromatography coupled to isotope ratio mass spectrometry). A new LC-IRMS method for the SMX metabolite, 3-amino-5-methylisoxazole (3A5MI), was established. Carbon isotope enrichment factors for SMX (ε(C)) were -0.6 ± 0.1‰ for biodegradation and -2.0 ± 0.1‰ and -3.0 ± 0.2‰ for direct photolysis, at pH 7.4 and pH 5, respectively. The corresponding apparent kinetic isotope effects (AKIE) for ipso-hydroxylation were 1.006 ± 0.001; these fall in the same range as AKIE in previously studied hydroxylation reactions. The differences in SMX and 3A5MI fractionation upon biotic and abiotic degradation suggest that compound specific stable isotope analysis (CSIA) is a suitable method to distinguish SMX reaction pathways. In addition, the study revealed that the extent of isotope fractionation during SMX photolytic cleavage is pH-dependent.

Reversible Supramolecular Surface Attachment of Enzyme-Polymer Conjugates for the Design of Biocatalytic Filtration Membranes.

To be used successfully in continuous reactor systems, enzymes must either be retained using filtration membranes or immobilized on a solid component of the reactor. Whereas the first approach requires large amounts of energy, the second approach is limited by the low temporal stability of enzymes under operational conditions. To circumvent these major stumbling blocks, we have developed a strategy that enables the reversible supramolecular immobilization of bioactive enzyme-polymer conjugates at the surface of filtration membranes. The polymer is produced through a reversible addition-fragmentation transfer method; it contains multiple adamantyl moieties that are used to bind the resulting conjugate at the surface of the membrane which has surface-immobilized cyclodextrin macrocycles. This supramolecular modification is stable under operational conditions and allows for efficient biocatalysis, and can be reversed by competitive host-guest interactions.

Recycling of indium from CIGS photovoltaic cells: potential of combining acid-resistant nanofiltration with liquid-liquid extraction.

Electronic consumer products such as smartphones, TV, computers, light-emitting diodes, and photovoltaic cells crucially depend on metals and metalloids. So-called "urban mining" considers them as secondary resources since they may contain precious elements at concentrations many times higher than their primary ores. Indium is of foremost interest being widely used, expensive, scarce and prone to supply risk. This study first investigated the capability of different nanofiltration membranes of extracting indium from copper-indium-gallium- selenide photovoltaic cell (CIGS) leachates under low pH conditions and low transmembrane pressure differences (<3 bar). Retentates were then subjected to a further selective liquid-liquid extraction (LLE). Even at very acidic pH indium was retained to >98% by nanofiltration, separating it from parts of the Ag, Sb, Se, and Zn present. LLE using di-(2-ethylhexyl)phosphoric acid (D2EHPA) extracted 97% of the indium from the retentates, separating it from all other elements except for Mo, Al, and Sn. Overall, 95% (2.4 g m(-2) CIGS) of the indium could be extracted to the D2EHPA phase. Simultaneously, by nanofiltration the consumption of D2EHPA was reduced by >60% due to the metal concentration in the reduced retentate volume. These results show clearly the potential for efficient scarce metal recovery from secondary resources. Furthermore, since nanofiltration was applicable at very low pH (≥ 0.6), it may be applied in hydrometallurgy typically using acidic conditions.