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Traditional methods to incorporate metals into graphene oxide (GO) usually require toxic reagents or high temperatures. This study proposes an innovative and sustainable method to incorporate silver (Ag) into graphene oxide using electron beam and evaluate its antibacterial activities. The method is based on green synthesis, without toxic reagents or hazardous wastes, and can be carried out at room temperature, in short reaction times. To synthesize the Ag/rGO nanocomposite, a water/isopropanol solution with dispersed graphene oxide and silver nitrate was submitted to a dose range from 150 to 400 kGy. The product was characterized by thermogravimetry analysis, X-ray diffraction and transmission electron microscopy. The antibacterial activity of Ag/rGO was observed against Gram-negative Escherichia coli by plate count method and atomic force microscopy. The results showed that concentrations as low as 100 μg/mL of produced Ag/rGO were enough to inactivate the cells.

Partial nitrification (PN) in sequencing batch reactors (SBR) was investigated in the presence of cadmium to simulate treatment of landfill leachate containing heavy metals. Different pH conditions were investigated in the SBR to determine the optimum pH to establish PN, which was 7.5. After successful establishment of the PN process, different concentrations of cadmium (Cd) were added into the SBR to evaluate the heavy metal influence on the PN process. The results showed that PN activities were not affected by low concentrations of Cd (

A combination of approaches, including volatile organic compound (VOC) metabolomics, enzymatic activity and morphological investigations, are employed to provide insights on the toxic interactions among carbon-based nanomaterials and fungi. The model fungi investigated are Aspergillus niger and Aspergillus flavus. Their exposure to graphene (G) and graphene oxide (GO) presents 62% reduction in biomass and abnormal hyphae aspect. The stress response of these microorganisms in the presence of these nanomaterials is further determined by overall cell changes through apoptosis analyses and production of reactive oxygen species (ROS) by the fungal hyphae. The ROS production by these microorganisms exposed to the nanomaterials confirms cellular oxidative stress, which explains the apoptotic-like cell death observed in the presence of the nanomaterials. This fungal response is also linked to lower production of several important enzymes involved in the catabolism of nutrients for cell growth, such as acid phosphatase, Naphthol-ASBI phosphohydrolase, β-glucosidase and β-galactosidase. The presence of these nanomaterials also triggers changes in the production of VOC by the fungi. These changes are indicative of stress conditions since these compounds have significant roles in fungal metabolism. Overall, A. niger was more sensitive to GO and A. flavus was more sensitive to G.

Background: Over the past several years, the scientific community has described the diversity of microbial communities in a variety of soils associated with plants, but at present, little is known about the specific diversity of the soil fungal microbiome involving bacteria colonizing the surface of fungi (i.e., exo-bacteria) or existing within fungal hypha (i.e., endobacteria). This study aimed to collect, identify, and characterize several fungi and their associated (endo- and exo-) microbiome obtained from the rhizosphere of six different plants. Microcosm devices called fungal highway columns, containing one of four plant-based media as attractants, were placed in the rhizosphere of six different plants. The isolated fungi and their associated endo- and exo- bacteria were identified by sequencing of the ITS (fungi) or 16S (bacteria) rRNA regions, followed by Scanning Electron Microscope (SEM), and fluorescence in situ hybridization (FISH) imaging.Results: Most of the fungi recovered are known plant pathogens, such as Fusarium, Pleosporales, and Cladosporium together with species associated with the soil, e.g. Kalmusia. The exo-bacteria recovered were previously described as plant promoters, such as Bacillus, Rhizobium, Acinetobacter or Ensifer. The interactions between fungi and exo-bacteria recovered from fungal highway columns were further investigated via confrontation assays. From the reconstruction of the potential co-occurring bacterial-fungal associations in the rhizosphere, we discovered that the most promiscuous exo-bacterium group (associated with diverse fungi) was Bacillus. From the study of the endobacterial community, emerged a core of shared endosymbionts with a potential implication in the nitrogen cycle.Conclusions: The present study demonstrated the importance of selecting and studying cultivable fungi and bacteria from the rhizosphere. Our findings demonstrated that at the rhizosphere level, the range of interactions between fungi and bacteria, both internal and external to the fungal hypha, could vary even among closely related species.

Bacteria–fungi interactions (BFIs) are essential in ecosystem functioning. These interactions are modulated not only by local nutritional conditions but also by the physicochemical constraints and 3D structure of the environmental niche. In soils, the unsaturated and complex nature of the substrate restricts the dispersal and activity of bacteria. Under unsaturated conditions, some bacteria engage with filamentous fungi in an interaction (fungal highways) in which they use fungal hyphae to disperse. Based on a previous experimental device to enrich pairs of organisms engaging in this interaction in soils, we present here the design and validation of a modified version of this sampling system constructed using additive printing. The 3D printed devices were tested using a novel application in which a target fungus, the common coprophilous fungus Coprinopsis cinerea, was used as bait to recruit and identify bacterial partners using its mycelium for dispersal. Bacteria of the genera Pseudomonas, Sphingobacterium and Stenotrophomonas were highly enriched in association with C. cinerea. Developing and producing these new easy-to-use tools to investigate how bacteria overcome dispersal limitations in cooperation with fungi is important to unravel the mechanisms by which BFIs affect processes at an ecosystem scale in soils and other unsaturated environments., Fungal highway columns produced by additive printing.

For the past two decades, microbial monitoring of the International Space Station (ISS) has relied on culture-dependent methods that require return to Earth for analysis. This has a number of limitations, with the most significant being bias towards the detection of culturable organisms and the inherent delay between sample collection and ground-based analysis. In recent years, portable and easy-to-use molecular-based tools, such as Oxford Nanopore Technologies&rsquo, MinIONTM sequencer and miniPCR bio&rsquo, s miniPCR&trade, thermal cycler, have been validated onboard the ISS. Here, we report on the development, validation, and implementation of a swab-to-sequencer method that provides a culture-independent solution to real-time microbial profiling onboard the ISS. Method development focused on analysis of swabs collected in a low-biomass environment with limited facility resources and stringent controls on allowed processes and reagents. ISS-optimized procedures included enzymatic DNA extraction from a swab tip, bead-based purifications, altered buffers, and the use of miniPCR and the MinION. Validation was conducted through extensive ground-based assessments comparing current standard culture-dependent and newly developed culture-independent methods. Similar microbial distributions were observed between the two methods, however, as expected, the culture-independent data revealed microbial profiles with greater diversity. Protocol optimization and verification was established during NASA Extreme Environment Mission Operations (NEEMO) analog missions 21 and 22, respectively. Unique microbial profiles obtained from analog testing validated the swab-to-sequencer method in an extreme environment. Finally, four independent swab-to-sequencer experiments were conducted onboard the ISS by two crewmembers. Microorganisms identified from ISS swabs were consistent with historical culture-based data, and primarily consisted of commonly observed human-associated microbes. This simplified method has been streamlined for high ease-of-use for a non-trained crew to complete in an extreme environment, thereby enabling environmental and human health diagnostics in real-time as future missions take us beyond low-Earth orbit.

The synthesis of biocompatible polymers for coating applications has gained significant attention in recent years due to the increasing spread of infectious diseases via contaminated surfaces. One strategy to combat this problem is to apply antimicrobial coatings to surfaces prone to microbial contamination. This study presents a series of biomimetic polymers that can be used as adhesives to immobilize known antimicrobial agents on the surfaces as coatings. Several polymers containing dopamine methacrylate as co-polymers were synthesized and investigated as adhesives for the deposition of an antimicrobial polymer (polyethyleneimine) and antimicrobial nanoparticles (graphene, graphene oxide and molybdenum trioxide) onto glass surfaces. The results showed that different antimicrobials required different types of adhesives for effective coating. Overall, the coatings fabricated from these composites were shown to inactivate E. coli and B. subtilis within 1 h. These coatings were also effective to prevent biofilm growth and demonstrated to be non-toxic to the human corneal epithelial cell line (htCEpi). Leaching tests of the coatings proved that the coatings were stable under biological conditions.

Sorghum is the fifth most cultivated and consumed grain in the world. However, this grain is frequently contaminated with toxins from fungi. The present study evaluated the effects of environmental factors on tenuazonic acid (TeA) production by Epicoccum sorghinum in the field and in controlled laboratory conditions. In this study, 50 sorghum grain samples were collected from summer and autumn growing seasons and analyzed for TeA contamination using LC-MS/MS. To further understand the ecophysiology of this fungus, an isolated strain of E. sorghinum from the field was investigated for its development and TeA production under controlled environmental conditions in the laboratory. In the ecophysiological investigation, the effects of water activity (0.90, 0.95, 0.99) and temperature (18, 22, 26 and 30 °C) were evaluated on the radial growth, enzymatic production and expression of TAS1, which is the gene involved in TeA production. Results showed that in the field, the summer season presented the highest TeA average level in the grains (587.8 μg/kg) compared to level found in the autumn (440.5 μg/kg). The ecophysiological investigation confirmed that E. sorghinum produces more actively TeA under environmental conditions simulating the summer season. Optimum growth, maximum TAS1 gene expression, and higher extracellular enzymatic production were observed at 26 °C with a water activity of 0.99. Pearson correlation analyses showed that the production of TeA highly correlates with fungal growth. The present study demonstrates that abiotic factors in a combined approach of field and laboratory conditions will assist in predicting the driving environmental factors that could affect growth of E. sorghinum and TeA production in sorghum grains.

Graphene’s unique physical structure, as well as its chemical and electrical properties, make it ideal for use in sensor technologies. In the past years, novel sensing platforms have been proposed with pristine and modified graphene with nanoparticles and polymers. Several of these platforms were used to immobilize biomolecules, such as antibodies, DNA, and enzymes to create highly sensitive and selective biosensors. Strategies to attach these biomolecules onto the surface of graphene have been employed based on its chemical composition. These methods include covalent bonding, such as the coupling of the biomolecules via the 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride and N-hydroxysuccinimide reactions, and physisorption. In the literature, several detection methods are employed; however, the most common is electrochemical. The main reason for researchers to use this detection approach is because this method is simple, rapid and presents good sensitivity. These biosensors can be particularly useful in life sciences and medicine since in clinical practice, biosensors with high sensitivity and specificity can significantly enhance patient care, early diagnosis of diseases and pathogen detection. In this review, we will present the research conducted with antibodies, DNA molecules and, enzymes to develop biosensors that use graphene and its derivatives as scaffolds to produce effective biosensors able to detect and identify a variety of diseases, pathogens, and biomolecules linked to diseases.

The present study reports two routes to modify reduced graphene oxide (rGO) nanosheets with polysulfone (PSU) brushes via nitrene chemistry. The PSU polymer is bonded to rGO at the extremity (rGO-PSU end) and at the middle of the PSU chain (rGO-PSU mid). The resulting rGO-PSU synthetic products are carefully characterized by Raman and FTIR spectroscopy, XPS, TEM, and thermogravimetric analysis, evidencing the successful grafting of PSU onto rGO surfaces. The long-term stability of these nanosheets is also determined in common solvents. The antibacterial properties of polymer-functionalized rGO against the planktonic Bacilus subtilis and Escherichia coli are also investigated. It is established that the antimicrobial properties of these nanocomposites are due to the production of reactive oxygen species. The results also demonstrate that rGO-PSU mid presents better antimicrobial properties due to shorter polymer chains, which improves the contact of the microorganisms with the graphene surface. This work was financially supported by the Spanish Ministry of Economy and Competitiveness (MAT2014-57557-R), and partially supported by the U.S. National Science Foundation Career Award (NSF Award #104093). R.O. would like acknowledge support from U.S. National Science Foundation (CMMI-1538730 and DUE-CMMI-1538730/1003574). Janire Peña wants to acknowledge mobility grant from Carlos III University and Instituto Tecnológico de Química y Materiales “Alonso Barba”. Authors acknowledge Dr. A. Esteban-Arranz for giving access to ATR equipment.

Nanocomposites containing graphene oxide (GO), polyethyleneimine (PEI), and chitosan (CS) were synthesized for chromium(VI) and copper(II) removal from water. Response surface methodology (RSM) was used for the optimization of the synthesis of the CS–PEI–GO beads to achieve simultaneous maximum Cr(VI) and Cu(II) removals. The RSM experimental design involved investigating different concentrations of PEI (1.0–2.0%), GO (500–1500 ppm), and glutaraldehyde (GLA) (0.5–2.5%), simultaneously. Batch adsorption experiments were performed to obtain responses in terms of percent removal for both Cr(VI) and Cu(II) ions. A second-order polynomial equation was used to model the relationship between the synthesis conditions and the adsorption responses. High R2 values of 0.9848 and 0.8327 for Cr(VI) and Cu(II) removal, respectively, were obtained from the regression analyses, suggesting good correlation between observed experimental values and predicted values by the model. The optimum bead composition contained 2.0% PEI, 1500 ppm GO, and 2.08% GLA, and allowed Cr(VI) and Cu(II) removals of up to 91.10% and 78.18%, respectively. Finally, characterization of the structure and surface properties of the optimized CS–PEI–GO beads was carried out using X-ray diffraction (XRD), porosity and BET surface area analysis, scanning electron microscopy (SEM), Fourier transform infrared spectroscopy (FTIR), and X-ray photoelectron spectroscopy (XPS), which showed favorable adsorbent characteristics as given by a mesoporous structure with high surface area (358 m2 g−1) and plenty of surface functional groups. Overall, the synthesized CS–PEI–GO beads were proven to be effective in removing both cationic and anionic heavy metal pollutants.

This study investigates the acute toxicity of graphene to sludge microbial communities. The acute toxicity was investigated with concentrations varying from low to relatively high concentrations of graphene (between 0 and 300 mg L−1) to understand the impact of different concentrations of graphene on the biological wastewater treatment process. The experiments were performed with a 10 h continuous aeration using batch reactors to simulate the wastewater biological treatment process. Results showed that increasing concentrations of graphene in the reactors led to decreasing COD, BOD5, ammonia and phosphate removals. In addition, abundances of ammonia oxidizing bacteria, ammonia monooxygenase and phosphate accumulating bacteria decreased along with the overall sludge microbial metabolic activity. The 16S rRNA deep sequencing of the sludge microbial community exposed to different concentrations of graphene showed that the abundances of the two most abundant phyla, i.e. Proteobacteria and Bacteriodetes, changed with increasing concentrations of graphene. The results also showed that releases of graphene concentrations at 10 mg L−1 and higher seem to present a short term impact in the wastewater treatment process.

Urea is an abundant nitrogen-containing compound found in urine of mammals and widely used in fertilizers. This compound is part of the nitrogen biogeochemical cycle and is easily biodegraded by ureolytic microorganisms that have the urease enzyme. Previous studies, with ureolytic isolates, have shown that some ureolytic microorganisms are able to sequester CO2 through a process called microbially-induced calcium carbonate precipitation. The present study investigates 15 ureolytic consortia obtained from the "Pamukkale travertines" and the "Cave Without A Name" using different growth media to identify the possible bacterial genera responsible for CO2 sequestration through the microbially-induced calcite precipitation (MICP). The community structure and diversity were determined by deep-sequencing. The results showed that all consortia presented varying CO2 sequestration capabilities and MICP rates. The CO2 sequestration varied between 0 and 86.4%, and it depended largely on the community structure, as well as on pH. Consortia with predominance of Comamonas, Plesiomonas and Oxalobacter presented reduced CO2 sequestration. On the other hand, consortia dominated by Sporosarcina, Sphingobacterium, Stenotrophomonas, Acinetobacter, and Elizabethkingia showed higher rates of CO2 uptake in the serum bottle headspace.

The present study compares for the first time the effects of h-MoO3 and α-MoO3 against two fungal strains: Aspergillus niger and Aspergillus flavus. The h-MoO3 nanoparticles were more toxic to both fungi than α-MoO3. The toxic effects of h-MoO3 were more pronounced toward A. flavus, which presented a growth inhibition of 67.4% at 200 mg L-1. The presence of the nanoparticles affected drastically the hyphae morphology by triggering nuclear condensation and compromising the hyphae membrane. Further analysis of the volatile organic compounds (VOCs) produced by both fungi in the presence of the nanomaterials indicated important metabolic changes related to programmed cell death. These nanomaterials induced the production of specific antifungal VOCs, such as β-Elemene and t-Cadinol, by the fungi. The production of essential enzymes involved in fungal metabolism, such as acid phosphatase, naphthol-As-BI-phosphohydrolase, β-galactosidase, β-glucosidase and N-acetyl-β-glucosaminidase, reduced significantly in the presence of the nanomaterials. The changes in enzymatic production and VOCs corroborate the fact that these nanoparticles, especially h-MoO3, exert changes in the fungal metabolism, triggering apoptotic-like cell death responses in these fungi.

We demonstrate for the first time that suspensions of single-layered MoS2 nanosheets can act as photocatalytic antimicrobial materials under visible light in the presence of ethylenediaminetetraacetic acid (EDTA) as an electron donor. The antimicrobial capacity of exfoliated MoS2 (Ex-MoS2) was found to be 5.7 times higher than that of annealed exfoliated MoS2 (Ae-MoS2) against planktonic cells in the presence of 40 ppm EDTA. This difference in the antimicrobial performance was attributed to the 1T-phase of Ex-MoS2, which presents higher electron conductivity than that of Ae-MoS2. This higher electron conductivity of Ex-MoS2 led to increase generation of reactive oxygen species (ROS), as observed by the superoxide anion and hydrogen peroxide production assays under visible light. Additionally, Ex-MoS2 could also inactivate 65% of mature E. coli K12 biofilms without significant cytotoxicity to mammalian fibroblast cells. The suspension of single-layered MoS2 nanosheets opens up new opportunities for the development of advanced functional nanomaterials for biomedical and environmental applications.

In this study, we present a simple, fast and cost-effective sonochemical growth method for the synthesis of zinc oxide (ZnO) nanorods. ZnO nanorods were grown on glass substrates at room temperature without the addition of surfactants. The successful coating of substrates with ZnO nanorods was demonstrated by Raman spectroscopy, X-ray photoelectron spectroscopy (XPS), scanning electron microscopy (SEM), and energy dispersive X-ray spectroscopy (EDS). The antimicrobial properties of ZnO nanorods against the planktonic Bacillus subtilis and Escherichia coli and their respective biofilms were investigated. The cytotoxicity of ZnO nanorods were evaluated using the NIH 3T3 mammalian fibroblast cell line. Moreover, to understand the possible mechanisms of ZnO nanorod toxicity, glutathione oxidation, superoxide production, and release of Zn2+ ions by the ZnO nanorods were determined, and the LIVE/DEAD assay was employed to investigate cell membrane damage. The results showed that sonochemically grown ZnO nanorods exhibited significant antimicrobial effects to both bacteria and prevented biofilm formation. ZnO nanorods did not present any significant toxicity to fibroblast cells. The main anti-microbial mechanisms of ZnO nanorods were determined to be H2O2 production and cell membrane disruption.

Adhesion of bacteria to interfaces is the first step in pathogenic infection, in biofilm formation, and in bioremediation of oil spills and other pollutants. Bacteria use a variety of surface structures to promote interfacial adhesion, with the level of expression of these structures varying in response to local conditions and environmental signals. Here, we investigated how overexpression of type 1 fimbriae, one such appendage, modifies the ability of Escherichia coli to adhere to solid substrates, via biofilm formation and yeast agglomeration, and to oil/water interfaces, via a microbial adhesion to hydrocarbon assay. A plasmid that enables inducible expression of E. coli MG1655 type 1 fimbriae was transformed into fimbriae-deficient mutant strain MG1655ΔfimA. The level of fimH gene expression in the engineered strain, measured using quantitative real-time PCR, could be tuned by changing the concentration of inducer isopropyl β-d-1-thiogalactopyranoside (IPTG), and was higher than that in strain MG1655. Increasing the degree of fimbriation only slightly modified the surface energy and zeta potential of the bacteria, but enhanced their ability to agglomerate yeast cells and to adhere to solid substrates (as measured by biofilm formation) and to oil/water interfaces. We anticipate that the tunable extent of fimbriation accessible with this engineered strain can be used to investigate how adhesin expression modifies the ability of bacteria to adhere to interfaces and to actively self-assemble there.

The present study investigates the chronic toxicity of graphene (G) and graphene oxide (GO) in activated sludge. Sequencing batch bioreactors were fed with influents containing 0, 1 and 5 mg L−1 of GO or G (12 h cycles) for ten days. Reduction in performance of the bioreactors in relation to chemical oxygen demand, ammonia and phosphate removals was observed after three days in the bioreactors fed with 5 mg L−1 of nanomaterials. After about eight days, these reactors reached a steady state nutrient removal, which corresponded to recovery of certain groups of ammonia oxidizing bacteria and phosphate accumulating bacteria despite the increasing accumulation of nanomaterials in the sludge. These results suggested that biological treatment can be affected transiently by initial exposure to the nanomaterials, but certain groups of microorganisms, less sensitive to these nanomaterials, can potentially strive in the presence of these nanomaterials. Results of 16S rRNA gene deep sequencing showed that G and GO affected differently the microbial communities in the activated sludge. Between the two nanomaterials investigated, GO presented the highest impact in nutrient removal, gene abundance and changes in microbial population structures.

The development of functionalized nanomaterials that leads to multi-functionality, such as the ability to adsorb heavy metals coupled with anti-microbial properties is very attractive for diverse applications. The present study evaluated for the first time the antimicrobial activity of graphene oxide silanized with N-(trimethoxysilylpropyl) ethylenediamine triacetic acid (GO–EDTA) against Gram-negative, Cupriavidus metallidurans CH4, and Gram-positive bacteria, Bacillus subtilis , as well as its cytotoxicity to human corneal epithelial cell line hTCEpi. The results show that GO–EDTA has improved anti-microbial properties when compared to graphene oxide (GO) alone, with 92.3 ± 10% and 99.1 ± 1.3% cell inactivation of B. subtilis and C. metallidurans , respectively. Bacterial inactivation was attributed to an oxidative stress mechanism towards the cells. No cytotoxicity was observed towards human corneal epithelial cell lines hTCEp after 24 h exposure to GO–EDTA, suggesting that this nanomaterial has the potential for applications that have human exposure. This work also evaluated GO–EDTA’s adsorption capacity for two heavy metals, Cu 2+ and Pb 2+ at different concentrations, varying pH and contact time. The maximum adsorption capacity of the GO–EDTA was determined to be 454.6 mg g −1 and 108.7 mg g −1 for Pb 2+ and Cu 2+ , respectively, exceeding the capacity of traditional adsorbent materials, such as activated carbon.

Absorption capacity of biochar for crude oil and the mineralization potential of the absorbed oil in seawater were determined in laboratory-scale experiments. Texas, South Louisiana, or Qua-Iboe (Nigeria) crude oils were contacted with each of four commercial hardwood biochars and six synthesized biochars in seawater from the Gulf of Mexico and Long Island Sound (U.S.). Synthesized biochars were made from maplewood anoxically at different heat treatment temperatures (HTT) from 300°C to 700°C. Oil absorption capacity of the biochars determined in dip tests using oil on seawater ranged from 3.6 to 6.3 g/g. Oil-imbibed biochar particles were buoyant. Seawater enhanced absorption capacity in relation to the H/C ratio. Oil was less effectively absorbed in the form of weathered water-in-oil microemulsion (“chocolate mousse”) than in as-received form. Absorption capacity peaked at HTT about 400°C and correlated poorly with %C, H/C ratio, O/C ratio, surface area, and porosity. It is proposed that swellin...

Urea is an abundant nitrogen-containing compound found in urine of mammals and widely used in fertilizers. This compound is part of the nitrogen biogeochemical cycle and is easily biodegraded by ureolytic microorganisms that have the urease enzyme. Previous studies, with ureolytic isolates, have shown that some ureolytic microorganisms are able to sequester CO

In this work, we demonstrate the non-synthetic surface modification of a co-extruded multilayer poly(methyl methacrylate) (PMMA)/poly(ϵ-caprolactone) (PCL) film with gas barrier properties through electrospinning of polystyrene (PS)/multi-walled carbon nanotube (MWNT) nanofibers. As produced by forced assembly layer multiplying co-extrusion, the heterogeneous nucleating crystallization of PCL was induced using the glassy confinement of the amorphous PMMA thus creating in-plane lamellae crystallization, which is impermeable to most gas molecules. To complement these intrinsic gas barrier properties of the multilayer film, electrospun PS/MWNT nanofibers were deposited onto the surface of the PMMA/PCL film, which increased the surface roughness and resulted in superhydrophobic behavior (water contact angles>150°). Furthermore, the inclusion of the MWNT in the matrix caused an increasing antibacterial efficacy, which was determined to reach 97% inactivation against gram-positive bacteria Bacillus subtilis. The fiber mats were further characterized using scanning electron microscopy (SEM), Raman spectroscopy, and thermogravimetric analysis (TGA).

We report the successful RAFT-mediated emulsion polymerization of styrene using a non-ionic surfactant (Brij98), the highly reactive 1-phenylethyl phenyldithioacetate (PEPDTA) RAFT agent, and water-soluble initiator ammonium persulfate (APS). The molar ratio of RAFT agent to APS was identical in all experiments. Most of the monomer was contained within the micelles, analogous to microemulsion or miniemulsion systems but without the need of shear, sonication, cosurfactant, or a hydrophobe. The number-average molecular weight increased with conversion and the polydispersity index was below 1.2. This ideal ‘living’ behavior was only found when molecular weights of 9000 and below were targeted. It was postulated that the rapid transportation of RAFT agent from the monomer swollen micelles to the growing particles was fast on the polymerization timescale, and most if not all the RAFT agent is consumed within the first 10% conversion. In addition, it was postulated that the high nucleation rate from the high rate of exit (of the R radical from the RAFT agent) and high entry rate from water-phase radicals (high APS concentration) reduced the effects of ‘superswelling’ and therefore a similar molar ratio of RAFT agent to monomer was maintained in all growing particles. The high polydispersity indexes found when targeting molecular weights greater than 9000 were postulated to be due to the lower nucleation rate from the lower weight fractions of both APS and RAFT agent. In these cases, ‘superswelling’ played a dominant role leading to a heterogeneous distribution of RAFT to monomer ratios among the particles nucleated at different times.