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We report an effective and environmentally sustainable water treatment approach using enzymes encapsulated in biogenic vault nanoparticles. Manganese peroxidase (MnP), whose stability was remarkably extended by encapsulating into vaults, rapidly catalyzed the biotransformation of endocrine-disrupting compounds, including bisphenol A (BPA), bisphenol F (BPF), and bisphenol AP (BPAP). The vault-encapsulated MnP (vMnP) treatment removed 80-95% of each of the tested bisphenols (BPs) at lower enzyme dosage, while free native MnP (nMnP) only resulted in a 19-36% removal, over a 24-h period. Treatment by vMnP and nMnP resulted in considerable disparities in product species and abundance, which were consistent with the observed changes in the estrogenic activities of BPs. To test if vMnP-catalyzed transformations generated toxic intermediates, we assessed biological hallmarks of BP toxicity, namely, the ability to disrupt reproductive processes. The toxicity of vMnP-treated samples, as measured in the model organism, Caenorhabditis elegans, was dramatically reduced for all three BPs, including the reproductive indicators of BPA exposure such as reduced fertility and increased germ cell death. Collectively, our results indicate that the vMnP system represents an efficient and safe approach for the removal of BPs and promise the development of vault-encapsulated customized enzymes for treating other targeted organic compounds in contaminated waters.

The influence of growth medium water chemistry, specifically carbon/nitrogen (C/N) molar ratios, on the characteristics and development of biofilms of the model microorganism Pseudomonas aeruginosa was investigated. C/N = 9 had a unique effect on biofilm composition as well as quorum sensing (QS) pathways, with higher concentrations of carbohydrates and proteins in the biofilm and a significant upregulation of the QS gene lasI in planktonic cells. The effect of C/N ratio on total attached biomass was negligible. Principal component analysis revealed a different behavior of most outputs such as carbohydrates and QS chemicals at C/N = 9, and pointed to correlations between parameters of biofilm formation and steady state distribution of cells and extracellular components. C/N ratio was also shown to influence organic compound utilization by both planktonic and sessile organisms, with a maximum chemical oxygen demand (COD) removal of 83% achieved by biofilms at C/N = 21. Planktonic cells achieved higher COD removal rates, but greater overall rates after six days occurred in biofilms. The development of a dual-species biofilm of P. aeruginosa and Nitrobacter winogradskyi was also influenced by C/N, with increase in the relative abundance of the slower-growing N. winogradskyi above C/N = 9. These results indicate that altering operational parameters related to C/N would be relevant for mitigating or promoting biofilm formation and function depending on the desired industrial application or treatment configuration.

Unlike growth on tissue, microbes can grow freely on implantable devices with minimal immune system intervention and often form resilient biofilms that continuously pump out pathogenic cells. The efficacy of antibiotics used to treat infection is declining due to increased rates of pathogenic resistance. A simple, one-step zwitterionic surface modification is developed to significantly reduce protein and microbial adhesion to synthetic materials and demonstrate the successful modification of several clinically relevant materials, including recalcitrant materials such as elastomeric polydimethylsiloxane. The treated surfaces exhibit robust adhesion resistance against proteins and microorganisms in both static and flow conditions. Furthermore, the surface treatment prevents the adhesion of mammalian fibroblast cells while displaying no cytotoxicity. To demonstrate the clinical efficacy of the novel technology in the real-world, a surface-treated, commercial silicone foley catheter is developed that is cleared for use by the U.S. Food and Drug Administration (K192034). 16long-term catheterized patients received surface-treated catheters and completed a Patient Global Impression of Improvement (PGI-I) questionnaire. 10out of 16patients described their urinary tract condition post implantation as "much better" or "very much better" and 72% (n=13) of patients desire to continue using the surface-treated catheter over conventional latex or silicone catheters.

Microbial enzymes catalyze various reactions inside and outside living cells. Among the widely studied enzymes, fungal enzymes have been used for some of the most diverse purposes, especially in bioremediation, biosynthesis, and many nature-inspired commercial applications. To improve their stability and catalytic ability, fungal enzymes are often immobilized on assorted materials, conventional as well as nanoscale. Recent advances in fungal enzyme immobilization provide effective and sustainable approaches to achieve improved environmental and commercial outcomes. This review aims to provide a comprehensive overview of commonly studied fungal enzymes and immobilization technologies. It also summarizes recent advances involving immobilized fungal enzymes for the degradation or assembly of compounds used in the manufacture of products, such as detergents, food additives, and fossil fuel alternatives. Furthermore, challenges and future directions are highlighted to offer new perspectives on improving existing technologies and addressing unexplored fields of applications.

The use of bioaugmented zeolite (bio-zeolite) can be an effective technology for irreversibly removing recalcitrant organic pollutants in aqueous mixtures. Removal of 1,4-dioxane by a bio-zeolite (Pseudonocardia dioxanivorans CB1190-bioaugmented ZSM-5) in the presence of several chlorinated volatile organic compounds (CVOCs) was superior to removal by adsorption using abiotic zeolite. Mixtures containing 1,1-dichloroethene (1,1-DCE) were an exception, which completely inhibited the bio-zeolite system. Specific adsorption characteristics were studied using adsorption isotherms in single-solute and bisolute systems accompanied by Polanyi theory-based Dubinin-Astakhov (DA) modeling. Adsorption behavior was examined using characteristic energy (Ea/H) from modified DA models and molecular dynamics simulations. While the tight-fit of 1,4-dioxane in the hydrophobic channels of ZSM-5 appears to drive 1,4-dioxane adsorption, the greater hydrophobicity of trichloroethene and cis-1,2-dichloroethene cause them have a greater affinity over 1,4-dioxane for adsorption sites on the zeolite. 1,4-Dioxane was desorbed and displaced by CVOCs except 1,1-DCE because of its low Ea/H value, explaining why bio-zeolite only biodegraded 1,4-dioxane in 1,1-DCE-free CVOC mixtures. Understanding the adsorption mechanisms of solutes in complex mixtures is crucial for the implementation of sorption-based treatment technologies for the removal of complex contaminant mixtures from aquatic environments.

1,4-Dioxane is a probable carcinogen and trace contaminant that is difficult to remove from water because of its high solubility and low volatility. Removal of 1,4-dioxane from aqueous solutions by a set of potential adsorbents was investigated using batch experiments that revealed Titanium Silicate-1 (TS-1) as a high-capacity adsorbent (85.17 mg/g) with rapid kinetics (2 min equilibration time). The specific adsorption mechanisms at the molecular level were investigated using multiple approaches including FTIR spectra and molecular dynamics simulation of 1,4-dioxane adsorption on TS-1, which imply that the shape and size of 1,4-dioxane fits tightly into the hydrophobic straight channels of TS-1 with a diameter of 0.56 nm, and thus TS-1 provides strong van der Waals attraction within the channels. Additionally, 1,4-dioxane-water complexation was observed to connect to the frame oxygens of TS-1 via hydrogen bonds inside the channels. The calculated adsorption free energy of 1,4-dioxane was between −24.59 and −27.17 kJ/mol, which was consistent with the value of −24.89 kJ/mol derived from the adsorption isotherms at 298 K. The strong adsorption competition from nonpolar compounds 1,1,1-trichloroethane (TCA) and benzene onto TS-1 further confirmed that the adsorption was attributed to a combined mechanism of host-guest interactions including van der Waals interactions, hydrophobic interactions, and hydrogen bonding. Furthermore, 1,4-dioxane-loaded TS-1 was found to be efficiently regenerated by an enriched bacterial consortium (BD1) with recovery efficiencies for three cycles of 88.2%, 96.47% and nearly 100%, suggesting that TS-1 holds promise as a cost-effective renewable absorbent for eliminating hydrophilic organics from contaminated water supplies.

The application of urban wastewater treatment plants (WWTPs) products to agricultural lands has contributed to the rising level of antibiotic resistance and drawn a critical public health concern. It has not been thoroughly investigated at which spatial scales a biosolid applied area as a potentially predominant source affects surrounding soil resistomes. This study investigated distribution and impact of WWTP biosolids treated with anaerobic digestion on an agricultural area. Heterotrophic plate counts (HPCs) and quantitative polymerase chain reaction (qPCR) were performed for detection of selected antibiotic-resistant bacteria (ARB), selected antibiotic resistance genes (ARGs), intI1 genes, and 16S rRNA genes. Biosolid samples contained significantly higher levels of selected ARGs than the raw agricultural soils (p0.05). The average relative abundances of intI1, sul1, bla

Eroded particles from the source zone could transport a high concentration of perfluoroalkyl acids (PFAAs) to sediments and water bodies. Yet, the contribution of suspended particles has not been systematically reviewed. Analyzing reported studies, we quantitatively demonstrate that suspended particles in surface water can contain significantly higher concentrations of PFAAs than the sediment below, indicating the source of suspended particles are not the sediment but particles eroded and carried from the source zone upstream. The affinity of PFAAs to particles depends on the particle composition, including organic carbon fraction and iron or aluminum oxide content. In soils, most PFAAs are retained within the top 5 m below the ground surface. The distribution of PFAAs in the subsurface varies based on site properties and local weather conditions. The depth corresponding to the maximum concentration of PFAA in soil decreases with an increase in soil organic carbon or rainfall amount received in the catchment areas. We attribute a greater accumulation of PFAAs near the upper layer of the subsurface to an increase in the accumulation of particles eroded from source zones upstream receiving heavy rainfall. Precursor transformation in the aerobic zone is significantly higher than in the anaerobic zone, thereby making the aerobic subsurface zone serve as a long-term source of groundwater pollution. Collectively, these results suggest that suspended particles, often an overlooked vector for PFAAs, can be a dominant pathway for the transport of PFAAs in environments.

Microbial community dynamics were characterized following combined oxidation and biodegradation treatment trains for mixtures of 1,4-dioxane and chlorinated volatile organic compounds (CVOCs) in laboratory microcosms. Bioremediation is generally inhibited by co-contaminate CVOCs; with only a few specific bacterial taxa reported to metabolize or cometabolize 1,4-dioxane being unaffected. Chemical oxidation by hydrogen peroxide (H2O2) as a non-selective treatment demonstrated 50-80% 1,4-dioxane removal regardless of the initial CVOC concentrations. Post-oxidation bioaugmentation with 1,4-dioxane metabolizer Pseudonocardia dioxanivorans CB1190 removed the remaining 1,4-dioxane. The intrinsic microbial population, biodiversity, richness, and biomarker gene abundances decreased immediately after the brief oxidation phase, but recovery of cultivable microbiomes and a more diverse community were observed during the subsequent 9-week biodegradation phase. Results generated from the Illumina Miseq sequencing and bioinformatics analyses established that generally oxidative stress tolerant genus Ralstonia was abundant after the oxidation step, and Cupriavidus, Pseudolabrys, Afipia, and Sphingomonas were identified as dominant genera after aerobic incubation. Multidimensional analysis elucidated the separation of microbial populations as a function of time under all conditions, suggesting that temporal succession is a determining factor that is independent of 1,4-dioxane and CVOCs mixtures. Network analysis highlighted the potential interspecies competition or commensalism, and dynamics of microbiomes during the biodegradation phase, in line with the shifts of predominant genera and various developing directions during different steps of the treatment train. Collectively, this study demonstrated that chemical oxidation followed by bioaugmentation is effective for treating 1,4-dioxane, even in the presence of high levels of CVOC mixtures and residual peroxide, a disinfectant, and enhanced our understanding of microbial ecological impacts of the treatment train. These results will be valuable for predicting treatment synergies that lead to cost savings and improved remedial outcomes in short-term active remediation as well as long-term changes to the environmental microbial communities.

The overuse of antibiotics and subsequent enrichment of antibiotic resistant microbes in the natural and built environments is a severe threat to global public health. In this study, a Phanerochaete chrysosporium fungal-luffa fiber system was found to efficiently biodegrade two sulfonamides, sulfadimethoxine (SDM) and sulfadizine (SDZ), in cow urine wastewater. Biodegradation pathways were proposed on the basis of key metabolites identified using high performance liquid chromatography coupled with quadrupole-time-of-flight mass spectrometry (HPLC-QqTOF-MS). Transcriptomic, metabolomic, and free radical analyses were performed to explore the functional groups and detailed molecular mechanisms of SDM and SDZ degradation. A total of 27 UniGene clusters showed significant differences between luffa fiber and luffa fiber-free systems, which were significantly correlated to cellulose catabolism, carbohydrate metabolism, and oxidoreductase activity. Carbohydrate-active enzymes and oxidoreductases appear to play particularly important roles in SDM and SDZ degradation. Electron paramagnetic resonance (EPR) spectroscopy revealed the generation and evolution of OH and R during the biodegradation of SDM and SDZ, suggesting that beyond enzymatic degradation, SDM and SDZ were also transformed through a free radical pathway. Luffa fiber also acts as a co-substrate to improve the activity of enzymes for the degradation of SDM and SDZ. This research provides a potential strategy for removing SDM and SDZ from agricultural and industrial wastewater using fungal-luffa fiber systems.

This study presents an eco-friendly and efficient technology, using immobilized enzymes, vault-encapsulated laccases (vlaccase), for decolorization and detoxification of dyes. Vault encapsulation remarkably improved the performance of laccase at industrially relevant conditions, including neutral to alkaline pH and relatively high temperatures. Two representative anthraquinone and azo dyes, Reactive Blue 19 and Acid Orange 7, respectively, were rapidly decolorized (72% and 80%) by vlaccase treatment while natural laccase (nlaccase) achieved 40% and 32% decolorization. The toxicity of treated and untreated dyes was tested on model bacterial, algal, and insect cells. The inhibitory effects of dyes towards selected bacteria were reduced in vlaccase-treated samples. The chlorophyll synthesis in algae was less inhibited by dyes after vlaccase treatment. Furthermore, the toxicity of dye degradation products to insect cells was significantly mitigated in the vlaccase group. Collectively, these results indicate that vlaccase is a stable and strong enzymatic system for removing dyes from waters.

We developed a microbial community capable of biodegrading mixtures of chlorinated ethenes and 1,4-dioxane under varying redox conditions. Achieving the removal of chlorinated ethenes as well as 1,4-dioxane to below health advisory levels in groundwater that has anaerobic and aerobic zones has proven to be challenging. However, our mixed community, composed of the anaerobic chlorinated ethene-degrading consortium KB-1 and aerobic bacterial strain, Pseudonocardia dioxanivorans CB1190, was able to reduce trichloroethene (TCE) in anaerobic environments and oxidize 1,4-dioxane in the presence of oxygen. Aerobic biodegradation of cis-1,2-dichloroethene by CB1190 was also confirmed, thus decreasing the accumulation of TCE transformation products, such as vinyl chloride. The results of this study demonstrate that the assembled microbial community not only survived significant redox changes but sequentially biodegraded chlorinated ethenes and 1,4-dioxane. This approach toward degrading mixed contaminants with a c...

Biodegradation of 1,4-dioxane was examined in packed quartz and soil column flow-through systems. The inhibitory effects of co-contaminants, specifically trichloroethene (TCE), 1,1-dichloroethene (1,1-DCE), and copper (Cu2+) ions, were investigated in the columns either with or without bioaugmentation with a 1,4-dioxane degrading bacterium Pseudonocardia dioxanivorans CB1190. Results indicate that CB1190 cells readily grew and colonized in the columns, leading to significant degradation of 1,4-dioxane under oxic conditions. Degradation of 1,4-dioxane was also observed in the native soil (without bioaugmentation), which had been previously subjected to enhanced reductive dechlorination treatment for co-contaminants TCE and 1,1-DCE. Bioaugmentation of the soil with CB1190 resulted in nearly complete degradation at influent concentrations of 3–10 mg L−1 1,4-dioxane and a residence reaction time of 40–80 h, but the presence of co-contaminants, 1,1-DCE and Cu2+ ions (up to 10 mg L−1), partially inhibited 1,4-dioxane degradation in the untreated and bioaugmented soil columns. However, the inhibitory effects were much less severe in the column flow-through systems than those previously observed in planktonic cultures, which showed near complete inhibition at the same co-contaminant concentrations. These observations demonstrate a low susceptibility of soil microbes to the toxicity of 1,1-DCE and Cu2+ in packed soil flow-through systems, and thus have important implications for predicting biodegradation potential and developing sustainable, cost-effective technologies for in situ remediation of 1,4-dioxane contaminated soils and groundwater.

Vault particles are the largest naturally occurring ribonucleoprotein complexes found in the cytoplasm. In all 78 copies of major vault protein (MVP) assemble on polyribosome templates, forming recombinant vault particles, which are of great interest as encapsulation carriers for therapeutics delivery and enzyme stabilization. Baculovirus-insect cell expression is the only system that has been developed for recombinant vault synthesis, but it has low scalability and slow production rate. In this study, we demonstrated the first use of yeast cells for the production of vault particles with full integrity and functionality solely by expressing the complementary DNA (cDNA) encoding MVP. Vaults synthesized in Pichia pastoris yeast cells are morphologically indistinguishable from recombinant vault particles produced in insect cells, and are able to package and stabilize enzymes resulting in improved longevity and catalytic efficiency. Thus, our results imply that the yeast system is an economical alternative to insect cells for the production of recombinant vaults. The consistency of vault morphology between yeast and insect cell systems also underlines that polyribosome templating may be conserved among eukaryotes, which promises the synthesis and assembly of recombinant human vault particles in other eukaryotic organisms.

Metallic nanoparticles (NPs), the most abundant nanomaterials in consumer and industrial products, are the most probable class to enter the environment. In this study, wetland-derived microcosms were incubated with copper nanoparticles (Cu-NP) and ionic CuCl2 to investigate acute (10 days) and chronic (100 days) exposure towards nitrogen cycling microorganisms. The microbial ecology of wetlands play a crucial role in balancing nitrogen in pristine environments as well as in areas impacted by high nutrient loads (e.g., at wastewater effluent discharges). Gene abundance and expression changes were monitored using the GeoChip 5.0 high throughput functional gene microarray and metatranscriptomic shotgun sequencing (RNA-seq), respectively. After 10 days, the Cu-NP impacted microbial communities experienced structural shifts within microorganisms associated with dissimilatory nitrogen reduction accompanied by lower nitrate removal as compared to the unexposed controls. By day 100, these differences were largely resolved and nitrate removal was similar to the unexposed control. Furthermore, the Cu-NP exposed microcosms tolerated copper and were more resilient and adaptive than the unexposed controls based on the abundance and expression of other functions, including electron transfer, metal homeostasis, and stress response. These findings suggest sudden influxes of Cu-NPs into wetland systems may impair nitrogen removal initially, but long-term microbial shifts and functional redundancy would promote the net flux of total nitrogen out of the wetlands.

Granular activated carbon (GAC) has been used to remove per- and polyfluoroalkyl substances (PFASs) from industrial or AFFF-impacted waters,but its effectiveness can be low because adsorption of short-chained PFASs is ineffective and its sites are exhausted rapidly by co-contaminants. To increase adsorption of anionic PFASs on GAC by electrostatic attractions,we modified GAC's surface with the cationic polymer poly diallyldimethylammonium chloride (polyDADMAC) and tested its capacity in complex water matrices containing dissolved salts and humic acid. Amending with concentrations of polyDADMAC as low as 0.00025% enhanced GAC's adsorption capacity for PFASs,even in the presence of competing ions. This suggests that electrostatic interactions with polyDADMAC's quaternary ammonium functional groups helped bind organic and inorganic ions as well as the headgroup of short-chain PFASs,allowing more overall PFAS removal by GAC. Evaluating the effect of polymer dose is important because excessive addition can block pores and reduce overall PFAS removal rather than increase it. To decrease the waste associated with this adsorption strategy by making the adsorbent viable for more than one saturation cycle,a regeneration method is proposed which uses low-power ultrasound to enhance the desorption of PFASs from the polyDADMAC- GAC with minimum disruption to the adsorbent's structure. Re-modification with the polymer after sonication resulted in a negligible decrease in the sorbent's capacity over four saturation rounds. These results support consideration of polyDADMAC-modified GAC as an effective regenerable adsorbent for ex-situ concentration step of both short and long-chain PFASs from real waters with high concentrations of competing ions and low PFAS loads.

Per- and polyfluoroalkyl substances (PFASs) are anthropogenic amphipathic heat-resistant materials found in some household products and Aqueous Film-Forming Foams (AFFFs) used to extinguish hydrocarbon fires. There is a critical need for developing destructive technologies for the treatment of PFAS-impacted waters because several PFASs are classified as probable carcinogens and endocrine disruptors. We tested the destruction of 7 individual PFASs, a mix of 24 native PFASs (24Mix), and samples of AFFF and concentrated Investigation Derived Waste (IDW) by high-frequency ultrasound (700 kHz – 1040 W) in deionized water, groundwater containing low total dissolved solids (TDS) (388 mg L−1), and high-TDS groundwater (10.2 g L−1). This study demonstrated the degradation of novel PFASs, hexafluoropropylene oxide dimer acid (HFPO-DA) and 6:2 fluorotelomer sulfonamidoalkyl betaine (6:2 FTAB), with near-stoichiometric fluoride release. The salts and surfactants affect the air-water partitioning coefficients of PFASs and their availability at the ultrasonic cavity, thereby affecting the degradation rates. The degradation rates of sulfonates and short-chain PFASs were 30% to 60% higher in low TDS groundwater than those in deionized water, while the rates were lower in high TDS groundwater. The degradation rates of sulfonates in AFFF were 40% to 60% higher as compared to the 24Mix. The treatment of concentrated, high-TDS IDW resulted in significant defluorination of 41 PFASs, consuming 3 kWh g−1 – 76 kWh g−1 whereas dilute AFFF degradation utilized 5900 kWh g−1. The results inform important parameters for designing and operating ultrasonic reactors for the degradation of PFASs and verify that sonolytic treatment of concentrated PFAS mixtures and AFFF IDWs can effectively degrade and defluorinate PFASs without the formation of disinfection by-products.

Bioremediation of soil and groundwater sites contaminated by petroleum hydrocarbons is known as a technically viable, cost-effective, and environmentally sustainable technology. The purpose of this study is to investigate laboratory-scale bioremediation of petroleum-hydrocarbon contaminated soil through development of eight bioreactors, two bioreactors for each bioremediation mode. The modes were: (1) natural attenuation (NA); (2) biostimulation (BS) with oxygen and nutrients; (3) bioaugmentation (BA) with hydrocarbon degrading isolates; (4) a combination of biostimulation and bioaugmentation (BS-BA). Total petroleum hydrocarbons (TPH) mass balance over the bioreactors showed about 2% of initial 20,000mgkg-soil-1 TPH was removed by advection due to synthetic groundwater which was flowing through the soil, and the rest of decrease in TPH was caused by biodegradation. The BS-BA mode showed the highest TPH biodegradation percentage (89.7±0.3%) compared to the NA (51.4±0.6%), BS (81.9±0.3%) and BA (62.9±0.5%) modes. Furthermore, an increase in microbial population was another evidence of TPH biodegradation by microorganism. Reaction rate data from each bioremediation mode were fitted with a first-order reaction rate model. The Monod kinetic constants including maximum specific growth rate of microorganisms (μmax) and substrate concentration at half-velocity constant (Ks) were estimated for each bioremediation modes.

Groundwater flow through aquifer soils or packed bed systems can fluctuate for various reasons, which could affect the concentration of natural colloids and per- and polyfluoroalkyl substances (PFAS) in the pore water. In such cases, PFAS concentration could either decrease due to matrix diffusion of PFAS or increase by the detachment of colloids carrying PFAS. Yet, the effect of flow fluctuation on PFAS transport or release in porous media has not been examined. To examine the relative importance of either process, we interrupted the flow during an injection of groundwater spiked with perfluorobutanoic acid (PFBA), perfluorooctanoic acid (PFOA), and bromide as conservative tracer through clay-rich soil, so that diffusive transport would be prominent during flow interruption. After flow interruption, the PFAS concentration did not decrease indicating an insignificant contribution of matrix diffusion. The concentration increased, potentially due to enhanced release of colloid-associated PFAS. Analysis of samples before and after flow interruption by particle size analysis and SEM confirmed an increase in soil colloid concentration after the flow interruption. XRD analysis of soil and the colloids proved that PFAS were associated with specific sites of the colloids. Due to a higher affinity of PFOA to soil colloids, the total PFOA concentration in the effluent samples increased more than PFBA after the flow interruption process. The results indicate that colloids may have a disproportionally higher role in the transport of PFAS in conditions that release colloids from porous media. Thus, fluctuations in groundwater flow can increase this colloid facilitated mobility of PFAS.

Microbial community dynamics were characterized following combined catalysis and biodegradation treatment trains for mixtures of 1,4-dioxane and chlorinated volatile organic compounds (CVOCs) in laboratory microcosms. Although a few specific bacterial taxa are capable of removing 1,4-dioxane and individual CVOCs, many microorganisms are inhibited when these contaminants are present in mixtures. Chemical catalysis by tungstated zirconia (WOx/ZrO2) and hydrogen peroxide (H2O2) as a non-selective treatment was designed to achieve nearly 20% 1,4-dioxane and over 60% trichloroethene and 50% dichloroethene removals. Post-catalysis, bioaugmentation with 1,4-dioxane metabolizing bacterial strain,Pseudonocardia dioxanivorans CB1190, removed the remaining 1,4-dioxane. The evolution of the microbial community under different conditions was time-dependent but relatively independent of the concentrations of contaminants. The compositions of microbiomes tended to be similar regardless of complex contaminant mixtures during the biodegradation phase, indicating a r-K strategy transition attributed to the shock experienced during catalysis and the subsequent incubation. The originally dominant genera Pseudomonas and Ralstonia were sensitive to catalytic oxidation, and were overwhelmed by Sphingomonas, Rhodococcus, and other catalyst-tolerant microbes, but microbes capable of biodegradation of organics thrived during the incubation. Methane metabolism, chloroalkane-, and chloroalkene degradation pathways appeared to be responsible for CVOC degradation, based on the identifications of haloacetate dehalogenases, 2-haloacid dehalogenases, and cytochrome P450 family. Network analysis highlighted the potential interspecies competition or commensalism, and dynamics of microbiomes during the biodegradation phase that were in line with shifting predominant genera, confirming the deterministic processes guiding the microbial assembly. Collectively, this study demonstrated that catalysis followed by bioaugmentation is an effective treatment for 1,4-dioxane in the presence of high CVOC concentrations, and it enhanced our understanding of microbial ecological impacts resulting from abiotic-biological treatment trains. These results will be valuable for predicting treatment synergies that lead to cost savings and improve remedial outcomes in short-term active remediation as well as long-term changes to the environmental microbial communities.

Increasing pollution of global water sources and challenges in rapid detection and treatment of the wide range of contaminants pose considerable burdens on public health. The issue is particularly critical in rural areas, where building of centralized water treatment systems and pipe infrastructure to connect dispersed populations is not always practical. Point-of-use (POU) water supply systems provide cost-effective and energy-efficient approaches to store, treat, and monitor the quality of water. Currently available POU systems have limited success in dealing with the portfolio of emerging contaminants, particularly those present at trace concentrations. A site-to-site variation in contaminant species and concentrations also requires versatile POU systems to detect and treat contaminants and provide on-demand clean water. Among different technologies for developing rapid and sensitive water purification processes and sensors, enzymes offer one of the potential solutions because of their strong activity and selectivity toward chemical substrates. Many enzyme-nanomaterial composites have recently been developed that enhance enzymes' stability and activity and expand their functionality, thus facilitating the application of nanosupported enzymes in advanced POU systems. In this Account, we highlight the strengths and limitations of nanosupported enzymes for their potential applications in POU systems for water treatment as well as detection of contaminants, even at trace levels. We first summarize the mechanisms by which silica, carbon, and metallic nanosupports improve enzyme stability, selectivity, and catalysis. The unique immobilization properties and potential advantages of novel bioderived nanosupports over non-bioderived nanomaterials are emphasized. We illustrate prospective applications of nanosupported enzymes in POU systems with different roles: water purification, disinfection, and contaminant sensing. For each type of application, nanosupported enzymes offer higher performance than free enzymes. Nanosupports prolong enzymes' lifetimes and improve the rates of contaminant removal by concentrating contaminants near the enzymes. Nanosupports also stabilize antimicrobial enzymes while facilitating their attachment to bacterial surfaces, thereby increasing their potential uses for disinfection and prevention of biofouling in water purification and storage devices. As enzyme-based electrochemical sensors rely on electrochemical reactions of enzymatically generated species, the ability of conductive nanosupports to enhance enzyme activity and stability and to promote transfer of electrons onto the electrode greatly improves the sensitivity and durability of electroenzymatic contaminant sensors. Despite the promising results in laboratory settings, the application of nanosupported enzymes in real-world POU systems requires the implementation of multiple enzyme combinations and strategies for minimizing health risks associated with unintended releases of nanomaterials. Finally, we identify multidisciplinary research gaps in the development of nanosupported enzyme treatment systems and provide frameworks for the early adopters to make informed decisions on whether and how to use such POU systems.

Silica materials are attractive protein supports for applications in catalysis, sensing, water purification, and protein therapy. However, current approaches for loading proteins in silica are limited by inefficient protein immobilization, unintended leaching, the requirement for large quantities of silica precipitants, and associated protein inactivation. Here, we report a highly efficient and robust strategy that directly synthesizes protein-embedded silica via a vault protein nanocapsule-templated biomimetic silicification route. The vaults serve as nucleation sites for the deposition and condensation of silica precursors to form vault/silica composites. Using vaults encapsulated with proteins as the templating agent enables direct embedding of proteins in silica composites with nearly 100% immobilization efficiency. The vaults also act as protective shelters to preserve high protein activity during silica condensation. With the dual advantages of vault templating and protection, the proteins immobilized via this strategy retained over 90% activity after 30 reuse cycles and exhibited high activity, minimal leaching, and improved stability, which would be valuable in a broad variety of medical, industrial, and environmental applications.

This study used integrated omics technologies to investigate the potential novel pathways and enzymes for 1,4-dioxane degradation by a consortium enriched from activated sludge of a domestic wastewater treatment plant. An unclassified genus belonging to Xanthobacteraceae increased significantly after magnetic nanoparticle-mediated isolation for 1,4-dioxane degraders. Species with relatively higher abundance (> 0.3%) were identified to present high metabolic activities in the biodegradation process through shotgun sequencing. The functional gene investigations revealed that Xanthobacter sp. 91, Xanthobacter sp. 126, and a Rhizobiales strain carried novel 1,4-dioxane-hydroxylating monooxygenase genes. Xanthobacter sp. 126 contained the genes coding for glycolate oxidase, which was the main enzyme responsible for utilization of 1,4-dioxane intermediates through the TCA cycle, and further proven by the specific glycolate oxidase inhibitor, α-hydroxy-2-pyridinemethanesulfonic acid. An expanded and detailed degradation pathway of 1,4-dioxane was proposed on the basis of the three major intermediates (2-hydroxy-1,4-dioxane, ethylene glycol, and oxalic acid) confirmed by metabolomics. These findings of microbial community and function as well as the novel pathway will be valuable in predicting natural attenuation or reconstruction of a bacterial consortium for enhanced remediation of 1,4-dioxane-contaminated sites as well as wastewater treatment.

Microbial community compositions and functional profiles were analyzed in microcosms established using aquifer materials from a former automobile factory site, where 1,4-dioxane was identified as the primary contaminant of concern. Propane or oxygen biostimulation resulted in limited 1,4-dioxane degradation, which was markedly enhanced with the addition of nutrients, resulting in abundant Mycobacterium and Methyloversatilis taxa and high expressions of propane monooxygenase gene, prmA. In bioaugmented treatments, Pseudonocardia dioxanivorans CB1190 or Rhodococcus ruber ENV425 strains dominated immediately after augmentation and degraded 1,4-dioxane rapidly which was consistent with increased representation of xenobiotic and lipid metabolism-related functions. Although the bioaugmented microbes decreased due to insufficient growth substrates and microbial competition, they did continue to degrade 1,4-dioxane, presumably by indigenous propanotrophic and heterotrophic bacteria, inducing similar community structures across bioaugmentation conditions. In various treatments, functional redundancy acted as buffer capacity to ensure a stable microbiome, drove the restoration of the structure and microbial functions to original levels, and induced the decoupling between basic metabolic functions and taxonomy. The results of this study provided valuable information for design and decision-making for ex-situ bioreactors and in-situ bioremediation applications. A metagenomics-based understanding of the treatment process will enable efficient and accurate adjustments when encountering unexpected issues in bioremediation.

Development of a multiple lines of evidence (MLOE) framework to evaluate the intrinsic biodegradation potential of 1,4-dioxane is vital to implementing management strategies at groundwater sites impacted by 1,4-dioxane. A comprehensive MLOE approach was formed to provide significant evidence of natural degradation of 1,4-dioxane comingled with tetrahydrofuran (THF) within a large, diffuse plume. State-of-the art molecular biological analyses and compound-specific isotope analysis (CSIA) were employed to support more traditional approaches for data analysis (concentration trend analyses, spatial distribution, temporal changes, geochemical biodegradation attenuation indicators, plume mass estimates, and fate and transport modeling). The molecular analyses demonstrated that microorganisms capable of both metabolic and cometabolic degradation of 1,4-dioxane were present throughout the groundwater plume, whereas the CSIA data provided supporting evidence of biodegradation. 1,4-Dioxane biomarkers were present and abundant throughout the 1,4-dioxane plume, and our biomarkers tracked the plume with reasonable accuracy. Evidence also suggests that THF-driven cometabolic biodegradation as well as catabolic 1,4-dioxane biodegradation were active at this site. These data supplemented the traditional lines of evidence approaches, which demonstrated that 1,4-dioxane attenuation was occurring across the groundwater plume and that nondestructive physical processes alone did not account for the observed 1,4-dioxane attenuation. This MLOE framework combining new and traditional analyses demonstrates that this site has a significant capacity for intrinsic biodegradation of 1,4-dioxane. ©2016 Wiley Periodicals, Inc.

Amino-aromatic compounds, 2-amino-4-nitrotoluene (ANT), and 2,4-diaminotoluene (DAT) are carcinogens and environmentally persistent pollutants. In this study, we investigated their degradation by natural manganese peroxidase (nMnP) derived from Phanerochaete chrysosporium and recombinant manganese peroxidase packaged in vaults (vMnP). Encapsulation of manganese peroxidase (MnP) in ribonucleoprotein nanoparticle cages, called vaults, was achieved by creating recombinant vaults in yeast Pichia pastoris. Vault packaging increased the stability of MnP by locally sequestering multiple copies of the enzyme. Within 96 h, both vMnP and nMnP catalyzed over 72% removal of ANT in-vitro, which indicates that vault packaging did not limit substrate diffusion. It was observed that vMnP was more efficient than nMnP and P. chrysosporium for the catalysis of target contaminants. Only 57% of ANT was degraded by P. chrysosporium even when MnP activity reached about 480 U L−1 in cultures. At 1.5 U L−1 initial activity, vMnP achieved 38% of ANT and 51% of DAT degradation, whereas even 2.7 times higher activity of nMnP showed insignificant biodegradation of both compounds. These results imply that due to protection by vault cages, vMnP has lower inactivation rates. Thus, it works effectively at lower dosage for a longer duration compared to nMnP without requiring frequent replenishment. Collectively, these results indicate that fungal enzymes packaged in vault nanoparticles are more stable and active, and they would be effective in biodegradation of energetic compounds in industrial processes, waste treatment, and contaminated environments.

Biological treatment of 1,4-dioxane, a probable human carcinogen and a recalcitrant contaminant of concern, is often complicated by the presence of inhibitory co-contaminants. Due to its use as a solvent, wetting agent, and stabilizer for chlorinated solvents employed in metal vapor degreasing, 1,4-dioxane has often been found to occur with a variety of co-contaminants, including heavy metals such as hexavalent chromium [Cr(VI)]. Cr(VI) also occurs naturally in groundwater due to geological formations, but also has sources that can coincide with 1,4-dioxane from anthropogenic activities such as metal vapor degreasing. Biodegradation of 1,4-dioxane can be accomplished by microbes that use it as a source of carbon or energy as well as those that cometabolize it after growth on other organic substrates. A propanotroph, Mycobacterium austroafricanum JOB5, was grown in planktonic pure cultures and biofilms to determine its ability to cometabolize 1,4-dioxane in the presence of varying concentrations of Cr(VI). 1,4-Dioxane cometabolism by JOB5 planktonic cells was uninhibited by Cr(VI) at levels up to 10 mg/L, while biofilms were only mildly inhibited at 10 mg/L. As an important part of the biofilms commonly found in subsurface aquifers and engineered systems, extracellular polymeric substances (EPS) were found to play an important role in preventing Cr(VI) exposure to cells. We observed that soluble EPS were able to bind to Cr(VI) and theorize that biofilm-associated EPS additionally served to impede penetration of the biofilm structure by Cr(VI), thus mitigating exposure and toxicity. These findings suggest that bioremediation would be a viable treatment strategy for 1,4-dioxane-contaminated waters that contain elevated levels of Cr(VI) in natural and built environments.

Post-treatment impacts of a novel combined hydrogen peroxide (H2O2) oxidation and WOx/ZrO2 catalysis used for the removal of 1,4-dioxane and chlorinated volatile organic compound (CVOC) contaminants were investigated in soil and groundwater microbial community. This treatment train removed ~90% 1,4-dioxane regardless of initial concentrations of 1,4-dioxane and CVOCs. The Illumina Miseq platform and bioinformatics were used to study the changes to microbial community structure. This approach determined that dynamic shifts of microbiomes were associated with conditions specific to treatments as well as 1,4-dioxane and CVOCs mixtures. The biodiversity was observed to decrease only after oxidation under conditions that included high levels of 1,4-dioxane and CVOCs, but increased when 1,4-dioxane was present without CVOCs. WOx/ZrO2 catalysis reduced biodiversity across all conditions. Taxonomic classification demonstrated oxidative tolerance for members of the genera Massilia and Rhodococcus, while catalyst tolerance was observed for members of the genera Sphingomonas and Devosia. Linear discriminant analysis effect size was a useful statistical tool to highlight representative microbes, while the multidimensional analysis elucidated the separation of microbiomes under the low 1,4-dioxane-only condition from all other conditions containing CVOCs, as well as the differences of microbial population among original, post-oxidation, and post-catalysis states. The results of this study enhance our understanding of microbial community responses to a promising chemical treatment train, and the metagenomic analysis will help practitioners predict the microbial community status during the post-treatment period, which may have consequences for long-term management strategies that include additional biodegradation treatment or natural attenuation.

Metallic nanoparticles (NPs), the most abundant nanomaterials in consumer and industrial products, are the most probable class to enter the environment. In this study, wetland-derived microcosms were incubated with copper nanoparticles (Cu-NP) and ionic CuCl(2) to investigate acute (10 days) and chronic (100 days) exposure towards nitrogen cycling microorganisms. The microbial ecology of wetlands play a crucial role in balancing nitrogen in pristine environments as well as in areas impacted by high nutrient loads (e.g., at wastewater effluent discharges). Gene abundance and expression changes were monitored using the GeoChip 5.0 high throughput functional gene microarray and metatranscriptomic shotgun sequencing (RNA-seq), respectively. After 10 days, the Cu-NP impacted microbial communities experienced structural shifts within microorganisms associated with dissimilatory nitrogen reduction accompanied by lower nitrate removal as compared to the unexposed controls. By day 100, these differences were largely resolved and nitrate removal was similar to the unexposed control. Furthermore, the Cu-NP exposed microcosms tolerated copper and were more resilient and adaptive than the unexposed controls based on the abundance and expression of other functions, including electron transfer, metal homeostasis, and stress response. These findings suggest sudden influxes of Cu-NPs into wetland systems may impair nitrogen removal initially, but long-term microbial shifts and functional redundancy would promote the net flux of total nitrogen out of the wetlands.

Natural vault nanoparticles are ribonucleoprotein particles with a mass of 13 MDa that have been found in a wide variety of eukaryotes. Empty recombinant vaults are assembled from heterologously expressed Major Vault Protein (MVP), forming the barrel-shaped vault shell. These structures are morphologically indistinguishable from natural vault particles. Here, we describe the packaging and purification of exogenous proteins into these recombinant vault particles by mixing with proteins attached to the INT domain that binds to MVP.

Vault particles are the largest naturally occurring ribonucleoprotein complexes found in the cytoplasm. In all 78 copies of major vault protein (MVP) assemble on polyribosome templates, forming recombinant vault particles, which are of great interest as encapsulation carriers for therapeutics delivery and enzyme stabilization. Baculovirus-insect cell expression is the only system that has been developed for recombinant vault synthesis, but it has low scalability and slow production rate. In this study, we demonstrated the first use of yeast cells for the production of vault particles with full integrity and functionality solely by expressing the complementary DNA (cDNA) encoding MVP. Vaults synthesized in Pichia pastoris yeast cells are morphologically indistinguishable from recombinant vault particles produced in insect cells, and are able to package and stabilize enzymes resulting in improved longevity and catalytic efficiency. Thus, our results imply that the yeast system is an economical alternative to insect cells for the production of recombinant vaults. The consistency of vault morphology between yeast and insect cell systems also underlines that polyribosome templating may be conserved among eukaryotes, which promises the synthesis and assembly of recombinant human vault particles in other eukaryotic organisms.

Proper characterization of nanoparticle (NP) interactions with environmentally relevant bacteria under representative conditions is necessary to enable their sustainable manufacture, use, and disposal. Previous nanotoxicology research based on planktonic growth has not adequately explored biofilms, which serve as the predominant mode of bacterial growth in natural and engineered environments. Copper nanoparticle (Cu-NP) impacts on biofilms were compared with respective planktonic cultures of the ammonium-oxidizing Nitrosomonas europaea, nitrogen-fixing Azotobacter vinelandii, and denitrifying Paracoccus denitrificans using a suite of independent toxicity diagnostics. Median inhibitory concentration (IC50) values derived from adenosine triphosphate (ATP) for Cu-NPs were lower in N. europaea biofilms (19.6 ± 15.3 mg/L) than in planktonic cells (49.0 ± 8.0 mg/L). However, in absorbance-based growth assays, compared with unexposed controls, N. europaea growth rates in biofilms were twice as resilient to inhibition than those in planktonic cultures. Similarly, relative to unexposed controls, growth rates and yields of P. denitrificans in biofilms exposed to Cu-NPs were 40-fold to 50-fold less inhibited than those in planktonic cells. Physiological evaluation of ammonium oxidation and nitrate reduction suggested that biofilms were also less inhibited by Cu-NPs than planktonic cells. Furthermore, functional gene expression for ammonium oxidation (amoA) and nitrite reduction (nirK) showed lower inhibition by NPs in biofilms relative to planktonic-grown cells. These results suggest that biofilms mitigate NP impacts, and that nitrogen-cycling bacteria in wastewater, wetlands, and soils might be more resilient to NPs than planktonic-based assessments suggest. Environ Toxicol Chem 2015;34:887–897. © 2014 SETAC

Natural vault nanoparticles are ribonucleoprotein particles with a mass of 13 MDa that have been found in a wide variety of eukaryotes. Empty recombinant vaults are assembled from heterologously expressed Major Vault Protein (MVP), forming the barrel-shaped vault shell. These structures are morphologically indistinguishable from natural vault particles. Here, we describe the packaging and purification of exogenous proteins into these recombinant vault particles by mixing with proteins attached to the INT domain that binds to MVP.

1,4-Dioxane is a probable human carcinogen and an emerging contaminant that has been detected in surface water and groundwater resources. Many conventional water treatment technologies are not effective for the removal of 1,4-dioxane due to its high water solubility and chemical stability. Biological degradation is a potentially low-cost, energy-efficient approach to treat 1,4-dioxane-contaminated waters. Two bacterial strains, Pseudonocardia dioxanivorans CB1190 (CB1190) and Mycobacterium austroafricanum JOB5 (JOB5), have been previously demonstrated to break down 1,4-dioxane through metabolic and co-metabolic pathways, respectively. However, both CB1190 and JOB5 have been primarily studied in laboratory planktonic cultures, while most environmental microbes grow in biofilms on surfaces. Another treatment technology, adsorption, has not historically been considered an effective means of removing 1,4-dioxane due to the contaminant's low Koc and Kow values. We report that the granular activated carbon (GAC), Norit 1240, is an adsorbent with high affinity for 1,4-dioxane as well as physical dimensions conducive to attached bacterial growth. In abiotic batch reactor studies, 1,4-dioxane adsorption was reversible to a large extent. By bioaugmenting GAC with 1,4-dioxane-degrading microbes, the adsorption reversibility was minimized while achieving greater 1,4-dioxane removal when compared with abiotic GAC (95–98% reduction of initial 1,4-dioxane as compared to an 85–89% reduction of initial 1,4-dioxane, respectively). Bacterial attachment and viability was visualized using fluorescence microscopy and confirmed by amplification of taxonomic genes by quantitative polymerase chain reaction (qPCR) and an ATP assay. Filtered samples of industrial wastewater and contaminated groundwater were also tested in the bioaugmented GAC reactors. Both CB1190 and JOB5 demonstrated 1,4-dioxane removal greater than that of the abiotic adsorbent controls. This study suggests that bioaugmented adsorbents could be an effective technology for 1,4-dioxane removal from contaminated water resources.