April Z. Gu

Aptamer-Based Optical Biosensor For Rapid and Sensitive Detection of 17β-Estradiol In Water Samples

Required routine monitoring of endocrine disrupting compounds (EDCs) in water samples, as posed by EPA Unregulated Contaminant Regulation (UCMR3), demands for cost-effective, reliable and sensitive EDC detection methods. This study reports a reusable evanescent wave aptamer-based biosensor for rapid, sensitive and highly selective detection of 17β-estradiol, an EDC that is frequently detected in environmental water samples. In this system, the capture molecular, β-estradiol 6-(O-carboxy-methyl)oxime-BSA, was covalently immobilized onto the optical fiber sensor surface. With an indirect competitive detection mode, samples containing different concentrations of 17β-estradiol were premixed with a given concentration of fluorescence-labeled DNA aptamer, which highly specifically binds to 17β-estradiol. Then, the sample mixture is pumped to the sensor surface, and a higher concentration of 17β-estradiol leads to less fluorescence-labeled DNA aptamer bound to the sensor surface and thus to lower fluorescence signal. The dose-response curve of 17β-estradiol was established and a detection limit was determined as 2.1 nM (0.6 ng mL(-1)). The high specificity and selectivity of the sensor were demonstrated by evaluating its response to a number of potentially interfering EDCs. Potential interference of real environmental sample matrix was assessed by spiked samples in several tertiary wastewater effluents. The sensor can be regenerated with a 0.5% SDS solution (pH 1.9) over tens of times without significant deterioration of the sensor performance. This portable sensor system can be potentially applied for on-site real-time inexpensive and easy-to-use monitoring of 17β-estradiol in environmental samples such as effluents or water bodies.

Mechanistic Toxicity Assessment of Nanomaterials by Whole-Cell-Array Stress Genes Expression Analysis

This study performed mechanistic toxicity assessment of nanosilver (nAg) and nanotitanium dioxide anatase (nTiO2_a) via toxicogenomic approach, employing a whole-cell-array library consisting of 91 recombinated Escherichia coli K12 strains with transcriptional GFP-fusions covering most known stress response genes. The results, for the first time, revealed more detailed transcriptional information on the toxic mechanism of nAg and nTiO2_a, and led to a better understanding of the mode of action (MOA) of metal and metal oxide nanomaterials (NMs). The detailed pathways network established for the oxidative stress system and for the SOS (DNA damage) repair system based on the temporal gene expression profiling data revealed the relationships and sequences of key genes involved in these toxin response systems. Both NMs were found to cause oxidative stress as well as cell membrane and transportation damage. Genotoxicity and DNA damage were also observed, although nTiO2_a induced SOS response via previously identified pathway and nAg seemed to induce DNA repair via a pathway different from SOS. We observed that the NMs at lower concentration tend to induce more chemical-specific toxicity response, while at higher concentrations, more general global stress response dominates. The information-rich real-time gene expression data allowed for identification of potential biomarkers that can be employed for specific toxin detection and biosensor developments. The concentration-dependent gene expression response led to the determination of the No Observed Transcriptional Effect Level (NOTEL) values, which can be potentially applied in the regulatory and risk assessment framework as an alternative toxicity assessment end point.

Impact of titanium dioxide nanomaterials on nitrogen fixation rate and intracellular nitrogen storage in Anabaena variabilis.

This study comprehensively investigated the impact of titanium dioxide nanomaterials (nTiO(2)) exposure on cell growth, nitrogen fixation activity, and nitrogen storage dynamics in the primary producer cyanobacteria Anabaena variabilis at various dose concentrations and exposure time lengths. The results indicated that both growth rate (EC(50)-96 h of 0.62 mgTiO(2)/L) and nitrogen fixation activity (EC(50)-96 h of 0.4 mgTiO(2)/L) were inhibited by nTiO(2) exposure. The Homs law (C(n)T(m)) was used as inactivation model to predict the concentration- and time-dependent inhibition of growth and nitrogen fixation activity. The kinetic parameters determined suggested that the time of exposure has a greater influence than the nTiO(2) concentration in toxicity. We observed, for the first time, that nTiO(2) induced a dose (concentration and time)-dependent increase in both the occurrence and intracellular levels of the nitrogen-rich cyanophycin grana proteins (CGPs). The results implied that CGPs may play an important role in the stress response mechanisms of nTiO(2) exposure and can serve as a toxicity assessment endpoint indicator. This study demonstrated that nitrogen-fixing activity could be hampered by the release of nTiO(2) in aquatic environments; therefore it potentially impacts important biogeochemical processes, such as carbon and nitrogen cycling.

Implication of using different carbon sources for denitrification in wastewater treatments.

Application of external carbon sources for denitrification becomes necessary for wastewater treatment plants that have to meet very stringent effluent nitrogen limits (e.g., 3 to 5 mgTN/L). In this study, we evaluated and compared three carbon sources--MicroC (Environmental Operating Solutions, Bourne, Massachusetts), methanol, and acetate-in terms of their denitrification rates and kinetics, effect on overall nitrogen removal performance, and microbial community structure of carbon-specific denitrifying enrichments. Denitrification rates and kinetics were determined with both acclimated and non-acclimated biomass, obtained from laboratory-scale sequencing batch reactor systems or full-scale plants. The results demonstrate the feasibility of the use of MicroC for denitrification processes, with maximum denitrification rates (k(dmax)) of 6.4 mgN/gVSSh and an observed yield of 0.36 mgVSS/mgCOD. Comparable maximum nitrate uptake rates were found with methanol, while acetate showed a maximum denitrification rate nearly twice as high as the others. The maximum growth rates measured at 20 degrees C for MicroC and methanol were 3.7 and 1.2 day(-1), respectively. The implications resulting from the differences in the denitrification rates and kinetics of different carbon sources on the full-scale nitrogen removal performance, under various configurations and operational conditions, were assessed using Biowin (EnviroSim Associates, Ltd., Flamborough, Ontario, Canada) simulations for both pre- and post-denitrification systems. Examination of microbial population structures using Automated Ribosomal Intergenic Spacer Analysis (ARISA) throughout the study period showed dynamic temporal changes and distinct microbial community structures of different carbon-specific denitrifying cultures. The ability of a specific carbon-acclimated denitrifying population to instantly use other carbon source also was investigated, and the chemical-structure-associated behavior patterns observed suggested that the complex biochemical pathways/enzymes involved in the denitrification process depended on the carbon sources used.

Functionally relevant microorganisms to enhanced biological phosphorus removal performance at full-scale wastewater treatment plants in the United States

The abundance and relevance ofAccumulibacter phosphatis (presumed to be polyphosphate-accumulating organisms [PAOs]), Competibacter phosphatis (presumed to be glycogen-accumulating organisms [GAOs]), and tetrad-forming organisms (TFOs) to phosphorus removal performance at six full-scale enhanced biological phosphorus removal (EBPR) wastewater treatment plants were investigated. Coexistence of various levels of candidate PAOs and GAOs were found at these facilities. Accumulibacter were found to be 5 to 20% of the total bacterial population, and Competibacter were 0 to 20% of the total bacteria population. The TFO abundance varied from nondetectable to dominant. Anaerobic phosphorus (P) release to acetate uptake ratios (P(rel)/HAc(up)) obtained from bench tests were correlated positively with the abundance ratio of Accumulibacter/(Competibacter +TFOs) and negatively with the abundance of (Competibacter +TFOs) for all plants except one, suggesting the relevance of these candidate organisms to EBPR processes. However, effluent phosphorus concentration, amount of phosphorus removed, and process stability in an EBPR system were not directly related to high PAO abundance or mutually exclusive with a high GAO fraction. The plant that had the lowest average effluent phosphorus and highest stability rating had the lowest P(rel)/HAc(up) and the most TFOs. Evaluation of full-scale EBPR performance data indicated that low effluent phosphorus concentration and high process stability are positively correlated with the influent readily biodegradable chemical oxygen demand-to-phosphorus ratio. A system-level carbon-distribution-based conceptual model is proposed for capturing the dynamic competition between PAOs and GAOs and their effect on an EBPR process, and the results from this study seem to support the model hypothesis.

IMPACT OF NANO TITANIUM DIOXIDE EXPOSURE ON CELLULAR STRUCTURE OF ANABAENA VARIABILIS AND EVIDENCE OF INTERNALIZATION

The present study investigated the impact of nano titanium dioxide (nTiO(2) ) exposure on the cellular structures of the nitrogen-fixing cyanobacteria Anabaena variabilis. Results of the present study showed that nTiO(2) exposure led to observable alteration in various intracellular structures and induced a series of recognized stress responses, including production of reactive oxygen species (ROS), appearance and increase in the abundance of membrane crystalline inclusions, membrane mucilage layer formation, opening of intrathylakoidal spaces, and internal plasma membrane disruption. The production of total ROS in A. variabilis cells increased with increasing nTiO(2) doses and exposure time, and the intracellular ROS contributed to only a small fraction (<10%) of the total ROS measured. The percentage of cells with loss of thylakoids and growth of membrane crystalline inclusions increased as the nTiO(2) dose and exposure time increased compared with controls, suggesting their possible roles in stress response to nTiO(2) , as previously shown for metals. Algal cell surface morphology and mechanical properties were modified by nTiO(2) exposure, as indicated by the increase in cell surface roughness and shifts in cell spring constant determined by atomic force microscopy analysis. The change in cell surface structure and increase in the cellular turgor pressure likely resulted from the structural membrane damage mediated by the ROS production. Transmission electron microscopy (TEM) analysis of nTiO(2) aggregates size distribution seems to suggest possible disaggregation of nTiO(2) aggregates when in close contact with microbial cells, potentially as a result of biomolecules such as DNA excreted by organisms that may serve as a biodispersant. The present study also showed, for the first time, with both TEM and Raman imaging that internalization of nTiO(2) particles through multilayered membranes in algal cells is possible. Environ. Toxicol. Chem. 2011; 30:861-869.

Reusable evanescent wave DNA biosensor for rapid, highly sensitive, and selective detection of mercury ions.

Mercury ions (Hg(2+)) are a highly toxic and ubiquitous pollutants requiring rapid and sensitive on-site detection methods in the environment and foods. Herein, we report an envanescent wave DNA-based biosensor for rapid and very sensitive Hg(2+) detection based on a direct structure-competitive detection mode. In this system, a DNA probe covalently immobilized onto a fiber optic sensor contains a short common oligonucleotide sequences that can hybidize with a fluorescently labeled complementary DNA. The DNA probe also comprises a sequence of T-T mismatch pairs that binds with Hg(2+) to form a T-Hg(2+)-T complex by folding of the DNA segments into a hairpin structure. With a structure-competitive mode, a higher concentration of Hg(2+) leads to less fluorescence-labeled cDNA bound to the sensor surface and thus to lower fluorescence signal. The total analysis time for a single sample, including the measurement and surface regeneration, was under 6 min with a Hg(2+) detection limit of 2.1 nM. The high specificity of the sensor was demonstrated by evaluating its response to a number of potentially interfering metal ions. The sensors surface can be regenerated with a 0.5% SDS solution (pH 1.9) over 100 times with no significant deterioration of performance. This platform is potentially applicable to detect other heavy metal ions or small-molecule analytes for which DNA/aptamers can be used as specific sensing probes.

Quantum Dot/Carrier−Protein/Haptens Conjugate as a Detection Nanobioprobe for FRET-Based Immunoassay of Small Analytes with All-Fiber Microfluidic Biosensing Platform

This study demonstrates the use of carrier-protein/haptens conjugate (e.g., BSA/2,4-dichlorophenoxyacetic acid, 2,4-D-BSA) for biological modification of quantum dots (QDs) for the detection of small analytes. Bioconjugated QDs, which are used as a detection nanoimmunoprobe, were prepared through conjugating carboxyl QDs with 2,4-D-BSA conjugate. Based on the principle of quantum dot-fluorescence resonance energy transfer (QD-FRET), an all-fiber microfluidic biosensing platform has been developed for investigating FRET efficiency, immunoassay mechanism and format, and binding kinetics between QD immunoprobe and fluorescence labeled anti-2,4-D monoclonal antibody. The structure of multiplex-haptens/BSA conjugate coupling to QD greatly improves the FRET efficiency and the sensitivity of the nanosensor. With a competitive detection mode, samples containing different concentrations of 2,4-D were incubated with a given concentration of QD immunoprobe and fluorescence-labeled antibody, and then detected by the all-fiber microfluidic biosensing platform. A higher concentration of 2,4-D led to less fluorescence-labeled anti-2,4-D antibody bound to the QD immunoprobe surface and, thus, a lower fluorescence signal. The quantification of 2,4-D over concentration ranges from 0.5 nM to 3 μM with a detection limit determined as 0.5 nM. The performance of the nanosensor with spiked real water samples showed good recovery, precision, and accuracy, indicating that it was less suspectable to water matrix effects. With the use of different QD nanobioprobes modified by other carrier-protein/haptens conjugates, this biosensing protocol based on QD-FRET can be potentially applied for on-site, real-time, inexpensive, and easy-to-use monitoring of other trace analytes.

Process optimization by decoupled control of key microbial populations: distribution of activity and abundance of polyphosphate-accumulating organisms and nitrifying populations in a full-scale IFAS-EBPR plant.

This study investigated the abundance and distribution of key functional microbial populations and their activities in a full-scale integrated fixed film activated sludge-enhanced biological phosphorus removal (IFAS-EBPR) process. Polyphosphate accumulating organisms (PAOs) including Accumulibacter and EBPR activities were predominately associated with the mixed liquor (>90%) whereas nitrifying populations and nitrification activity resided mostly (>70%) on the carrier media. Ammonia oxidizer bacteria (AOB) were members of the Nitrosomonas europaea/eutropha/halophila and the Nitrosomonas oligotropha lineages, while nitrite oxidizer bacteria (NOB) belonged to the Nitrospira genus. Addition of the carrier media in the hybrid activated sludge system increased the nitrification capacity and stability; this effect was much greater in the first IFAS stage than in the second one where the residual ammonia concentration becomes limiting. Our results show that IFAS-EBPR systems enable decoupling of solid residence time (SRT) control for nitrifiers and PAOs that require or prefer conflicting SRT values (e.g. >15 days required for nitrifiers and <5 days preferred for PAOs). Allowing the slow-growing nitrifiers to attach to the carrier media and the faster-growing phosphorus (P)-removing organisms (and other heterotrophs, e.g. denitrifiers) to be in the suspended mixed liquor (ML), the EBPR-IFAS system facilitates separate SRT controls and overall optimization for both N and P removal processes.

UV inactivation and resistance of rotavirus evaluated by integrated cell culture and real-time RT-PCR assay.

Rotaviruses are double-stranded RNA viruses which are among the most resistant waterborne enteric viruses to UV disinfection. An integrated cell culture and real-time RT-PCR (ICC real-time RT-PCR) assay was developed to detect the infectivity of rotaviruses in water, which uses real-time RT-PCR to detect RNA produced by infectious rotaviruses during replication in host cells. Detection of rotaviral RNA in host cells provides direct evidence of the presence of infectious rotavirus rather than just the presence of rotavirus RNA. Using this newly developed method, the inactivation and resistance of rotavirus to UV treatments at various doses was evaluated. With an initial concentration of 2 x 10(4)PFU/ml simian rotavirus (SA11), a first-order linear relationship was obtained at UV dose range of 0-120 mJ cm(-2), and the inactivation rate constant was estimated to be 0.0343 cm(2) mJ(-1) (R(2)=0.966). The dose-inactivation curve tailed off and reached plateau as the UV dose increased from 120 to 360 mJ cm(-2), indicating resistance phenomena of sub-populations of SA11 at very high UV doses. A maximal reduction of 4.8 log(10) was observed. Through parallel comparison with traditional culture assay, the ICC real-time RT-PCR method demonstrated more effective, sensitive and faster infectivity detection of rotavirus and, the results reveal that rotaviruses are more resistant to UV irradiation than previously reported with traditional cell culture assays.