Pilot scale microbial fuel cells using air cathodes for producing electricity while treating wastewater.

Microbial fuel cells (MFCs) can generate electrical energy from the oxidation of the organic matter, but they must be demonstrated at large scales, treat real wastewaters, and show the required performance needed at a site to provide a path forward for this technology. Previous pilot-scale studies of MFC technology have relied on systems with aerated catholytes, which limited energy recovery due to the energy consumed by pumping air into the catholyte. In the present study, we developed, deployed, and tested an 850 L (1400 L total liquid volume) air-cathode MFC treating domestic-type wastewater at a centralized wastewater treatment facility. The wastewater was processed over a hydraulic retention time (HRT) of 12 h through a sequence of 17 brush anode modules (11 m2 total projected anode area) and 16 cathode modules, each constructed using two air-cathodes (0.6 m2 each, total cathode area of 20 m2) with the air side facing each other to allow passive air flow. The MFC effluent was further treated in a biofilter (BF) to decrease the organic matter content. The field test was conducted for over six months to fully characterize the electrochemical and wastewater treatment performance. Wastewater quality as well as electrical energy production were routinely monitored. The power produced over six months by the MFC averaged 0.46 ± 0.35 W (0.043 W m-2 normalized to the cross-sectional area of an anode) at a current of 1.54 ± 0.90 A with a coulombic efficiency of 9%. Approximately 49 ± 15 % of the chemical oxygen demand (COD) was removed in the MFC alone as well as a large amount of the biochemical oxygen demand (BOD5) (70%) and total suspended solid (TSS) (48%). In the combined MFC/BF process, up to 91 ± 6 % of the COD and 91 % of the BOD5 were removed as well as certain bacteria (E. coli, 98.9%; fecal coliforms, 99.1%). The average effluent concentration of nitrate was 1.6 ± 2.4 mg L-1, nitrite was 0.17 ± 0.24 mg L-1 and ammonia was 0.4 ± 1.0 mg L-1. The pilot scale reactor presented here is the largest air-cathode MFC ever tested, generating electrical power while treating wastewater.

Comparison of Estrogenic, Spectroscopic, and Toxicological Analyses of Pilot-Scale Water, Wastewaters, and Processed Wastewaters at Select Military Installations.

Reuse of water requires the removal of contaminants to ensure human health. We report the relative estrogenic activity (REA) of reuse treatment design scenarios for water, wastewaters, and processed wastewaters before and after pilot-scale treatment systems tested at select military facilities. The comparative relationships between REA, several composite toxicological endpoints, and spectroscopic indicators were evaluated for different reuse treatment trains. Four treatment processes including conventional and advanced treatments reduced the estrogenicity by at least 33%. Biologically based methods reduced estrogenicity to below detection levels. Conventional treatment scenarios led to significantly less reduction of adverse biological endpoints compared to the advanced treatment scenarios. Incorporating the anaerobic membrane bioreactor reduced more endpoints with higher reduction percentages compared to the sequencing batch reactor design. Membrane technology and advanced oxidation generated reductions across all biological endpoints, from 65% (genotoxicity) to 100% (estrogenicity). The design scenarios featuring a low-cutoff mechanical screen filter, intermittent activated carbon biofilter, and membrane filtration achieved the highest percent reduction and produced water with the lowest negative biological endpoints. Spectroscopic indicators demonstrated case-specific relationships with estrogenicity and toxicity. Estrogenicity consistently correlated with cytotoxicity and thiol reactivity, indicating the potential for preliminary estrogenicity screening using thiol reactivity.

Toxicological Comparison of Water, Wastewaters, and Processed Wastewaters.

Drinking water utilities will increasingly rely on alternative water sources in the future, including wastewater reuse. Safety must be assured in the application of advanced oxidation processes (AOPs) and supporting treatments for wastewater effluent reuse. This study developed toxicological profiles for source and tap waters, wastewaters, and treated effluents by different processes from four military installation locations. The objective of this study was to evaluate the toxicity of extracted organics from diverse source waters and after reuse treatments. The toxicity analyses included thiol reactivity, mammalian cell cytotoxicity, and genotoxicity. Differences in toxicity between source or tap waters and effluents from wastewater treatment processes supported AOP treatment to reduce risks of potable reuse. An anoxic and aerobic activated sludge process followed by sand filtration controlled toxicity to levels similar to a municipal drinking water. An anaerobic membrane bioreactor process exceeded the toxicity levels of a typical drinking water. Two AOP processes (ultraviolet (UV) + reverse osmosis (RO) + chlorination (NaOCl) or RO + UV-H2O2 + NaOCl) significantly reduced toxicity. The integration of the wastewater systems with ultrafiltration, AOP, and RO was effective to reduce the toxicity to levels comparable to, or better than, tap water samples.

Evaluating a multi-panel air cathode through electrochemical and biotic tests.

To scale up microbial fuel cells (MFCs), larger cathodes need to be developed that can use air directly, rather than dissolved oxygen, and have good electrochemical performance. A new type of cathode design was examined here that uses a "window-pane" approach with fifteen smaller cathodes welded to a single conductive metal sheet to maintain good electrical conductivity across the cathode with an increase in total area. Abiotic electrochemical tests were conducted to evaluate the impact of the cathode size (exposed areas of 7 cm2, 33 cm2, and 6200 cm2) on performance for all cathodes having the same active catalyst material. Increasing the size of the exposed area of the electrodes to the electrolyte from 7 cm2 to 33 cm2 (a single cathode panel) decreased the cathode potential by 5%, and a further increase in size to 6200 cm2 using the multi-panel cathode reduced the electrode potential by 55% (at 0.6 A m-2), in a 50 mM phosphate buffer solution (PBS). In 85 L MFC tests with the largest cathode using wastewater as a fuel, the maximum power density based on polarization data was 0.083 ±â€¯0.006 W m-2 using 22 brush anodes to fully cover the cathode, and 0.061 ±â€¯0.003 W m-2 with 8 brush anodes (40% of cathode projected area) compared to 0.304 ±â€¯0.009 W m-2 obtained in the 28 mL MFC. Recovering power from large MFCs will therefore be challenging, but several approaches identified in this study can be pursued to maintain performance when increasing the size of the electrodes.

Thiol Reactivity Analyses To Predict Mammalian Cell Cytotoxicity of Water Samples.

An in chemico high throughput assay based on N-acetylcysteine was developed and used in conjunction with previous and new mammalian cell cytotoxicity data. Our objective was to derive an empirical equation with confidence levels for mammalian cell cytotoxicity prediction. Modeling data included 16 unique sources of waters and wastewaters of distinct water qualities to encompass a wide range of real environmental samples. This approach provides a quick screen to identify those water and wastewaters that could be prioritized for in depth analytical biological analyses and toxicity. The resulting model can serve as a preliminary convenient tool to screen for potential mammalian cell cytotoxicity in organic extracts of a wide variety of water samples.

Rapid Detection of Viable Bacillus anthracis Spores in Environmental Samples by Using Engineered Reporter Phages.

Bacillus anthracis, the causative agent of anthrax, was utilized as a bioterrorism agent in 2001 when spores were distributed via the U.S. postal system. In responding to this event, the Federal Bureau of Investigation used traditional bacterial culture viability assays to ascertain the extent of contamination of the postal facilities within 24 to 48 h of environmental sample acquisition. Here, we describe a low-complexity, second-generation reporter phage assay for the rapid detection of viableB. anthracis spores in environmental samples. The assay uses an engineered B. anthracis reporter phage (Wß::luxAB-2) which transduces bioluminescence to infected cells. To facilitate low-level environmental detection and maximize the signal response, expression of luxABin an earlier version of the reporter phage (Wß::luxAB-1) was optimized. These alterations prolonged signal kinetics, increased light output, and improved assay sensitivity. Using Wß::luxAB-2, detection of B. anthracis spores was 1 CFU in 8 h from pure cultures and as low as 10 CFU/g in sterile soil but increased to 10(5)CFU/g in unprocessed soil due to an unstable signal and the presence of competing bacteria. Inclusion of semiselective medium, mediated by a phage-expressed antibiotic resistance gene, maintained signal stability and enabled the detection of 10(4)CFU/g in 6 h. The assay does not require spore extraction and relies on the phage infecting germinating cells directly in the soil sample. This reporter phage displays promise for the rapid detection of low levels of spores on clean surfaces and also in grossly contaminated environmental samples from complex matrices such as soils.

Characterization of the activity of the spore cortex lytic enzyme CwlJ1.

The germination enzyme CwlJ1 plays an important role in degrading the cortex during the germination of Bacillus anthracis spores. However, the specific function and catalytic activity of CwlJ1 remain elusive. Here we report for the first time a detailed in vitro mechanistic study of CwlJ1 expressed in Escherichia coli and its activity against the spore cortical fragments of B. anthracis when added exogenously. CwlJ1 was active on both decoated spores and spore cortical fragments. Through liquid chromatography-mass spectrometry analysis of the digested cortical fragments, we determined that CwlJ1 was a thermostable N-acetylmuramoyl-L-alanine amidase. CwlJ1 mainly recognized large segments of glycan chains in the cortex instead of the minimal structural unit tetrasaccharide, with specificity for muramic acid-δ-lactam-containing glycan chains and preference for the tetrapeptide side chain. Unlike most amidases, CwlJ1 did not appear to contain a divalent cation, as it retained its activity in the presence of EDTA. This study shines some light on the mechanism of spore germination, and provides increased insight into the development of sporicidal enzyme systems for decontamination of B. anthracis and other related bacteria.

Characterization of AmiBA2446, a novel bacteriolytic enzyme active against Bacillus species.

There continues to be a need for developing efficient and environmentally friendly treatments for Bacillus anthracis, the causative agent of anthrax. One emerging approach for inactivation of vegetative B. anthracis is the use of bacteriophage endolysins or lytic enzymes encoded by bacterial genomes (autolysins) with highly evolved specificity toward bacterium-specific peptidoglycan cell walls. In this work, we performed in silico analysis of the genome of Bacillus anthracis strain Ames, using a consensus binding domain amino acid sequence as a probe, and identified a novel lytic enzyme that we termed AmiBA2446. This enzyme exists as a homodimer, as determined by size exclusion studies. It possesses N-acetylmuramoyl-l-alanine amidase activity, as determined from liquid chromatography-mass spectrometry (LC-MS) analysis of muropeptides released due to the enzymatic digestion of peptidoglycan. Phylogenetic analysis suggested that AmiBA2446 was an autolysin of bacterial origin. We characterized the effects of enzyme concentration and phase of bacterial growth on bactericidal activity and observed close to a 5-log reduction in the viability of cells of Bacillus cereus 4342, a surrogate for B. anthracis. We further tested the bactericidal activity of AmiBA2446 against various Bacillus species and demonstrated significant activity against B. anthracis and B. cereus strains. We also demonstrated activity against B. anthracis spores after pretreatment with germinants. AmiBA2446 enzyme was also stable in solution, retaining its activity after 4 months of storage at room temperature.

Inactivation of bacteriophage MS2 with potassium ferrate(VI).

Ferrate [Fe(VI); FeO(4)(2-)] is an emerging oxidizing agent capable of controlling chemical and microbial water contaminants. Here, inactivation of MS2 coliphage by Fe(VI) was examined. The inactivation kinetics observed in individual batch experiments was well described by a Chick-Watson model with first-order dependences on disinfectant and infective phage concentrations. The inactivation rate constant k(i) at a Fe(VI) dose of 1.23 mgFe/L (pH 7.0, 25 °C) was 2.27(±0.05) L/(mgFe × min), corresponding to 99.99% inactivation at a Ct of ~4 (mgFe × min)/L. Measured k(i) values were found to increase with increasing applied Fe(VI) dose (0.56-2.24 mgFe/L), increasing temperature (5-30 °C), and decreasing pH conditions (pH 6-11). The Fe(VI) dose effect suggested that an unidentified Fe byproduct also contributed to inactivation. Temperature dependence was characterized by an activation energy of 39(±6) kJ mol(-1), and k(i) increased >50-fold when pH decreased from 11 to 6. The pH effect was quantitatively described by parallel reactions with HFeO(4)(-) and FeO(4)(2-). Mass spectrometry and qRT-PCR analyses demonstrated that both capsid protein and genome damage increased with the extent of inactivation, suggesting that both may contribute to phage inactivation. Capsid protein damage, localized in the two regions containing oxidant-sensitive cysteine residues, and protein cleavage in one of the two regions may facilitate genome damage by increasing Fe(VI) access to the interior of the virion.

Mechanistic aspects of adenovirus serotype 2 inactivation with free chlorine.

Free chlorine is an effective disinfectant for controlling adenoviruses in drinking water, but little is known about the underlying inactivation mechanisms. The objective of this study was to elucidate the molecular components of adenovirus type 2 (Ad2) targeted by free chlorine during the inactivation process. The effects of free chlorine treatment on several Ad2 molecular components and associated life cycle events were compared to its effect on the ability of adenovirus to complete its life cycle, i.e., viability. Free chlorine treatment of Ad2 virions did not impair their ability to interact with monoclonal antibodies specific for hexon and fiber proteins of the Ad2 capsid, as measured by enzyme-linked immunosorbent assays, nor did it impair their interaction with recombinant, purified Coxsackie-adenovirus receptor (CAR) proteins in vitro. Free chlorine-treated Ad2 virions also retained their ability to bind to CAR receptors on A549 cell monolayers, despite being unable to form plaques, suggesting that free chlorine inactivates Ad2 by inhibiting a postbinding event of the Ad2 life cycle. DNA isolated from Ad2 virions that had been inactivated by free chlorine was able to be amplified by PCR, indicating that genome damage was not the cause of inactivation. However, inactivated Ad2 virions were unable to express E1A viral proteins during infection of A549 host cells, as measured by using immunoblotting. Collectively, these results indicate that free chlorine inactivates adenovirus by damaging proteins that govern life cycle processes occurring after host cell attachment, such as endocytosis, endosomal lysis, or nuclear delivery.

Kinetics of adenovirus type 2 inactivation with free chlorine.

The objective of this study was to elucidate the effects of pH, temperature, and other relevant water quality parameters on the kinetics of adenovirus serotype 2 inactivation with free chlorine. Over a pH range of 6.5-10, a temperature range of 1-30 degrees C, and in a variety of water types, free chlorine was highly effective against adenovirus type 2. Its disinfection efficacy decreased with increasing pH and decreasing temperature, yet was unaffected by hardness and buffering species. Under the most challenging conditions investigated in this study (pH 10, 1 degrees C), a four-log reduction of adenovirus viability would be achieved at a CT value of 2.6mgCl(2)min/L. The inactivation kinetics was characterized by three phases of inactivation under most conditions. The first phase resulted from a reaction involving primarily the hypochlorous acid species and was characterized by rapid inactivation of viruses to a limit that increased with decreasing pH and increasing temperature. After reaching this limit, adenovirus exhibited two subsequent phases of inactivation at lesser rates that were not affected by temperature or pH. As with the first phase of kinetics, a limit of inactivation was approached in the second phase that decreased with increasing pH, and after which the kinetics was characterized by a third and final phase. An inactivation model consistent with these observations was found to provide adequate representation for the free chlorine inactivation of adenovirus serotype 2 as well as that reported in the literature for other adenovirus serotypes.

Treatment of coliphage MS2 with palladium-modified nitrogen-doped titanium oxide photocatalyst illuminated by visible light.

A palladium-modified nitrogen-doped titanium oxide (TiON/-PdO) photocatalytic fiber was synthesized on a mesoporous activated carbon fiber template by a sol-gel process. Calcination of the coated fibers resulted in a macroporous interfiber structure and mesoporous photocatalyst coating. Atomic ratios of major photocatalyst constituents determined by X-ray photoelectron spectroscopy analyses were N/Ti approximately equal to 0.1 and Pd/-Ti approximately equal to 0.03. X-ray diffraction analyses revealed that the photocatalyst had an anatase structure and palladium additive was present as PdO. Triplicate batch experiments performed with MS2 phage (average initial concentration of 3 x 10(8) plaque forming units/mL) and TiON/PdO photocatalyst at a dose of 0.1 g/L under dark conditions revealed the occurrence of virus adsorption on the photocatalyst fibers at a rate that resulted in equilibrium within 1 h of contact time with corresponding virion removals of 95.4-96.7%. Subsequent illumination of the dark-equilibrated samples with visible light (wavelengths greater than 400 nm and average intensity of 40 mW/cm2) resulted in additional virus removal of 94.5-98.2% within 1 h of additional contact time. By combining adsorption and visible-light photocatalysis, TiON/PdO fibers reached final virus removal rates of 99.75-99.94%. Spin trapping electron paramagnetic resonance (EPR) measurements confirmed the production of *OH radicals by TiON/PdO under visible light illumination, which provided indirect evidence about MS2 phage being potentially inactivated.