Effect of free ammonia concentration on monochloramine penetration within a nitrifying biofilm and its effect on activity, viability, and recovery.

Chloramine has replaced free chorine for secondary disinfection at many water utilities because of disinfection by-product (DBP) regulations. Because chloramination provides a source of ammonia, there is a potential for nitrification when using chloramines. Nitrification in drinking water distribution systems is undesirable and may result in degradation of water quality and subsequent non-compliance with existing regulations. Thus, nitrification control is a major issue and likely to become increasingly important as chloramine use increases. In this study, monochloramine penetration and its effect on nitrifying biofilm activity, viability, and recovery was investigated and evaluated using microelectrodes and confocal laser scanning microscopy (CLSM). Monochloramine was applied to nitrifying biofilm for 24 h at two different chlorine to nitrogen (Cl(2):N) mass ratios (4:1 [4.4 mg Cl(2)/L] or 1:1 Cl(2):N [5.3 mg Cl(2)/L]), resulting in either a low (0.23 mg N/L) or high (4.2 mg N/L) free ammonia concentration. Subsequently, these biofilm samples were allowed to recover without monochloramine and receiving 4.2 mg N/L free ammonia. Under both monochloramine application conditions, monochloramine fully penetrated into the nitrifying biofilm within 24 h. Despite this complete monochloramine penetration, complete viability loss did not occur, and both biofilm samples subsequently recovered aerobic activity when fed only free ammonia. When monochloramine was applied with a low free ammonia concentration, dissolved oxygen (DO) fully penetrated, but with a high free ammonia concentration, complete cessation of aerobic activity (i.e., oxygen utilization) did not occur and subsequent analysis indicated that oxygen consumption still remained near the substratum. During the ammonia only recovery phase, different spatial recoveries were seen in each of the samples, based on oxygen utilization. It appears that the presence of higher free ammonia concentration allowed a larger biomass to remain active during monochloramine application, particularly the organisms deeper within the biofilm, leading to faster recovery in oxygen utilization when monochloramine was removed. These results suggest that limiting the free ammonia concentration during monochloramine application will slow the onset of nitrification episodes by maintaining the biofilm biomass at a state of lower activity.

Persistence of non-native spore forming bacteria in drinking water biofilm and evaluation of decontamination methods.

Persistence of Bacillus globigii spores, a surrogate for Bacillus anthracis, was studied on biofouled concrete-lined slides in drinking water using biofilm annular reactors. Reactors were inoculated with B. globigii spores and persistence was monitored in the bulk and biofilm phases, first in dechlorinated water and later with free chlorine concentrations of 1 and 5 mg/L. In the dechlorinated study, a steady state population of spores developed on the slides. The addition of free chlorine at 5 mg/L decreased the adhered spore density by 2-logs within 4 hours and spores were not detected after 67 and 49 hours in the presence of 1 and 5 mg/L free chlorine, respectively. This suggests that adhered spores can persist in non-chlorinated conditions, but detach and/or are inactivated upon addition of free chlorine. When injected into a chlorinated reactor, adhered spore density continually decreased and spores were either undetectable or unquantifiable by 48 hours for both 1 and 5 mg/L chlorine concentrations. Results from these experiments suggest that the presence of a free chlorine residual limits adherence of viable spores to biofouled concrete-lined pipe walls by inactivating spores before they have attached. Both free chlorine concentrations (1 and 5 mg/L) were equally effective at inactivating spores in terms of log reduction, but the higher concentrations yielded faster rates of log reduction.

Free chlorine and monochloramine application to nitrifying biofilm: comparison of biofilm penetration, activity, and viability.

Biofilm in drinking water systems is undesirable. Free chlorine and monochloramine are commonly used as secondary drinking water disinfectants, but monochloramine is perceived to penetrate biofilm better than free chlorine. However, this hypothesis remains unconfirmed by direct biofilm monochloramine measurement. This study compared free chlorine and monochloramine biofilm penetration into an undefined mixed-culture nitrifying biofilm by use of microelectrodes and assessed the subsequent effect on biofilm activity and viability by use of dissolved oxygen (DO) microelectrodes and confocal laser scanning microscopy (CLSM) with LIVE/DEAD BacLight. For equivalent chlorine concentrations, monochloramine initially penetrated biofilm 170 times faster than free chlorine, and even after subsequent application to a monochloramine penetrated biofilm, free chlorine penetration was limited. DO profiles paralleled monochloramine profiles, providing evidence that either the biofilm was inactivated with monochloramine's penetration or its persistence reduced available substrate (free ammonia). While this research clearly demonstrated monochloramine's greater penetration, this penetration did not necessarily translate to immediate viability loss. Even though free chlorine's penetration was limited compared to that of monochloramine, it more effectively (on a cell membrane integrity basis) inactivated microorganisms near the biofilm surface. Limited free chlorine penetration has implications when converting to free chlorine in full-scale chloraminated systems in response to nitrification episodes.

Potentiometric and voltammetric polymer lab chip sensors for determination of nitrate, pH and Cd(II) in water.

Due to their toxicity to humans and animals, heavy metals and nitrate in groundwater are of particular concern. The combination of high toxicity and widespread occurrence has created a pressing need for effective monitoring and measurement of nitrate and heavy metals in soil pore water and groundwater at shallow depths. In this work, a new electrochemical sensing platform with the self-assembly nanobeads-packed (nBP) hetero columns has been developed for the pH and nitrate measurements. In addition, for on-site determination of cadmium (Cd(II)), a bismuth (Bi(III)) based polymer lab chip sensor using the square-wave anodic stripping voltammetry (SWASV) sensing principle has been designed, fabricated and successfully characterized. Factors affecting sensitivity and precision of the sensor, including deposition potential and deposition time, were studied. Miniaturized electrochemical lab chip sensors could be very valuable in environmental monitoring area due to their many benefits, such as greatly reduced sensing cost, sensing system portability, and ease of use.

Fabrication, calibration and evaluation of a phosphate ion-selective microelectrode.

To conduct the micro-environment study of flocs in an enhanced biological phosphorus removal (EBPR) process, a phosphate ion-selective microelectrode was developed. The cobalt-based microelectrodes have tip diameters of 5-20µm and respond to all the three forms of phosphate ions, namely, H(2)PO(4)(-), HPO(4)(2-), and PO(4)(3-). The calibration curve at pH 7.5 had a slope of 31.5mV per decade change of concentration and a R(2) value of 0.99. Other characteristics of this microelectrode, such as response time, interferences from pH, ion strength, DO and other anions were also evaluated.

Kinetics determination of electrogenerated hydrogen peroxide (H2O2) using carbon fiber microelectrode in electroenzymatic degradation of phenolic compounds.

The kinetics of electrogenerated hydrogen peroxide (H(2)O(2)), which can activate peroxidases in an electroenzymatic process, was examined by an amperometric technique using a carbon fiber microelectrode that was modified by polyaniline (PAn) film and platinum particles. The electrogeneration of H(2)O(2) was found to be dependent on the pH and applied potential, and resulting in a variable current response of the carbon fiber microelectrode. The highest amount of H(2)O(2) was electrogenerated when 2.3 V was applied between the Pt/Ti anode and a reticulated vitreous carbon (RVC) cathode at pH 6.0, with a current response of 0.0190 microA min(-1). Phenol was completely degraded by the electroenzymatic reaction of the immobilized horseradish peroxidase (HRP), and the time required for the electrogeneration of H(2)O(2) increased according to the initial concentration of phenol. The degradation stoichiometric ratio between the electrogenerated H(2)O(2) and the aqueous phenol under HRP immobilized on RVC was found to be 1:1.

Biological application of micro-electro mechanical systems microelectrode array sensors for direct measurement of phosphate in the enhanced biological phosphorous removal process.

The determination of phosphate has been of great importance in the fields of clinical, environmental, and horticultural analysis for over three decades. New cobalt-based micro-electro mechanical systems (MEMS) microelectrode array (MEA) sensors for direct measurement of phosphate in small environmental samples, such as microbial aggregates, has been introduced and applied here for in situ measurement of phosphate within activated sludge flocs in the enhanced biological phosphorus removal process. The MEMS technologies offer the advantages of accurate fabrication methods, reduced complexity of the fabrication process, mass production, low cost, and increased reliability. Well-defined phosphate profiles across the flocs were observed under anaerobic conditions, during which, phosphate was released from the flocs, using the MEMS MEA sensor. The microprofiles were compared with the microprofiles measured using conventional phosphate microelectrodes. The developed MEMS MEA sensors were useful tools for the in situ measurement of phosphate in small aggregates.

The application of a mulch biofilm barrier for surfactant enhanced polycyclic aromatic hydrocarbon bioremediation.

Lab scale mulch biofilm barriers were constructed and tested to evaluate their performance for preventing the migration of aqueous and surfactant solubilized PAHs. The spatial distribution of viable PAH degrader populations and resultant biofilm formation were also monitored to evaluate the performance of the biobarrier and the prolonged surfactant effect on the PAH degrading microorganism consortia in the biobarrier. Sorption and biodegradation of PAHs resulted in stable operation of the system for dissolved phenanthrene and pyrene during 150 days of experimentation. The nonionic surfactant could increase the solubility of phenanthrene and pyrene significantly. However, the biobarrier itself couldn't totally prevent the migration of micellar solubilized phenanthrene and pyrene. The presence of surfactant and the resultant highly increased phenanthrene or pyrene concentration didn't appear to cause toxic effects on the attached biofilm in the biobarrier. However, the presence of surfactant did change the structural composition of the biofilm.

Characteristics of a cobalt-based phosphate microelectrode for in situ monitoring of phosphate and its biological application.

The in situ monitoring of phosphate has been of great importance in many environmental applications, particularly those involving biological treatment processes and eutrophication monitoring. A microelectrode with small tip size (~10 mum) was fabricated with cobalt wire, characterized and evaluated for in situ and in vivo environmental analysis of phosphate in biological applications. The electrochemical performance of this cobalt-based microelectrode was fully examined for its characteristics, including detection limit, response time, selectivity, reproducibility, life time, interference with pH, ions and dissolved oxygen (DO), and the stirring effect. The microelectrode showed excellent selectivity for the orthophosphate ions (HPO(4) (2-), H(2)PO(4) (-)) in various environmental conditions. Alkalinity and DO were found to interfere with electrode response to phosphate. The phosphate microelectrode was also evaluated with Scanning Electron Microscopy (SEM) and Cyclic Voltammetry (CV). The developed microelectrode was used for in situ monitoring of phosphate in microbial flocs. Well-defined phosphate profiles across the flocs were observed under anaerobic (phosphorus release) conditions. This full characterization and successful application showed that the cobalt-based phosphate microelectrodes can be a very useful tool for in situ measurement of phosphate in various environmental conditions, including within microbial flocs.

The monitoring of biofilm formation in a mulch biowall barrier and its effect on performance.

Lab scale mulch biofilm biowall barriers were constructed and tested to monitor the effect of biofilm formation on the performance of the biobarrier. Naphthalene, a two-ring polycyclic aromatic hydrocarbon (PAH), was used as the model compound. With column reactors, the amounts of viable naphthalene degraders and biofilm formation were monitored, as was the performance of the biobarrier. The sorption capacity of the mulch, the increase in biomass and the extracellular polymeric substance (EPS) content of the biofilm created a strong affinity for naphthalene and induced an increase in the number of slowly growing hydrocarbon degraders, resulting in a higher degradation rate and more stable PAH removal. Concentration profiles of pore water naphthalene and electron acceptors indicated that dissolved oxygen (DO) was preferentially used as the electron acceptor, and the greatest removal occurred at the inlet to the column reactor where DO was highest. However, when using nitrate as an alternative electron acceptor, both biofilm formation and continual degradation of naphthalene also occurred. Microprofiles of DO in the biofilm revealed that oxygen transport in the biofilm was limited, and there might be sequential utilization of nitrate for naphthalene removal in the anoxic zones of the biofilm. These results provide insight into the distribution of viable biomass and biofilm EPS production in engineered permeable reactive mulch biobarriers.

MEMS needle-type sensor array for in situ measurements of dissolved oxygen and redox potential.

Microelectrodes are among the most accurate and reliable monitoring devices for measuring the dynamics of biofilm processes. This paper describes a novel needle-type microelectrode array (MEA) for simultaneous in situ measurements of dissolved oxygen (DO) and oxidation reduction potential (ORP) fabricated using microelectromechanical systems (MEMS) technologies. The MEA exhibits fast response times for both DO and ORP measurements and shows a substantial increase in DO sensitivity. To demonstrate the versatility of the new sensor, it was applied to the measurement of DO and ORP microprofiles in a multispecies biofilm. This work demonstrates that the MEA is able to monitor local concentration changes with a high spatial resolution and provide the versatility of the microelectrode technique needed for biofilm studies as well as the capability for repetitive measurements. In addition, the use of MEMS technologies and batch fabrication approaches enables integration, high consistency, high yields, and mass production. With further development, it may be possible to add additional sensors to the MEA (e.g., pH, phosphate) and integrate them with a reference electrode.

Influence of nonionic surfactant on attached biofilm formation and phenanthrene bioavailability during simulated surfactant enhanced bioremediation.

Surfactant effects on preformed attached biofilm formation and the biodegradation of soil-sorbed phenanthrene were monitored using a continuously flowing flowcell system. Thirty-two flowcell reactors were constructed to monitor phenanthrene degradation and biofilm formation under five different surfactant concentrations. Initially, all flowcell reactors were operated without surfactant for 25 days to allow for the formation of an attached bacterial biofilm on phenanthrene spiked soil; after that, a model nonionic surfactant (Triton X-100) was applied to the flowcell reactors at five different concentrations (0, 100, 200, 500, and 1000 mg/L), which represent concentrations below (0 and 100 mg/L) and above (200, 500, and 1000 mg/L) the critical micelle concentration (cmc). The results obtained in this study reveal that bacterial biofilm formation with extracellular polymeric substances was the strategy of bacteria to utilize partially soluble PAHs. However, in the presence of surfactant, this strategy was modified. The presence of surfactant at all concentrations changed the physiological aspects of the attached biofilm, and the bioavailability of the phenanthrene increased with the addition of surfactant above the cmc.

Persistence and decontamination of Bacillus atrophaeus subsp. globigii spores on corroded iron in a model drinking water system.

Persistence of Bacillus atrophaeus subsp. globigii spores on corroded iron coupons in drinking water was studied using a biofilm annular reactor. Spores were inoculated at 10(6) CFU/ml in the dechlorinated reactor bulk water. The dechlorination allowed for observation of the effects of hydraulic shear and biofilm sloughing on persistence. Approximately 50% of the spores initially adhered to the corroded iron surface were not detected after 1 month. Addition of a stable 10 mg/liter free chlorine residual after 1 month led to a 2-log(10) reduction of adhered B. atrophaeus subsp. globigii, but levels on the coupons quickly stabilized thereafter. Increasing the free chlorine concentration to 25 or 70 mg/liter had no additional effect on inactivation. B. atrophaeus subsp. globigii spores injected in the presence of a typical distribution system chlorine residual (approximately 0.75 mg/liter) resulted in a steady reduction of adhered B. atrophaeus subsp. globigii over 1 month, but levels on the coupons eventually stabilized. Adding elevated chlorine levels (10, 25, and 70 mg/liter) after 1 month had no effect on the rate of inactivation. Decontamination with elevated free chlorine levels immediately after spore injection resulted in a 3-log(10) reduction within 2 weeks, but the rate of inactivation leveled off afterward. This indicates that free chlorine did not reach portions of the corroded iron surface where B. atrophaeus subsp. globigii spores had adhered. B. atrophaeus subsp. globigii spores are capable of persisting for an extended time in the presence of high levels of free chlorine.

A disposable on-chip phosphate sensor with planar cobalt microelectrodes on polymer substrate.

Disposable microsensors on polymer substrates consisting of fully integrated on-chip planar cobalt (Co) microelectrodes, Ag/AgCl reference electrodes, and microfluidic channels have been designed, fabricated, and characterized for phosphate concentration measurement in aqueous solution. The planar Co microelectrode shows phosphate-selective potential response over the range from 10(-5) to 10(-2)M in acidic medium (pH 5.0) for both inorganic (KH(2)PO(4)) and organic (adenosine 5'-triphosphate (ATP) and adenosine 5'-diphosphates (ADP)) phosphate compounds. This microfabricated sensor also demonstrates significant reproducibility with a small repeated sensing deviation (i.e. relative standard deviation (R.S.D.)<1%) on a single chip and a small chip-to-chip deviation (i.e. R.S.D.<2.5%). Specifically, while keeping the high selectivity, sensitivity, and stability of a conventional bulk Co-wire electrode, the proposed phosphate sensor yields advantages such as ease of use, cost effectiveness, reduced analyte consumption, and ease of integrating into disposable polymer lab-on-a-chip devices. The capability to sense both inorganic and organic phosphate compounds makes this sensor applicable in diverse areas such as environmental monitoring, soil extract analysis, and clinical diagnostics.

Persistence of Klebsiella pneumoniae on simulated biofilm in a model drinking water system.

Persistence of Klebsiella pneumoniae on corroded iron surfaces in drinking water was studied using biofilm annular reactors operated under oligotrophic conditions. Reactors were inoculated with K. pneumoniae, and persistence was monitored in the bulk and biofilm phases. Initial cell concentration of 10(6) MPN/mL in the bulkwater phase resulted in significantly longer adhesion than initial concentrations 1 and 2 orders of magnitude lower. K. pneumoniae cultured in low nutrient growth medium persisted longer in dechlorinated tap water than those cultured in full strength medium. Cell surface charge was more negative under low nutrient conditions, and this influenced electrostatic attraction between the cells and the oxidized iron surface. Cells grown in full strength media persisted longer in water with both low (<0.2 mg/L) and high (>0.5 mg/L) free chlorine residuals. Growth media injected with the cells dechlorinated the water allowing adhesion without inactivation. Microelectrode measurements showed a 40-70% drop in free chlorine from the bulk to the coupon surface, which decreased disinfectant potency against adhered cells. Growth and injection conditions clearly influenced cell adhesion and persistence, but permanent colonization of the corroded iron surface by K. pneumoniae was not observed.

Biological phosphate uptake and release: effect of pH and magnesium ions.

Enhanced biological phosphorus removal (EBPR) is based on poly-phosphate accumulating organisms' (PAOs) unique features of "luxury" phosphate uptake during aerobic conditions and phosphate release in anaerobic conditions. It is believed that poly-phosphate accumulation is accompanied by the uptake and accumulation of potassium ions (K+) and magnesium ions (Mg2+). The release of phosphate under anaerobic conditions is also accompanied by the release of both cations. The objective of this research was to evaluate the effect of pH and Mg2+ on the biological phosphate uptake and release behavior of activated sludge mixed liquor during aeration and sedimentation. Research results indicate that Mg2+, supplied either by magnesium chloride (MgCl2) or magnesium hydroxide [Mg(OH)2], stimulated phosphate uptake during the aeration period, while pH increase, caused by the application of Mg(OH)2, enhanced phosphate release during the sedimentation period. It is also noted in our experiments with MgCl2 that Mg2+ slightly inhibited anaerobic phosphate release.

Measurement of chlorine dioxide penetration in dairy process pipe biofilms during disinfection.

Biofilms are considered a significant health risk in the food and dairy industries because they can harbor pathogens, and direct contact with them can lead to food contamination. Biofilm control is often performed using strong oxidizing agents like chlorine and peracetic acid. Although chlorine dioxide (ClO2) is being used increasingly to control microbiological growth in a number of different industries, not much is known about disinfection in biofilms using chlorine dioxide. In this study, a microelectrode originally made for chlorine detection was modified to measure the profiles of chlorine dioxide in biofilm as a function of depth into the biofilm. In addition, discarded microelectrodes proved useful for in situ direct measurement of biofilm thicknesses. The chlorine dioxide microelectrode had a linear response when calibrated up to a ClO2 concentration of 0.4 mM. ClO2 profiles showed depletion of disinfectant at 100 microm in the biofilm depth, indicating that ClO2 may not reach bacteria in a biofilm thicker than this using a 25 mg/l solution.

Stabilization of mercury-containing wastes using sulfide.

This paper summarizes the findings of our studies on mercury stabilization using sulfide. Primary stabilization variables such as stabilization pH and sulfide/mercury molar ratio were tested. Mercury stabilization effectiveness was evaluated using the Toxicity Characteristic Leaching Procedure (TCLP) and the constant pH leaching tests. The influence of interfering ions on mercury immobilization was also tested. The experimental results indicate that the sulfide-induced treatment technology is an effective way to minimize mercury leaching. It was found that the most effective mercury stabilization occurs at pH 6 combined with a sulfide/mercury molar ratio of 1. The combined use of increased dosage of sulfide and ferrous ions ([S]/[Hg]=2 and [Fe]/[Hg]=3 at pH=6) can significantly reduce interferences by chloride and/or phosphate during sulfide-induced mercury immobilization. The sulfide-treated waste stabilization efficiency reached 98%, even with exposure of the wastes to high pH leachants.

Miniaturized redox potential probe for in situ environmental monitoring.

The need for accurate, robust in situ microscale monitoring of oxidation-reduction potentials (ORP) is required for continuous soil pore water quality monitoring. We are developing a suite of self-contained microelectrodes that can be used in the environment, such as at Superfund sites, to monitor ORP in contaminated soils and sediments. This paper presents details on our development of microelectrode sensor arrays for ORP measurements. The electrochemical performance of these ORP electrodes was fully characterized by measuring redox potentials in standard solutions. It found that the newly developed integrated ORP microelectrodes produced a very stable voltage response (the corresponding rate of the integrated microelectrode potential change was in the range of 0.6-1.1 mV/min), even when the measurement was carried out outside of a Faraday cage where signals from most conventional microelectrodes are usually inhibited by external electrical nose. These new microelectrodes were easier to fabricate and were more robust than conventional microelectrodes. The tip size of the integrated ORP microelectrode was approximately 200 nm square, with a taper angle of approximately 20 degrees and a length of 57 microm. The integrated ORP microelectrode exhibited better signal stability and substantially shorter response times (from less than a few milliseconds to 30 s, depending on the standard solution used) than the commercial millielectrode (a few minutes). Compared with the slope of the commercial millelectrode, the slope of the integrated microelectrode (61.5 mV/pH) was closerto the ideal slope against quinhydrone calibration solutions. Therefore, it is to be expected that the newly developed ORP microelectrode may have wider applications in contaminated soils, biofilms, and sediments.

A strategy for controlling deposition of struvite in municipal wastewater treatment plants.

This paper presents strategies to reduce the risk of struvite deposition by controlling its location of formation. Two technical routes were investigated: (1) to fix the phosphate into the dewatered sludge cake, and (2) to remove phosphate from centrate or filtrate. Chemicals used include magnesium hydroxide [Mg(OH)2], both of reagent grade and reclaimed from a flue gas desulfurization system, magnesium chloride (MgCl2), calcium hydroxide [Ca(OH)2], ferric chloride (FeCl3) and aluminum sulfate [Al2(SO4)3]. Research results indicate that (1) for anaerobically well-digested sludge, Mg(OH)2 is effective in fixing phosphate into sludge cake and improving sludge dewaterability, and (2) adding Mg(OH)2 into a reactor, located between the sludge dewatering facilities and the centrate or filtrate discharge line, and using air for mixing and carbon dioxide stripping, proves feasible in reducing struvite deposition in centrate or filtrate discharge lines and can generate a potentially valuable plant fertilizer--struvite.