Simplified Mercury Extraction from Coal Fly Ash for Quantification of Total Mercury by ELISA-based Immunoassay.

A simplified two-step mercury extraction procedure enabled the selective and reproducible mercury recovery from actual coal fly ash (CFA). The optimized extraction procedure involving conventional enzyme-linked immunosorbent assay (ELISA)-based immunoassay allowed the ultra-sensitive quantification of total mercury content in CFA. The total mercury content of 41 CFA samples were successfully determined using the above-mentioned method, and the results were in agreement with those obtained by standard instrumental analysis (thermal decomposition atomic absorption spectrometry) within a 15% coefficient of variation. Our method for total mercury quantification is not only simple but suitable for management of the mercury content at coal-fired electric power plants and landfill sites, which deal with large amounts of waste CFA.

Development and verification of a model for estimating the screening utility in the detection of PCBs in transformer oil.

A simple new model for estimating the screening performance (false positive and false negative rates) of a given test for a specific sample population is presented. The model is shown to give good results on a test population, and is used to estimate the performance on a sampled population. Using the model developed in conjunction with regulatory requirements and the relative costs of the confirmatory and screening tests allows evaluation of the screening test's utility in terms of cost savings. Testers can use the methods developed to estimate the utility of a screening program using available screening tests with their own sample populations.

Label-free impedimetric immunoassay for trace levels of polychlorinated biphenyls in insulating oil.

A rapid, ultrasensitive, and practical label-free impedimetric immunoassay for measuring trace levels of total polychlorinated biphenyls (PCBs) in insulating oil was developed. First, we developed a novel monoclonal antibody (RU6F9) for PCBs by using a designed immunogen and characterized its binding affinity for a commercial mixtures of PCBs and its main congeners. A micro comblike gold electrode was fabricated, and the antibody was covalently immobilized on the electrode through a self-assembled monolayer formed by dithiobis-N-succinimidyl propionate. The antigen-binding event on the surface of the functionalized electrode was determined as the change in charge transfer resistance by using electrochemical impedance spectroscopy. The resulting impedimetric immunoassay in aqueous solution achieved a wide determination range (0.01-10 µg/L) and a low detection limit (LOD) of 0.001 µg/L, which was 100-fold more sensitive than a conventional flow-based immunoassay for PCBs. By combining the impedimetric immunoassay with a cleanup procedure for insulating oil utilizing a multilayer cleanup column followed by DMSO partitioning, an LOD of 0.052 mg/kg-oil was achieved, which satisfied the Japanese regulation criterion of 0.5 mg/kg-oil. Finally, the immunoassay was employed to determine total PCB levels in actual used insulating oils (n = 33) sampled from a used transformer containing trace levels of PCBs, and the results agreed well with the Japanese official method (HRGC/HRMS).

New antibody and immunoassay pretreatment strategy to screen polychlorinated biphenyls in Korean transformer oil.

Development and modifications are described that expand the application of an immunoassay from the detection of Kanechlors (Japanese technical PCBs mixtures) to the detection of Aroclors (U. S. technical PCB mixtures, used in Korea) in contaminated Korean transformer oil. The first necessary modification was the development of a new antibody with a reactivity profile favorable for Aroclors. The second modification was the addition of a second column to the solid-phase extraction method to reduce assay interference caused by the Korean oil matrix. The matrix interference is suspected to be caused by the presence of synthetic oils (or similar materials) present as contaminants. The modified assay was validated by comparison to high-resolution gas chromatography/high-resolution mass spectrometry analysis, and was shown to be tolerant of up to 10% of several common synthetic insulating oils. Finally the screening performance of the modified assay was evaluated using 500 used transformer oil samples of Korean origin, and was shown to have good performance in terms of false positive and false negative rates. This report provides evidence for the first establishment of immunoassay screening for Aroclor based PCB contamination in Korean transformer oil.

Increased growth of a hydrogenotrophic methanogen in co-culture with a cellulolytic bacterium under cathodic electrochemical regulation.

Bioelectrochemical (-0.8 V, -0.3 V, and +0.6 V vs. Ag/AgCl) and non-bioelectrochemical co-cultures of a hydrogenotrophic methanogen and a cellulolytic bacterium were conducted. Unlike non-bioelectrochemical co-cultures, a cathodic reaction (-0.8 V) increased the growth of the hydrogenotrophic methanogen and the cellulolytic bacterium, by 6.0- and 2.2-fold respectively, and increased cellulose degradation. In contrast, anodic reactions (-0.3 V, +0.6 V) influenced them negatively.

Pretreatment method for immunoassay of polychlorinated biphenyls in transformer oil using multilayer capillary column and microfluidic liquid-liquid partitioning.

Polychlorinated biphenyls (PCBs) are persistent organic pollutants that are present in the insulating oil inside a large number of transformers. To aid in eliminating PCB-contaminated transformers, PCBs in oil need to be measured using a rapid and cost-effective analytical method. We previously reported a pretreatment method for the immunoassay of PCBs in oil using a large-scale multilayer column and a microchip with multiple microrecesses, which permitted concentrated solvent extraction. In this paper, we report on a more rapid and facile pretreatment method, without an evaporation process, by improving the column and the microchip. In a miniaturized column, the decomposition and separation of oil were completed in 2 min. PCBs can be eluted from the capillary column at concentrations seven-times higher than those from the previous column. The total volume of the microrecesses was increased by improving the microrecess structure, the enabling extraction of four-times the amount of PCBs achieved with the previous system. By interfacing the capillary column with the improved microchip, PCBs in the eluate from the column were extracted into dimethyl sulfoxide in microrecesses with high enrichment and without the need for evaporation. Pretreatment was completed within 20 min. The pretreated oil was analyzed using a flow-based kinetic exclusion immunoassay. The limit of detection of PCBs in oil was 0.15 mg kg(-1), which satisfies the criterion set in Japan of 0.5 mg kg(-1).

Efficient production of methane from artificial garbage waste by a cylindrical bioelectrochemical reactor containing carbon fiber textiles.

A cylindrical bioelectrochemical reactor (BER) containing carbon fiber textiles (CFT; BER + CFT) has characteristics of bioelectrochemical and packed-bed systems. In this study, utility of a cylindrical BER + CFT for degradation of a garbage slurry and recovery of biogas was investigated by applying 10% dog food slurry. The working electrode potential was electrochemically regulated at -0.8 V (vs. Ag/AgCl). Stable methane production of 9.37 L-CH4 · L-1 · day-1 and dichromate chemical oxygen demand (CODcr) removal of 62.5% were observed, even at a high organic loading rate (OLR) of 89.3 g-CODcr · L-1 · day-1. Given energy as methane (372.6 kJ · L-1 · day-1) was much higher than input electric energy to the working electrode (0.6 kJ · L-1 · day-1) at this OLR. Methanogens were highly retained in CFT by direct attachment to the cathodic working electrodes (52.3%; ratio of methanogens to prokaryotes), compared with the suspended fraction (31.2%), probably contributing to the acceleration of organic material degradation and removal of organic acids. These results provide insight into the application of cylindrical BER + CFT in efficient methane production from garbage waste including a high percentage of solid fraction.

Operation of a cylindrical bioelectrochemical reactor containing carbon fiber fabric for efficient methane fermentation from thickened sewage sludge.

A bioelectrochemical reactor (BER) containing carbon fiber fabric (CFF) (BER+CFF) enabled efficient methane fermentation from thickened sewage sludge. A cylindrical BER+CFF was proposed and scaled-up to a volume of 4.0-L. Thickened sewage sludge was treated using three types of methanogenic reactors. The working electrode potential in the BER+CFF was regulated at -0.8 V (vs. Ag/AgCl). BER+CFF showed gas production of 3.57 L L(-1) day(-1) at a hydraulic retention time (HRT) of 4.0 days; however, non-BER+CFF showed a lower gas production rate (0.83 L L(-1) day(-1)) at this HRT, suggesting positive effects of electrochemical regulation. A stirred tank reactor (without CFF) deteriorated at an HRT of 10 days, suggesting positive effects of CFF. 16S rRNA gene analysis showed that the BER+CFF included 3 kinds of hydrogenotrophic methanogens and 1 aceticlastic methanogen. These results demonstrate the effectiveness of the BER+CFF for scale-up and flexibility of this technology.

The membraneless bioelectrochemical reactor stimulates hydrogen fermentation by inhibiting methanogenic archaea.

The membraneless bioelectrochemical reactor (Ml-BER) is useful for dark hydrogen fermentation. The effect of the electrochemical reaction on microorganisms in the Ml-BER was investigated using glucose as the substrate and compared with organisms in a membraneless non-bioelectrochemical reactor (Ml-NBER) and bioelectrochemical reactor (BER) with a proton exchange membrane. The potentials on the working electrode of the Ml-BER and BER with membrane were regulated to -0.9 V (versus Ag/AgCl) to avoid water electrolysis with a carbon electrode. The Ml-BER showed suppressed methane production (19.8 ± 9.1 mg-C·L(-1)·day(-1)) and increased hydrogen production (12.6 ± 3.1 mg-H·L(-1)·day(-1)) at pHout 6.2 ± 0.1, and the major intermediate was butyrate (24.9 ± 2.4 mM), suggesting efficient hydrogen fermentation. In contrast, the Ml-NBER showed high methane production (239.3 ± 17.9 mg-C·L(-1)·day(-1)) and low hydrogen production (0.2 ± 0.0 mg-H·L(-1)·day(-1)) at pHout 6.3 ± 0.1. In the cathodic chamber of the BER with membrane, methane production was high (276.3 ± 20.4 mg-C·L(-1)·day(-1)) (pHout, 7.2 ± 0.1). In the anodic chamber of the BER with membrane (anode-BER), gas production was low because of high lactate production (43.6 ± 1.7 mM) at pHout 5.0 ± 0.1. Methanogenic archaea were not detected in the Ml-BER and anode-BER. However, Methanosarcina sp. and Methanobacterium sp. were found in Ml-NBER. Prokaryotic copy numbers in the Ml-BER and Ml-NBER were similar, as were the bacterial community structures. Thus, the electrochemical reaction in the Ml-BER affected hydrogenotrophic and acetoclastic methanogens, but not the bacterial community.

Trace-level mercury ion (Hg2+) analysis in aqueous sample based on solid-phase extraction followed by microfluidic immunoassay.

Mercury is considered the most important heavy-metal pollutant, because of the likelihood of bioaccumulation and toxicity. Monitoring widespread ionic mercury (Hg(2+)) contamination requires high-throughput and cost-effective methods to screen large numbers of environmental samples. In this study, we developed a simple and sensitive analysis for Hg(2+) in environmental aqueous samples by combining a microfluidic immunoassay and solid-phase extraction (SPE). Using a microfluidic platform, an ultrasensitive Hg(2+) immunoassay, which yields results within only 10 min and with a lower detection limit (LOD) of 0.13 µg/L, was developed. To allow application of the developed immunoassay to actual environmental aqueous samples, we developed an ion-exchange resin (IER)-based SPE for selective Hg(2+) extraction from an ion mixture. When using optimized SPE conditions, followed by the microfluidic immunoassay, the LOD of the assay was 0.83 µg/L, which satisfied the guideline values for drinking water suggested by the United States Environmental Protection Agency (USEPA) (2 µg/L; total mercury), and the World Health Organisation (WHO) (6 µg/L; inorganic mercury). Actual water samples, including tap water, mineral water, and river water, which had been spiked with trace levels of Hg(2+), were well-analyzed by SPE, followed by microfluidic Hg(2+) immunoassay, and the results agreed with those obtained from reduction vaporizing-atomic adsorption spectroscopy.

Optimization of a commercial biosensor for polychlorinated biphenyls and evaluation of its utility for screening.

We previously described our systematic progress that eventually resulted in a commercially available immunoassay based biosensor (PCB biosensor) for detecting PCBs in oil. However, IC50 of the commercialized PCB biosensor was approximately 2 ppb for PCBs, and did not achieve the theoretical detection limit (TDL) which would represent an IC50 of approximately 0.5 ppb. In this study, we characterize the effects of the antibody concentration, flow volume and flow rate on the PCB biosensor's response. Using the optimum operating conditions, the PCB biosensor achieved the TDL and its performance as a screening test was improved. Working at the stringent maximum residue limit specified by Japanese law (0.5 ppm total PCBs), the optimized biosensor exhibited excellent performance (0% false negatives and 7% false positives) in the screening of 110 samples of used Japanese transformer oil. The general approach for optimization described here is expected to benefit immunoassay researchers attempting to achieve optimum performance.

Acceleration of cellulose degradation and shift of product via methanogenic co-culture of a cellulolytic bacterium with a hydrogenotrophic methanogen.

Although the effects of syntrophic relationships between bacteria and methanogens have been reported in some environments, those on cellulose decomposition using cellulolytic bacteria from methanogenic reactors have not yet been examined. The effects of syntrophic co-culture on the decomposition of a cellulosic material were investigated in a co-culture of Clostridium clariflavum strain CL-1 and the hydrogenotrophic methanogen Methanothermobacter thermautotrophicus strain ΔH and a single-culture of strain CL-1 under thermophilic conditions. In this study, strain CL-1 was newly isolated as a cellulolytic bacterium from a thermophilic methanogenic reactor used for degrading garbage slurry. The degradation efficiency and cell density of strain CL-1 were 2.9- and 2.7-fold higher in the co-culture than in the single-culture after 60 h of incubation, respectively. Acetate, lactate and ethanol were the primary products in both cultures, and the concentration of propionate was low. The content of acetate to total organic acids plus ethanol was 59.3% in the co-culture. However, the ratio decreased to 24.9% in the single-culture, although acetate was the primary product. Therefore, hydrogen scavenging by the hydrogenotrophic methanogen strain ΔH could shift the metabolic pathway to the acetate production pathway in the co-culture. Increases in the cell density and the consequent acceleration of cellulose degradation in the co-culture would be caused by increases in adenosine 5'-triphosphate (ATP) levels, as the acetate production pathway includes ATP generation. Syntrophic cellulose decomposition by the cellulolytic bacteria and hydrogenotrophic methanogens would be the dominant reaction in the thermophilic methanogenic reactor degrading cellulosic materials.

Construction of hydrogen fermentation from garbage slurry using the membrane free bioelectrochemical system.

The aim of this study was to show the effectiveness of the membrane free bioelectrochemical system (BES) using three electrodes on inhibition of methanogenesis and construction of hydrogen fermentation from the artificial garbage slurry. The electrical redox-potential on the working electrode was adjusted to -1.0V (vs. Ag/AgCl) that has positive effect on methanogenesis. The redox-potential on the counter electrode was measured to be 1.6V. The pH in the effluents was 5.5-6.4. Hydrogen production rate at the cathode side was similar to that at the anode side and much higher than that calculated from current, and reached a maximum of 2445±815 (average±standard deviation) mL L(-1) d(-1) at an organic loading rate of 58.7g dichromate chemical oxygen demand per L d(-1). Methane production was negligible throughout the experiment. Acetate and butyrate were the main products of the fermentation using a BES; these offered favorable conditions for hydrogen production. The bacterial community in the bioelectrochemical hydrogen fermentor differed from that in the methanogenic seed sludge and included hitherto unknown species. These results show that high redox-potential on the anodic electrode and acidic pH in the membrane free BES can be utilized for hydrogen fermentation from the artificial garbage slurry by avoiding methanogenesis.

Bioelectrochemical regulation accelerates facultatively syntrophic proteolysis.

Bioelectrochemical systems can affect microbial metabolism by controlling the redox potential. We constructed bioelectrochemical cultures of the proteolytic bacterium, Coprothermobacter proteolyticus strain CT-1, both as a single-culture and as a co-culture with the hydrogenotrophic methanogen, Methanothermobacter thermautotrophicus strain ∆H, to investigate the influences of bioelectrochemical regulation on facultatively syntrophic proteolysis. The co-culture and single-culture were cultivated at 55°C with an anaerobic medium containing casein as the carbon source. The working electrode potential of the bioelectrochemical system was controlled at -0.8V (vs. Ag/AgCl) for bioelectrochemical cultures and was not controlled for non-bioelectrochemical cultures. The cell densities of hydrogenotrophic methanogen and methane production in the bioelectrochemical co-culture were 3.6 and 1.5 times higher than those in the non-bioelectrochemical co-culture after 7 days of cultivation, respectively. Contrastingly, the cell density of Coprothermobacter sp. in the bioelectrochemical co-culture was only 1.3 times higher than that in the non-bioelectrochemical co-culture. The protein decomposition rates were nearly proportional to the cell density of Coprothermobacter sp. in the all types of cultures. These results indicate that bioelectrochemical regulation, particularly, affected the carbon fixation of the hydrogenotrophic methanogen and that facultatively syntrophic proteolysis was accelerated as a result of hydrogen consumption by the methanogens growing well in bioelectrochemical co-cultures.

Microfluidic heavy metal immunoassay based on absorbance measurement.

A simple and rapid flow-based multioperation immunoassay for heavy metals using a microfluidic device was developed. The antigen-immobilized microparticles in a sub-channel were introduced as the solid phase into a main channel structures through a channel flow mechanism and packed into a detection area enclosed by dam-like structures in the microfluidic device. A mixture of a heavy metal and a gold nanoparticle-labeled antibody was made to flow toward the corresponding metal through the main channel and make brief contact with the solid phase. A small portion of the free antibody was captured and accumulated on the packed solid phase. The measured absorbance of the gold label was proportional to the free antibody portion and, thus, to the metal concentration. Each of the monoclonal antibodies specific for cadmium-EDTA, chromium-EDTA, or lead-DTPA was applied to the single-channel microfluidic device. Under optimized conditions of flow rate, volume, and antibody concentration, the theoretical (antibody K(d)-limited) detection levels of the three heavy metal species were achieved within only 7 min. The dynamic range for cadmium, chromium, and lead was 0.57-60.06 ppb, 0.03-0.97 ppb, and 0.04-5.28 ppb, respectively. An integrated microchannel device for simultaneous multiflow was also successfully developed and evaluated. The multiplex cadmium immunoassay of four samples was completed within 8 min for a dynamic range of 0.42-37.48 ppb. Present microfluidic heavy metal immunoassays satisfied the Japanese environmental standard for cadmium, chromium and, lead, which provided in the soil contamination countermeasures act.

Analysis of polychlorinated biphenyls in transformer oil by using liquid-liquid partitioning in a microfluidic device.

Polychlorinated biphenyls (PCBs) that are present in transformer oil are a common global problem because of their toxicity and environmental persistence. The development of a rapid, low-cost method for measurement of PCBs in oil has been a matter of priority because of the large number of PCB-contaminated transformers still in service. Although one of the rapid, low-cost methods involves an immunoassay, which uses multilayer column separation, hexane evaporation, dimethyl sulfoxide (DMSO) partitioning, antigen-antibody reaction, and a measurement system, there is a demand for more cost-effective and simpler procedures. In this paper, we report a DMSO partitioning method that utilizes a microfluidic device with microrecesses along the microchannel. In this method, PCBs are extracted and enriched into the DMSO confined in the microrecesses under the oil flow condition. The enrichment factor was estimated to be 2.69, which agreed well with the anticipated value. The half-maximal inhibitory concentration of PCBs in oil was found to be 0.38 mg/kg, which satisfies the much stricter criterion of 0.5 mg/kg in Japan. The developed method can realize the pretreatment of oil without the use of centrifugation for phase separation. Furthermore, the amount of expensive reagents required can be reduced considerably. Therefore, our method can serve as a powerful tool for achieving a simpler, low-cost procedure and an on-site analysis system.

Syntrophic degradation of proteinaceous materials by the thermophilic strains Coprothermobacter proteolyticus and Methanothermobacter thermautotrophicus.

Protein is a major component of organic solid wastes, and therefore, it is necessary to further elucidate thermophilic protein degradation process. The effects of hydrogenotrophic methanogens on protein degradation were investigated using the proteolytic bacterial strain CT-1 that was isolated from a methanogenic thermophilic (55°C) packed-bed reactor degrading artificial garbage slurry. Strain CT-1 was closely related to Coprothermobacter proteolyticus, which is frequently found in methanogenic reactors degrading organic solid wastes. Strain CT-1 was cultivated in the absence or presence of Methanothermobacter thermautotrophicus by using 3 kinds of proteinaceous substrates. Degradation rates of casein, gelatin, and bovine serum albumin were higher in co-cultures than in monocultures. Strain CT-1 showed faster growth in co-cultures than in monocultures. M. thermautotrophicus comprised 5.5-6.0% of the total cells in co-culture. Increased production of ammonia and acetate was observed in co-cultures than in monocultures, suggesting that addition of M. thermautotrophicus increases the products of protein degradation. Hydrogen produced in the monocultures was converted to methane in co-cultures. These results suggest that thermophilic proteolytic bacteria find it favorable to syntrophically degrade protein in a methanogenic environment, and that it is important to retain hydrogen-scavenging methanogens within the reactor.

A bioelectrochemical reactor containing carbon fiber textiles enables efficient methane fermentation from garbage slurry.

A packed-bed system includes supporting materials to retain microorganisms and a bioelectrochemical system influences the microbial metabolism. In our study, carbon fiber textiles (CFT) as a supporting material was attached onto a carbon working electrode in a bioelectrochemical reactor (BER) that degrades garbage slurry to methane, in order to investigate the effect of combining electrochemical regulation and packing CFT. The potential on the working electrode in the BER containing CFT was set to -1.0 V or -0.8 V (vs. Ag/AgCl). BERs containing CFT exhibited higher methane production, elimination of dichromate chemical oxygen demand, and the ratio of methanogens in the suspended fraction than reactors containing CFT without electrochemical regulation at an organic loading rate (OLR) of 27.8 gCODcr/L/day. In addition, BERs containing CFT exhibited higher reactor performances than BERs without CFT at this OLR. Our results revealed that the new design that combined electrochemical regulation and packing CFT was effective.

Decreasing ammonia inhibition in thermophilic methanogenic bioreactors using carbon fiber textiles.

Ammonia accumulation is one of the main causes of the loss of methane production observed during fermentation. We investigated the effect of addition of carbon fiber textiles (CFT) to thermophilic methanogenic bioreactors with respect to ammonia tolerance during the process of degradation of artificial garbage slurry, by comparing the performance of the reactors containing CFT with the performance of reactors without CFT. Under total ammonia-N concentrations of 3,000 mg L(-1), the reactors containing CFT were found to mediate stable removal of organic compounds and methane production. Under these conditions, high levels of methanogenic archaea were retained at the CFT, as determined by 16S rRNA gene analysis for methanogenic archaea. In addition, Methanobacterium sp. was found to be dominant in the suspended fraction, and Methanosarcina sp. was dominant in the retained fraction of the reactors with CFT. However, the reactors without CFT had lower rates of removal of organic compounds and production of methane under total ammonia-N concentrations of 1,500 mg L(-1). Under this ammonia concentration, a significant accumulation of acetate was observed in the reactors without CFT (130.0 mM), relative to the reactors with CFT (4.2 mM). Only Methanobacterium sp. was identified in the reactors without CFT. These results suggest that CFT enables stable proliferation of aceticlastic methanogens by preventing ammonia inhibition. This improves the process of stable garbage degradation and production of methane in thermophilic bioreactors that include high levels of ammonia.