Development of an Improved Sulfur-Oxidizing Bacteria-Based Ecotoxicity Test for Simple and Rapid On-Site Application.

Microbial toxicity tests are considered efficient screening tools for the assessment of water contamination. The objective of this study was to develop a sulfur-oxidizing bacteria (SOB)-based ecotoxicity test with high sensitivity and reproducibility for simple and rapid on-site application. To attain this goal, we developed a 25 mL vial-based toxicity kit and improved our earlier SOB toxicity test technique. The current study applied a suspended form of SOB and shortened the processing time to 30 min. Moreover, we optimized the test conditions of the SOB toxicity kit in terms of initial cell density, incubating temperature, and mixing intensity during incubation. We determined that 2 × 105 cells/mL initial cell density, 32 °C incubating temperature, and 120 rpm mixing intensity are the optimal test conditions. Using these test conditions, we performed SOB toxicity tests for heavy metals and petrochemicals, and obtained better detection sensitivity and test reproducibility, compared to earlier SOB tests. Our SOB toxicity kit tests have numerous advantages, including a straightforward test protocol, no requirement of sophisticated laboratory equipment, and no distortion of test results from false readings of end-points and properties of test samples, making it suitable for simple and rapid on-site application.

Evaluation of phytoplankton-stimulating potency of effluent nitrogen depending on its chemical forms: A comparison between inorganic and organic nitrogen.

Wastewater-originated nitrogen (N) is considered a primary N source in urban waters. In order to mitigate eutrophication in such waters, decreasing N discharges from wastewater treatment plants (WWTPs) is necessary. Upgrading WWTPs from conventional activated sludge (CAS) to biological nutrient removal (BNR) is the most common measure to lower levels of effluent N. However, in spite of successful N reduction through such upgrades, eutrophication persists in numerous urban waters. In this study, we investigated why decreased N discharge resulting from upgrading CAS to BNR, particularly predenitrification BNR, cannot necessarily alleviate eutrophication. Our laboratory reactor study demonstrated that compared to CAS effluent N, predenitrification BNR effluent N contains less dissolved inorganic N (DIN) but more dissolved organic N (DON), especially low molecular weight DON (LMW-DON). Bioassay-based experimental and numerical analyses found that effluent N has dissimilar phytoplankton-stimulating potency depending on its chemical forms. In particular, effluent LMW-DON showed significantly greater potency than effluent DIN. This difference in potency makes predenitrification BNR effluent N more productive for causing primary production than CAS effluent N. These results indicate that the impact of effluent N on eutrophication should be evaluated based on not only the total quantity but also the qualitative aspect of N.

Evaluation of joint toxicity of BTEX mixtures using sulfur-oxidizing bacteria.

Benzene (B), toluene (T), ethylbenzene (E), and xylenes (X) are petrochemicals vital in various industrial and commercial processing but identified as priority pollutants due to their high toxicity. The objective of this study was to investigate the toxicological nature of BTEX mixtures under controlled laboratory aquatic conditions using sulfur-oxidizing bacteria (SOB). Results from individual BTEX tests demonstrated that the order of toxicity among BTEX was X ≥ E > T > B. Comparisons of dose-effect curves for BTEX suggest that the biochemical mode of action of B in SOB was different from those of T, E, and X. Toxicological interactions of BTEX in mixtures were studied using concentration addition (CA), independent action (IA), and combination index (CI)-isobologram models. The CI model approximated the actual toxicity of BTEX mixtures better than the CA and IA models. In most cases, BTEX induced synergistic interactions in mixtures. However, in some B-containing mixtures, antagonism was observed at low effective levels. The effective level (fa)-CI plots and polygonograms illustrate that synergistic interactions of BTEX became stronger with an increase in effective levels. In addition, ternary and quaternary mixtures were found to provoke stronger synergism than binary mixtures. The present study suggests that the CI-isobologram model is a suitable means to evaluate diverse toxicological interactions of contaminants in mixtures.

Investigation of characteristics of effluent DON derived from conventional activated sludge (CAS) and predenitrification biological removal (BNR): In terms of proteins and humic substances.

Upgrading wastewater treatment plants (WWTPs) from conventional activated sludge (CAS) to predenitrification biological nutrient removal (BNR) results in improved removal of dissolved inorganic nitrogen (DIN) from wastewater. However, changes in dissolved organic nitrogen (DON) with these WWTP upgrades and their potential impacts on receiving waters have been little researched. In this study, we investigated characteristics of effluent DON derived from CAS and predenitrification BNR, paying special attention to proteins and humic substances. Through a lab-scale reactor study and analysis of full-scale WWTP effluents, we found that in predenitrification BNR effluent, proteins are much more dominant than humic substances, whereas in CAS effluent, proteins and humic substances are similarly abundant. In terms of molecular weight, the majority of proteins were present in the effluent's low molecular weight (LMW) fraction (<1 kDa), while humic substances were found mostly in the effluent's high molecular weight (HMW) fraction (0.45 µm-1 kDa). Determination of dissolved organic carbon (DOC)/DON ratios in effluents supports that proteins (and LMW-DON) were most likely microbial-derived organic N produced during treatment processing, whereas humic substances (and HMW-DON) more likely originated outside of treatment systems. Bioassay tests demonstrated that effluent DON derived from predenitrification BNR was more bioavailable than that derived from CAS. We also found that LMW-DON and proteins were highly bioavailable DON compared to HMW-DON and humic substances. The results of this study suggest that upgrading CAS to predenitrification BNR makes effluent DON to become more conducive to phytoplankton blooms in receiving waters.

A simple and rapid algal assay kit to assess toxicity of heavy metal-contaminated water.

This study presents a novel algal-based toxicity test suitable for simple and rapid assessment of heavy metal (Hg2+, Cr6+, Cd2+, Pb2+, or As3+)-contaminated water. A closed-system kit-type algal assay was developed using Chlorella vulgaris. Toxicity was assessed by oxygen evolution in the gaseous phase of the assay kits, which was measured via a needle-type oxygen sensor. Initial cell density, light intensity, and exposure time that enabled favorable test performance for the algal assay kits were 103 cells/mL, 250 µmol m-2s-1, and 18 h, respectively. Results from the heavy metal toxicity tests demonstrate that Hg2+, Cr6+, Cd2+, and Pb2+ are more toxic in inhibiting algal photosynthetic activity than As3+. The 18 h half-maximum effective concentrations (EC50) for Hg2+, Cr6+, Cd2+, Pb2+, and As3+ were determined to be 31.3 ± 0.5, 179.6 ± 7.5, 301.3 ± 6.1, 476.1 ± 10.5, and 2184.1 ± 31.1 µg/L, respectively. A strong correlation between oxygen concentrations in the headspace of the assay kits and chlorophyll a production indicates that oxygen evolution in the gaseous phase is able to represent algal photosynthetic activity and serve as the end-point in algal toxicity tests. High test sensitivity and reproducibility as well as an easy test protocol and rapid processing time make the algal assay kit a suitable tool for simple and rapid toxicity testing of heavy metal-contaminated water.

Real-time biomonitoring of oxygen uptake rate and biochemical oxygen demand using a novel optical biogas respirometric system.

In response to the ever-increasing need for monitoring-based process control of wastewater treatment plants, an online applicable respirometer shows great promise for real-time measurement of oxygen uptake rate (OUR) and biochemical oxygen demand (BOD) measurements as a surrogate of the biodegradability of wastewater. Here, we have developed a photosensor-assisted real-time respirometric system equipped with bubble counting sensors for accurate measurement of microbial oxygen consumption in a bottle. This system can measure OUR and BOD in a bottle equipped with a tube containing NaOH solution to absorb carbon dioxide and supplied with continuous atmospheric oxygen to the bottle, which reliably supplies non-limiting dissolved oxygen (DO) for aerobic biodegradation even at high organic loads. These technical improvements allow a sensitive and rapid analytical tool offering real-time profiles of oxygen uptake rate as well as BOD measurements with an extended measurable range (0-420 mg O2/L), enabling significant reduction or elimination of dilution steps. The respirometric system was used to elucidate the biodegradable kinetics of domestic and swine wastewaters as a function of the type and concentration of organic matters, depending on source characteristics including rapidly or slowly oxidizable organic substances by bacteria. Compared with conventional and manometric BOD methods, our method is reliable and accurate.

Improved toxicity analysis of heavy metal-contaminated water via a novel fermentative bacteria-based test kit.

The objective of this study was development of a simple and reliable microbial toxicity test based on fermentative bacteria to assess heavy metal (Hg2+, Cu2+, Cr6+, Ni2+, As5+, or Pb2+)-contaminated water. The dominant species of test organisms used in this study was a spore-forming fermentative bacterium, Clostridium guangxiense. Toxicity of water was assessed based on inhibition of fermentative gas production of the test organisms, which was analyzed via a syringe method. Overall, the fermentative bacteria-based test kits satisfactorily identified increased toxicity of water as water was contaminated with high amounts of heavy metals; however, levels of inhibition were dissimilar depending on the species of metals. Inhibitory effects of Hg2+, Cu2+, Cr6+, and Ni2+ were considerably greater than those of As5+ and Pb2+. The 24 h half-maximum effective concentrations (EC50) for Hg2+, Cu2+, Cr6+, Ni2+, As5+, and Pb2+ were analyzed to be 0.10, 0.51, 1.09, 3.61, 101.33, and 243.45 mg/L, respectively, confirming that Hg2+, Cu2+, Cr6+, and Ni2+ are more toxic to fermentative gas production than As5+ and Pb2+. The fermentative bacteria-based toxicity test represents an improvement over other existing toxicity tests because of ease of end-point measurement, high reproducibility, and favorable on-site field applicability. These advantages make the fermentative bacteria-based test suitable for simple and reliable toxicity screening for heavy metal-contaminated water.

A Microbial Bioassay for Direct Contact Assessment of Soil Toxicity Based on Oxygen Consumption of Sulfur Oxidizing Bacteria.

A new direct contact assessment of soil toxicity using sulfur oxidizing bacteria (SOB) is proposed for analyzing the toxicity of soils. The proposed method is based on the ability of SOB to oxidize elemental sulfur to sulfuric acid in the presence of oxygen. Since sulfate ions are produced from sulfur by SOB oxidation activity, changes in electrical conductivity (EC) serve as a proxy to assess toxicity in water. However, in soil medium, EC values are not reliable due to the adsorption of SO4 2- ions by soils. Here, we suggest a new parameter which measures oxygen consumption by SOB for 6 hours to assess soil toxicity by using a lubricated glass syringe method. The proposed method is rapid, simple, cost- effective as well as sensitive and capable of assessing direct contact soil toxicity.

Rapid detection of heavy metal-induced toxicity in water using a fed-batch sulfur-oxidizing bacteria (SOB) bioreactor.

A fed-batch bioreactor based on sulfur-oxidizing bacteria (SOB) was tested for rapid detection of heavy metal-induced toxicity in water. For this evaluation, SOB were exposed to water contaminated by selenium, mercury, hexavalent chromium, arsenic, cyanide, cadmium, and lead for 2 h and their inhibition rates were analyzed based on changes in electrical conductivity (EC). The results demonstrate that SOB were highly inhibited by selenium, mercury, hexavalent chromium, and arsenic but not by cyanide, cadmium, and lead. The 2 h half maximum effective concentrations (EC50) of SOB for selenium, mercury, hexavalent chromium, and arsenic were estimated to be 0.33, 0.89, 1.18, and 0.24 mg/L, respectively, which are comparable or lower than earlier reports in the literature. However, the EC50 or EC20 values of SOB for cyanide, cadmium, and lead were notably higher compared to findings from previous toxicity tests that employed other microorganisms. The findings from the current study suggest that the fed-batch SOB bioreactor is suitable for rapid detection of toxicity induced by selenium, mercury, hexavalent chromium, and arsenic in water.

Formation of Low-Molecular-Weight Dissolved Organic Nitrogen in Predenitrification Biological Nutrient Removal Systems and Its Impact on Eutrophication in Coastal Waters.

To alleviate eutrophication in coastal waters, reducing nitrogen (N) discharge from wastewater treatment plants (WWTPs) by upgrading conventional activated sludge (CAS) to biological nutrient removal (BNR) processes is commonplace. However, despite numerous upgrades and successful reduction of N discharge from WWTPs, eutrophication problems persist. These unexpected observations raise the possibility that some aspects of BNR yield environmental responses as yet overlooked. Here, we report that one of the most common BNR processes, predenitrification, is prone to the production of low-molecular-weight dissolved organic N (LMW-DON), which is highly bioavailable and stimulates phytoplankton blooms. We found that in predenitrification BNR, LMW-DON is released during the post-aerobic step following the preanoxic step, which does not occur in CAS. Consequently, predenitrification systems produced larger amount of LMW-DON than CAS. In estuarine bioassays, predenitrification BNR effluents produced more phytoplankton biomass than CAS effluents despite lower N concentrations. This was also supported by stronger correlations found between phytoplankton biomass and LMW-DON than other N forms. These findings suggest that WWTPs upgraded to predenitrification BNR reduce inorganic N discharge but introduce larger quantities of potent LMW-DON into coastal systems. We suggest reassessing the N-removal strategy for WWTPs to minimize the eutrophication effects of effluents.

Development of a hybrid microbial fuel cell (MFC) and fuel cell (FC) system for improved cathodic efficiency and sustainability: the M2FC reactor.

In an effort to improve the efficiency and sustainability of microbial fuel cell (MFC) technology, a novel MFC reactor, the M2FC, was constructed by combining a ferric-based MFC with a ferrous-based fuel cell (FC). In this M2FC reactor, ferric ion, the catholyte in the MFC component, is regenerated by the FC system with the generation of additional electricity. When the MFC component was operated separately, the electricity generation was maintained for only 98 h due to the depletion of ferric ion in the catholyte. In combination with the fuel cell, however, the production of power was sustained because ferric ion was continually replenished from ferrous ion in the FC component. Moreover, the regeneration process of ferric ion by the FC produced additional energy. The M2FC reactor yielded a power density of up to 2 W m(-2) (or time-averaged value of approximately 650 mW m(-2)), density up to 20 times (or approximately six times based on time-averaged value) higher than the corresponding MFC system.