Daming Yong

Cell-based biosensor for measurement of phenol and nitrophenols toxicity

A cost-effective whole cell biosensor based on electrochemical technique to detect toxicities of phenol and nitrophenols has been developed. This method relied on the inhibition effect for respiratory chain activity of microorganism by toxicant, which was measured by chronoamperometry using mediator (ferricyanide). The current signals produced by suspended microorganisms and reoxidation of ferrocyanide were transformed to inhibiting efficiency directly, and 50% inhibiting concentration (IC(50)) was chosen as the quantitative standard of toxicity. The test microorganisms used here consist of three bacilli (Escherichia coli, Enterobacter cloacae and Alcaligenes faecalis), two pseudomonas (Pseudomonas fluorescens and Pseucomonas putida) and one fungus (Trichosporon cutaneum). 3,5-Dichlorophenol (DCP) was taken as the reference toxicant. The results showed that the microorganisms which belong to the same bacterial family had similar trends of inhibitions on respiratory activity and similar IC50 values. By comparing the IC(50) values, P. fluorescens was the most sensitive one to DCP toxicity, its IC(50) was estimated to be 4.2mg/L. pH 7.0 and together with the standard glucose-glutamic acid (GGA) as an exogenous material were taken for optimum conditions in this study. Here, P. fluorescens as model test microorganism was employed to assess toxicities of phenol and nitrophenols under the optimum conditions. IC(50) values of 291.4 mg/L for phenol, 64.1mg/L for 2-NP, 71.4 mg/L for 3-NP and 14.0mg/L for 4-NP were determined at 60 min, respectively. Comparison with the results of published data has confirmed that this cell biosensor is a sensitive and rapid alternative to toxicity screening of chemicals.

A sensitive, rapid and inexpensive way to assay pesticide toxicity based on electrochemical biosensor

We reported a rapid toxicity assay method using electrochemical biosensor for pesticides, Escherichia coli (E. coli) was taken as a model microorganism for test. In this method, we adopted ferricyanide instead of natural electron acceptor O(2), and then microbial oxidation was substantially accelerated. Toxicity assays measured the effect of toxic materials on the metabolic activity of microorganisms. The current signal of ferrocyanide produced from the metabolism was proven to be directly related to the toxicity, which could be amplified by ultramicroelectrode array (UMEA). The ratio of the electrochemical signals, recorded in the presence and absence of toxin, provided an index of inhibition. Accordingly, a direct toxicity assessment (DTA) based on chronoamperometry was proposed to detect the effect of toxic chemicals on microorganisms. 3,5-Dichlorophenol (DCP) was taken as the reference toxicant, its IC50 was estimated to be 8.0mg/L. Three pesticides were examined using this method. IC50 values of 6.5mg/L for Ametryn, 22 mg/L for Fenamiphos and 5.7 mg/L for Endosulfan were determined and in line with EC50 values reported in the literature. Atomic force microscopy (AFM) was also used for morphology characterization of E. coli induced by three pesticides. These results confirmed the present electrochemical method used is reliable. In addition, the electrochemical method is a sensitive, rapid and inexpensive way for toxicity assays of pesticides.

Development of a simple method for biotoxicity measurement using ultramicroelectrode array under non-deaerated condition.

In this paper, a mediated method by using ferricyanide under non-deaerated condition for biotoxicity measurement was proposed. Ultramicroelectrode array (UMEA) was employed for effectively amplify the electrochemical signal from the total limiting currents to distinguish a little change in toxicity. Five species of microorganisms including two bacilli (Escherichia coli and Enterobacter cloacae), two pseudomonas (Pseudomonas fluorescens and Pseucomonas putida) and one fungus (Trichosporon cutaneum) were employed. 3,5-dichlorophenol (DCP) was taken as the reference toxicant. The IC50 values we obtained were similar with the values obtained using in the deaerated method. E. coli was used as model test microorganism. The final concentration of ferricyanide is 45 mM, E. coli OD600 8 and 1 h incubation were taken in optimum conditions in this study. Four heavy metal ions (Cr(6+), Hg(2+), Cd(2+) and Bi(3+)) were examined under the optimum conditions. Comparison with the results reported previously has confirmed that this method provided a simple and rapid alternative to toxicity screening of chemicals, especially advantageous for in situ monitoring of water system.

Novel Environmental Analytical System based on Combined Biodegradation and Photoelectrocatalytic Detection Principles for Rapid Determination of Organic Pollutants in Wastewaters

This work describes the development of a novel biofilm reactor-photoelectrocatalytic chemical oxygen demand (BFR-PeCOD) analytical system for rapid online determination of biodegradable organic matters (BOMs). A novel air bubble sample delivery approach was developed to dramatically enhance the BFRs biodegradation efficiency and extend analytical linear range. Because the air bubble sample delivery invalidates the BOD quantification via the determination of oxygen consumption using dissolved oxygen probe, the PeCOD technique was innovatively utilized to resolve the BOD quantification issue under air bubble sample delivery conditions. The BFR was employed to effectively and efficiently biodegrade organic pollutants under oxygen-rich environment provided by the air bubbles. The BOD quantification was achieved by measuring the COD change (Δ[COD]) of the original sample and the effluent from BFR using PeCOD technique. The measured Δ[COD] was found to be directly proportional to the BOD5 values of the original sample with a slope independent of types and concentrations of organics. The slope was used to convert Δ[COD] to BOD5. The demonstrated analytical performance by BFR-PeCOD system surpasses all reported systems in many aspects. It has demonstrated ability to near real-time, online determining the organic pollution levels of wide range wastewaters without the need for dilution and ongoing calibration. The system possesses the widest analytical liner range (up to 800 mg O2 L(-1)) for BOD analysis, superior long-term stability, high accuracy, reliability, and simplicity. It is an environmentally friendly analytical system that consumes little reagent and requires minimal operational maintenance.

Native biofilm cultured under controllable condition and used in mediated method for BOD measurement

In this article, we developed a native biofilm (NBF) bioreactor used for biochemical oxygen demand mediated method (BOD(Med)). There were two innovations differed from previous BOD(Med) assay. Firstly, the immobilization of microorganisms was adopted in BOD(Med). Secondly, the NBF was introduced for BOD measurement. The NBF bioreactor has been characterized by optical microscopy. A culture condition of NBF with 24h, 35°C and pH 7 was optimized. Furthermore, a measuring condition with 35°C, pH 7 and 55 mM ferricyanide in 1h incubation were optimized. Based on the optimized condition, the real wastewater samples from local sewage treatment plant had been measured. Performances of the NBFs proposed at different culture conditions were recorded for 110 d, and the results indicated that long-term storage stability was obtained. With the proposed method, an uncontaminated native microbial source solution can be obtained from a wastewater treatment plant. In this way, we can ensure that the microbial species of all in the NBF are same as that in the target to be measured.

A reagent-free tubular biofilm reactor for on-line determination of biochemical oxygen demand

We reported a reagent-free tubular biofilm reactor (BFR) based analytical system for rapid online biochemical oxygen demand (BOD) determination. The BFR was cultivated using microbial seeds from activated sludge. It only needs tap water to operate and does not require any chemical reagent. The analytical performance of this reagent-free BFR system was found to be equal to or better than the BFR system operated using phosphate buffer saline (PBS) and high purity deionized water. The system can readily achieve a limit of detection of 0.25 mg O2 L(-1), possessing superior reproducibility, and long-term operational and storage stability. More importantly, we confirmed for the first time that the BFR system is capable of tolerating common toxicants found in wastewaters, such as 3,5-dichlorophenol and Zn(II), Cr(VI), Cd(II), Cu(II), Pb(II), Mn(II) and Ni(II), enabling the method to be applied to a wide range of wastewaters. The sloughing and clogging are the important attributes affecting the operational stability, hence, the reliability of most online wastewater monitoring systems, which can be effectively avoided, benefiting from the tubular geometry of the reactor and high flow rate conditions. These advantages, coupled with simplicity in device, convenience in operation and minimal maintenance, make such a reagent-free BFR analytical system promising for practical BOD online determination.

Toxicity detection of sodium nitrite, borax and aluminum potassium sulfate using electrochemical method

Based on the inhibition effect on the respiratory chain activity of microorganisms by toxicants, an electrochemical method has been developed to measure the current variation of a mediator in the presence of microorganisms contacted with a toxicant. Microelectrode arrays were adopted in this study, which can accelerate the mass transfer rate of an analyte to the electrode and also increase the total current signal, resulting in an improvement in detection sensitivity. We selected Escherichia coli as the testee and the standard glucose-glutamic acid as an exogenous material. Under oxygen restriction, the experiments in the presence of toxicant were performed at optimum conditions (solution pH 7.0, 37 degrees C and reaction for 3 hr). The resulting solution was then separated from the suspended microorganisms and was measured by an electrochemical method, using ferricyanide as a mediator. The current signal obtained represents the reoxidation of ferrocyanide, which was transformed to inhibiting efficiency, IC50, as a quantitative measure of toxicity. The IC50 values measured were 410, 570 and 830 mg/L for sodium nitrite, borax and aluminum potassium sulfate, respectively. The results show that the toxicity sequence for these three food additives is consistent with the value reported by other methods. Furthermore, the order of damage degree to the microorganism was also observed to be: sodium nitrite > borax > aluminum potassium sulfate > blank, according to the atomic force microscopy images of E. coli after being incubated for 3 hr with the toxic compound in buffer solutions. The electrochemical method is expected to be a sensitive and simple alternative to toxicity screening for chemical food additives.

Neutral red based colorimetric microorganism bioassay for direct toxicity assessment of toxic chemicals in water

In this work, we develop a reconfigured colorimetric bioassay for direct toxicity assessment (DTA) of toxic chemicals in water based on neutral red (NR) as a colorimetric indicator. Confocal laser scanning microscopy and UV-vis absorption spectroscopy are employed to prove the feasibility of the proposed assays using P. fluorescens as the model microorganism. The presence of toxicants can damage cells, and this results in an enhanced adsorption of NR by the cells. This fact provides a new strategy for DTA of toxic chemicals by comparing the absorption peak intensities of residual NR after incubation in the presence of toxicants. Four important toxicants such as 3,5-dichlorophenol (DCP), As3+, Hg2+ and Cr6+ are tested, and the toxicity order is DCP > Hg2+ > Cr6+ > As3+, which is identical with that obtained by the ferricyanide-mediated toxicity alerter (FM-tox) assay. The RC50 value of DCP is estimated to be 12.5 mg L−1, which is comparable with that of other DTA assays. The responses of E. coli and BOD seed (multi-species cultures) to DCP are further examined successfully, suggesting this NR based colorimetric assay may be a general one to measure the response of microorganisms to toxic chemicals. The low cost of NR, easy and simple operation procedure and reliable results make this assay a promising one in DTA.

Detecting total toxicity in water using a mediated biosensor system with flow injection

A novel total toxicity detection method based on a mediated biosensor system with flow injection (MB-FI) was developed to rapidly and reliably detect respiration inhibitors (i.e., As2O3, KCN, salicylic acid (SA), 2,4-dintirophenol (DNP)) in water. The mediated biosensor toxicity assessment using microorganisms immobilized in calcium alginate filaments can greatly simplify the testing process and save time. In the MB-FI system, ferricyanide together with a respiration inhibitor was injected into the bioreactor, inhibiting the respiration of the immobilized microorganisms. The degree of inhibition was measured by determining the ferrocyanide generated in the effluent, expressed as the 50% inhibition concentration (IC50). The IC50 values for the four respiration inhibitors obtained using this method were comparable to those obtained using the classic method, confirming that this approach is an alternative alert method. More importantly, this constructed biosensor system with flow injection will facilitate the application and commercialization of this toxicity monitoring technology.