Bioaugmentation for low C/N ratio wastewater treatment by combining endogenous partial denitrification (EPD) and denitrifying phosphorous removal (DPR) in the continuous A2/O - MBBR system.

Endogenous partial denitrification (EPD) and denitrifying phosphorous removal (DPR) were combined in a novel A2/O - MBBR (Anaerobic Anoxic Oxic - Moving Bed Biofilm Reactor) system for low carbon/nitrogen (C/N) ratio wastewater treatment. The DPR performance was compared and the nutrient metabolism was elucidated based on the optimization of hydraulic retention time (HRT, 4-12 h) and nitrate recycling (R, 200%-600%). In the continuous-flow, the nitrate (NO3-) denitrification accompanied by nitrite (NO2-, via EPD) accumulation with the nitrate-to-nitrite transformation ratio (NTR) of 35.87%-43.31% in the anoxic zones. At HRT of 12 h with R of 500%, batch test initially revealed the DPR mechanism using both NO3- and NO2- as electron acceptor, where denitrifying phosphorus accumulation organisms (DPAOs) and denitrifying glycogen accumulation organisms (DGAOs) were the main contributors for EPD with incomplete denitrification (NO3- → NO2-). Furthermore, stoichiometry-based functional bacteria analysis displayed that higher bioactivity of DPAOs (NO2-→N2, 57.30%; NO3-→N2, 35.85%) over DGAOs (NO3-→N2, 6.85%) facilitated the anoxic NO3- reduction. Microbial community analysis suggested that Cluster I of Defluviicoccus-GAO group (∼4%) was responsible for stable NO2- accumulation performance via EPD, while increased Accumulibacter-PAO group (by ∼15%) contributed to the advanced nutrient removal. Based on the achievement of NO2- accumulation, the application feasibility of integrated EPD - DPR - Anammox for deep-level nutrient removal was discussed.

A universal approach for the reversible phase transfer of hydrophilic nanoparticles.

The ability to engineer the surface properties of magnetic nanoparticles is important for their various applications, as numerous physical and chemical properties of nanoscale materials are seriously affected by the chemical constitution of their surfaces. For some specific applications, nanoparticles need to be transferred from a polar to a nonpolar environment (or vice versa) after synthesis. In this work we have developed a universal method for the phase transfer of magnetic nanoparticles that preserves their shape and size. Octadecyltrimethoxysilane was used to cap the surfaces of the aqueous magnetic nanoparticles, thereby allowing their transfer into nonpolar solution. The resulting hydrophobic magnetic nanoparticles were transferred back into aqueous solution by subsequently covering them with an egg-PC lipid monolayer. The superparamagnetic properties of the particles were retained after the phase transfer. The maximum transfer yields are dependent on their particle size with a maximum value of 93.16 ± 4.75% for magnetic nanoparticles with a diameter of 100 nm. The lipid-modified magnetic particles were stable over 1 week, and thus they have potential applications in the field of biomedicine. This work also provides a facile strategy for the controllable engineering of the surface properties of nanoparticles.

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 BFR's 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.

A rapid and sensitive p-benzoquinone-mediated bioassay for determination of heavy metal toxicity in water.

Determining and monitoring toxicity of chemicals in water are very important for human health and country security. Electrochemical measurement of respiratory chain activity is a rapid and reliable screening of the toxicity towards microorganisms. Here, we report a rapid and sensitive toxicity bioassay using p-benzoquinone as the artificial electron mediator and Escherichia coli as the test organism. Four heavy metal ions, Cu(2+), Ag(+), Hg(2+) and Co(2+), are tested as the model toxicants, and the corresponding 50% respiration inhibition concentrations (IC50) are determined to be 0.95, 8.14, 11.69 and 42.76 mg L(-1), respectively. Based on the IC50 values, the descending order of toxicity is: Cu(2+) > Ag(+) > Hg(2+) > Co(2+). The presented bioassay not only provides a good foundation for further toxicity tests using E. coli, but also the potential for expanding the technique to utilize other bacteria with complementary toxicity responses, thereby allowing use of the bioassay in a wide range of applications.

A novel colorimetric biosensor for monitoring and detecting acute toxicity in water.

This work presents a new colorimetric microorganism biosensor for monitoring and detecting acute toxicity in water, where prussian blue (PB) is used as the colorimetric indicator and E. coli as the model bacterial. In this biosensor, the electron mediator, ferricyanide, accepts electrons from E. coli during respiration to produce ferrocyanide, which subsequently reacts with ferric ions to yield PB, a famous material with a blue color. Since toxicants can inhibit the respiratory activity of E. coli and then reduce the ferrocyanide and consequent PB production, toxicity can be easily detected by measuring the decrease in the production of PB induced by toxicants. Three important toxicants, 3,5-dichlorophenol (DCP), As(3+), Cr(6+) are tested and the detection limits are 3.2, 25, and 3.2 ppm, respectively. Moreover, we could identify the yellow green to dark green color change by naked eye even at concentrations as low as 12.5 ppm for both DCP and Cr(6+). Subsequently, the acute toxicities of groundwater and south lake water are successfully determined by this sensor. This biosensor is rapid, sensitive and cost-effective, and can thus be regarded as a promising biosensor for giving an early warning of acute water toxicity.

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.

Elucidating nitrifying performance, nitrite accumulation and microbial community in a three-stage plug flow moving bed biofilm reactor (PF - MBBR).

A three-stage plug flow moving bed biofilm reactor (PF - MBBR, consisting of three identical chambers of N1, N2 and N3) was proposed for nitrifier enrichment using synthetic wastewater. During the stable operation, the average NH4+-N effluent was 0.67 mg/L and NH4+-N removal was as high as 97.19% with the nitrite accumulation ratio (NAR) of 54.23%, although the biofilm thickness and biomass both presented downward trends from N1 (296 µm, 2280 mg/L), N2 (248 µm, 1850 mg/L) to N3 (198 µm, 1545 mg/L). Particularly, the comparative results of three stages revealed that N2 showed the optimum NH4+-N removal (77.27%) and NAR (75.21%) in the continuous-flow, while NAR of N3 unexpectedly maintained a high level of 65.83% in the batch test, suggesting that ammonia oxidizing bacteria (AOB) accounted for absolute advantage over nitrite oxidizing bacteria (NOB). High-throughput sequencing initially verified different distribution of bacterial community structure, where N2 was far away from N1 and N3 with the lowest community richness and community diversity (operational taxonomic units (OTUs): 454(N2)<527(N3)<621(N1)). Proteobacteria (77.60%-83.09%), Bacteroidetes (1.66%-3.66%), Acidobacteria (2.28%-4.67%), and Planctomycetes (1.19%-6.63%) were the major phyla. At the genus level, AOB (mainly Nitrosomonas) accounted for 5.08% (N1), 20.74% (N2) and 14.24% (N3) while NOB (mainly Nitrospira) increased from 0.14% (N1), 7.06% (N2) to 4.91% (N3) with the total percentages of 5.22%, 27.80% and 19.15%. Finally, the application feasibility of MBBR optimization linked with nitrite (NO2--N) accumulation for deep-level nutrient removal was discussed.

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.

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.

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.

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 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.

Viable but nonculturable cells used in biosensor fabrication for long-term storage stability.

In this paper, we first reported the viable but nonculturable (VBNC) cells used for fabricating biosensor. The organic-inorganic hybrid material composed of silica and the grafting copolymer of poly(vinyl alcohol) and 4-vinylpyridine (PVA-g-P(4-VP)) was used to immobilize microbial cells for biosensor fabrication. The VBNC cells were formed after the hybrid material dried, showing the cell walls were sacrificed. With the intracellular enzymes as core and the "sacrificed" cell walls as shell, the present VBNC cells maybe considered as a core/shell structure. The extracellular material worked as the scaffold for core/shell structure. The core/shell structure and the scaffold structure were demonstrated by single-cell level image analysis using confocal laser scanning microscopy (CLSM). The electrochemical method was adopted for further examining the enzyme activity of VBNC cells. The VBNC cells did not need nutrient treatment and other physicochemical factors for cell growth, which is a significant contribution for storing biosensor. A glucose-glutamic acid biosensor fabricated by the VBNC cells exhibited long-term storage stability for 100 days.