Improving the practicality of recombinant Escherichia coli biosensor in detecting trace Cr(VI) by modifying the cryogenic storage conditions of biosensors and applying simple pretreatment.

Hexavalent chromium (Cr(VI)) is a global environmental pollutant. To reduce the risk caused by Cr(VI), a simple, accurate, reproducible, and inexpensive method for quantifying Cr(VI) in water and soil should be developed. In this study, three types of recombinant Escherichia coli biosensors (namely T7-lux-E. coli, T3-lux-E. coli, and SP6-lux-E. coli biosensor) containing promoters (T7, T3, and SP6), chromate-sensing regulator chrB, and the reporter gene luxAB were constructed. This study investigated the effects of cryogenic freezing temperature and time on trace Cr(VI) measurement by using recombinant E. coli biosensors. The results indicated that the activity of thawed frozen SP6-lux-E. coli cells stored at -20 °C for 270 days did not differ from that of freshly prepared cells. Turbidity and conductivity in water samples and organic matter in soil interfered with Cr(VI) measurement using the biosensor. The SP6-lux-E. coli biosensor exhibited a wide measurement range and a low deviation of <5% for measuring Cr(VI) in various Cr(VI)-contaminated water and soil samples and required only a simple pretreatment or extraction process even after 270-day storage at -20 °C. To the best of our knowledge, this is the first study to report the use of recombinant biosensors for accurately measuring Cr(VI) in both water and soil.

Highly Sensitive Luminescent Bioassay Using Recombinant Escherichia coli Biosensor for Rapid Detection of Low Cr(VI) Concentration in Environmental Water.

In this study, we constructed a recombinant Escherichia coli strain with different promoters inserted between the chromate-sensing regulator chrB and the reporter gene luxAB to sense low hexavalent chromium (Cr(VI)) concentrations (<0.05 mg/L); subsequently, its biosensor characteristics (sensitivity, selectivity, and specificity) for measuring Cr(VI) in various water bodies were evaluated. The luminescence intensity of each biosensor depended on pH, temperature, detection time, coexisting carbon source, coexisting ion, Cr(VI) oxyanion form, Cr(VI) concentration, cell type, and type of medium. Recombinant lux-expressing E. coli with the T7 promoter (T7-lux-E. coli, limit of detection (LOD) = 0.0005 mg/L) had the highest luminescence intensity or was the most sensitive for Cr(VI) detection, followed by E. coli with the T3 promoter (T3-lux-E. coli, LOD = 0.001 mg/L) and that with the SP6 promoter (SP6-lux-E. coli, LOD = 0.005 mg/L). All biosensors could be used to determine whether the Cr(VI) standard was met in terms of water quality, even when using thawing frozen cells as biosensors after 90-day cryogenic storage. The SP6-lux-E. coli biosensor had the shortest detection time (0.5 h) and the highest adaptability to environmental interference. The T7-lux-E. coli biosensor-with the optimal LOD, a wide measurement range (0.0005-0.5 mg/L), and low deviation (-5.0-7.9%) in detecting Cr(VI) from industrial effluents, domestic effluents, and surface water-is an efficient Cr(VI) biosensor. This unprecedented study is to evaluate recombinant lux E. coli with dissimilar promoters for their possible practice in Cr(VI) measurement in water bodies, and the biosensor performance is clearly superior to that of past systems in terms of detection time, LOD, and detection deviation for real water samples.

Analysis of bioavailable toluene by using recombinant luminescent bacterial biosensors with different promoters.

In this study, we constructed recombinant luminescent Escherichia coli with T7, T3, and SP6 promoters inserted between tol and lux genes as toluene biosensors and evaluated their sensitivity, selectivity, and specificity for measuring bioavailable toluene in groundwater and river water. The luminescence intensity of each biosensor depended on temperature, incubation time, ionic strength, and concentrations of toluene and coexisting organic compounds. Toluene induced the highest luminescence intensity in recombinant lux-expressing E. coli with the T7 promoter [T7-lux-E. coli, limit of detection (LOD) = 0.05 µM], followed by that in E. coli with the T3 promoter (T3-lux-E. coli, LOD = 0.2 µM) and SP6 promoter (SP6-lux-E. coli, LOD = 0.5 µM). Luminescence may have been synergistically or antagonistically affected by coexisting organic compounds other than toluene; nevertheless, low concentrations of benzoate and toluene analogs had no such effect. In reproducibility experiments, the biosensors had low relative standard deviation (4.3-5.8%). SP6-lux-E. coli demonstrated high adaptability to environmental interference. T7-lux-E. coli biosensor-with low LOD, wide measurement range (0.05-500 µM), and acceptable deviation (- 14.3 to 9.1%)-is an efficient toluene biosensor. This is the first study evaluating recombinant lux E. coli with different promoters for their potential application in toluene measurement in actual water bodies.

Three-Stage Single-Chambered Microbial Fuel Cell Biosensor Inoculated with Exiguobacterium aestuarii YC211 for Continuous Chromium (VI) Measurement.

Chromium (VI) [Cr(VI)] compounds display high toxic, mutagenic, and carcinogenic potential. Biological analysis techniques (e.g., such as enzyme-based or cell-based sensors) have been developed to measure Cr(VI); however, these biological elements are sensitive to the environment, limited to measuring trace Cr(VI), and require deployment offsite. In this study, a three-stage single-chambered microbial fuel cell (SCMFC) biosensor inoculated with Exiguobacterium aestuarii YC211 was developed for in situ, real-time, and continuous Cr(VI) measurement. A negative linear relationship was observed between the Cr(VI) concentration (5⁻30 mg/L) and the voltage output using an SCMFC at 2-min liquid retention time. The theoretical Cr(VI) measurement range of the system could be extended to 5⁻90 mg/L by connecting three separate SCMFCs in series. The three-stage SCMFC biosensor could accurately measure Cr(VI) concentrations in actual tannery wastewater with low deviations (<7%). After treating the wastewater with the SCMFC, the original inoculated E. aestuarii remained dominant (>92.5%), according to the next-generation sequencing analysis. The stable bacterial community present in the SCMFC favored the reliable performance of the SCMFC biosensor. Thus, the three-stage SCMFC biosensor has potential as an early warning device with wide dynamic range for in situ, real-time, and continuous Cr(VI) measurement of tannery wastewater.

Comparison of biofunctional activity of Asparagus cochinchinensis (Lour.) Merr. Extract before and after fermentation with Aspergillus oryzae.

Asparagus cochinchinensis root (ACR) is used in traditional Chinese medicine. In this study, ACR was first extracted with 25% ethyl acetate (EA) and then fermented by Aspergillus oryzae to enhance its antioxidant activity and evaluate its potential antityrosinase activity. The physiological activity and cytotoxicity of A. oryzae-fermented ACR extract, along with its antityrosinase activity and effects on melanogenic factor levels in human epidermal melanocytes (HEMs), were analyzed and compared with those of the unfermented extract. The results showed that the physiological activity of the fermented extract in vitro or in cells was significantly higher than that of the unfermented extract. The IC50 values for 2,2-diphenyl-1-picrylhydrazine radical scavenging activity, reducing power, and antityrosinase activity in vitro for the fermented extract were 250.6 ± 32.5, 25.7 ± 3.5, and 50.6 ± 3.1 mg/L, respectively. The fermented extract favored cellular antityrosinase activity with low melanin production in human melanoma cells compared with the unfermented extract. The inhibitory mechanism of melanin synthesis by unfermented extract was independent of the tested melanogenesis-related proteins. However, the inhibitory mechanism of the fermented extract was possibly caused by synergistic inhibition of these proteins. Thus, A. oryzae-fermented ACR extract may be used for developing new health food or cosmetic ingredients.

Evaluation of tyrosinase inhibitory and antioxidant activities of Angelica dahurica root extracts for four different probiotic bacteria fermentations.

Angelica dahurica root (ADR), which shows strong antioxidant activity, is used in Chinese medicine. This study evaluated the tyrosinase inhibitory and antioxidant activities of ADR extracts fermented by four different probiotic bacteria: Bifidobacterium bifidum, Bifidobacterium lactis, Lactobacillus acidophilus, and Lactobacillus brevis. The ADR was first extracted using distilled water, 70% ethanol, and ethyl acetate, and then fermented by probiotic bacteria. The physiological characteristics of these fermented extracts, namely the antityrosinase activity, antioxidant activity, phenolic composition, and phenolic content, were evaluated and compared with those of unfermented extracts. Results showed that the water extracts after fermentation by probiotic bacteria exhibited the most favorable physiological characteristics. Among the extracts fermented by these probiotic bacteria, L. acidophilus-fermented ADR extract showed the most favorable physiological characteristics. The optimal IC50 values for antityrosinase activity, DPPH radical scavenging activity, and reducing power for L. acidophilus-fermented ADR extract were 0.07 ± 0.03, 0.12 ± 0.01, and 0.68 ± 0.06 mg/mL, respectively. Furthermore, the physiological activities of fermented extracts were considerably higher than those of unfermented extracts. The tyrosinase inhibition and melanin content of B16F10 melanoma cells, and cytotoxicity effects of the fermented ADR extracts on B16F10 cells were also evaluated. We found that the L. acidophilus-fermented ADR extract at 1.5 mg/mL showed significant cellular antityrosinase activity with low melanin production in B16F10 cells and was noncytotoxic to B16F10 cells. Among all probiotic bacteria, water-extracted ADR fermented by L. acidophilus for 48 h was found to be the best skincare agent or antioxidant agent.

Utility of Ochrobactrum anthropi YC152 in a Microbial Fuel Cell as an Early Warning Device for Hexavalent Chromium Determination.

Fast hexavalent chromium (Cr(VI)) determination is important for environmental risk and health-related considerations. We used a microbial fuel cell-based biosensor inoculated with a facultatively anaerobic, Cr(VI)-reducing, and exoelectrogenic Ochrobactrum anthropi YC152 to determine the Cr(VI) concentration in water. The results indicated that O. anthropi YC152 exhibited high adaptability to pH, temperature, salinity, and water quality under anaerobic conditions. The stable performance of the microbial fuel cell (MFC)-based biosensor indicated its potential as a reliable biosensor system. The MFC voltage decreased as the Cr(VI) concentration in the MFC increased. Two satisfactory linear relationships were observed between the Cr(VI) concentration and voltage output for various Cr(VI) concentration ranges (0.0125-0.3 mg/L and 0.3-5 mg/L). The MFC biosensor is a simple device that can accurately measure Cr(VI) concentrations in drinking water, groundwater, and electroplating wastewater in 45 min with low deviations (<10%). The use of the biosensor can help in preventing the violation of effluent regulations and the maximum allowable concentration of Cr(VI) in water. Thus, the developed MFC biosensor has potential as an early warning detection device for Cr(VI) determination even if O. anthropi YC152 is a possible opportunistic pathogen.

Hexavalent chromium removal and bioelectricity generation by Ochrobactrum sp. YC211 under different oxygen conditions.

Bioremediation is an environmentally friendly method of reducing heavy metal concentration and toxicity. A chromium-reducing bacterial strain, isolated from the vicinity of an electroplate factory, was identified as Ochrobactrum sp. YC211. The efficiency and capacity per time of Ochrobactrum sp. YC211 for hexavalent chromium (Cr(VI)) removal under anaerobic conditions were superior to those under aerobic conditions. An acceptable removal efficiency (96.5 ± 0.6%) corresponding to 30.2 ± 0.8 mg-Cr (g-dry cell weight-h)(-1) was achieved by Ochrobactrum sp. YC211 at 300 mg L(-1) Cr(VI). A temperature of 30°C and pH 7 were the optimal parameters for Cr(VI) removal. By examining reactivated cells, permeabilized cells, and cell-free extract, we determined that Cr(VI) removal by Ochrobactrum sp. YC211 under anaerobic conditions mainly occurred in the soluble fraction of the cell and can be regarded as an enzymatic reaction. The results also indicated that an Ochrobactrum sp. YC211 microbial fuel cell (MFC) with an anaerobic anode was considerably superior to that with an aerobic anode in bioelectricity generation and Cr(VI) removal. The maximum power density and Cr(VI) removal efficiency of the MFC were 445 ± 3.2 mW m(-2) and 97.2 ± 0.3%, respectively. Additionally, the effects of coexisting ions (Cu(2+), Zn(2+), Ni(2+), SO4(2-), and Cl(-)) in the anolyte on the MFC performance and Cr(VI) removal were nonsignificant (P > 0.05). To our knowledge, this is the first report to compare Cr(VI) removal by different cells and MFC types under aerobic and anaerobic conditions.

Effects of Operating Parameters on Measurements of Biochemical Oxygen Demand Using a Mediatorless Microbial Fuel Cell Biosensor.

The conventional Biochemical Oxygen Demand (BOD) method takes five days to analyze samples. A microbial fuel cell (MFC) may be an alternate tool for rapid BOD determination in water. However, a MFC biosensor for continuous BOD measurements of water samples is still unavailable. In this study, a MFC biosensor inoculated with known mixed cultures was used to determine the BOD concentration. Effects of important parameters on establishing a calibration curve between the BOD concentration and output signal from the MFC were evaluated. The results indicate monosaccharides were good fuel, and methionine, phenylalanine, and ethanol were poor fuels for electricity generation by the MFC. Ions in the influent did not significantly affect the MFC performance. CN(-) in the influent could alleviate the effect of antagonistic electron acceptors on the MFC performance. The regression equation for BOD concentration and current density of the biosensor was y = 0.0145x + 0.3317. It was adopted to measure accurately and continuously the BOD concentration in actual water samples at an acceptable error margin. These results clearly show the developed MFC biosensor has great potential as an alternative BOD sensing device for online measurements of wastewater BOD.

Decolorization characteristics and mechanism of Victoria Blue R removal by Acinetobacter calcoaceticus YC210.

Acinetobacter calcoaceticus YC210 has been isolated and its ability to remove Victoria Blue R (VBR) from aqueous solution was assessed. The effects of various factors on decolorization efficiency were investigated in a batch system. The decolorization efficiency was found to be optimal within a pH of 5-7 and increased with VBR concentration up to 450 mg/l with high efficiency (94.5%) in a short time. The decolorization efficiency was significantly affected by cell concentrations. The decolorization of VBR by A. calcoaceticus YC210 followed first order kinetics. The apparent kinetic parameters of the Lineweaver-Burk equation, R(VBR,max) and K(m), were calculated as 6.93 mg-VBR/g-cell/h and 175.8 mg/l, respectively. Based on the biodegradation products, VBR degradation by A. calcoaceticus YC210 involves a stepwise demethylation process to yield partially dealkylated VBR species. To our knowledge, this is the first report using microbes to remove VBR. It clearly demonstrates the dealkylation pathway of VBR degradation.

Structure of the bacterial community in a biofilter during dimethyl sulfide (DMS) removal processes.

We report here both the successful treatment of DMS in a biofilter and the dynamic changes that occur in the composition of the bacterial community of the biofilter during this process. Denaturing gradient gel electrophoresis of eubacterial 16S rDNA samples taken from packing material at different DMS removal stages revealed 11 distinct bands. Phylogenetic analysis showed that the sequences of these bands were closest to sequences of species of the Proteobacteria, Firmicutes, and Actinobacteria. The specific occurrence of these bacterial species varied mainly with DMS load, but it was also affected by the addition of glucose and by ambient temperature. Based on the characteristics of the identified species, the system is conducive for such processes as sulfur oxidation, sulfate reduction, carbon oxidation, and fermentation. The strains identified in this study are potential candidates for purifying waste gas effluents containing DMS gas.

Comparisons of Vibrio fischeri, Photobacterium phosphoreum, and recombinant luminescent using Escherichia coli as BOD measurement.

To shorten the time needed to measure biochemical oxygen demand (BOD) in water samples and to provide a rapid feedback of the real condition of water quality, we tested and evaluated the validity and reliability of luminescent bacteria Vibrio fischeri, Photobacterium phosphoreum, and recombinant Escherichia coli as potential indicators of BOD in the domestic wastewaters. The results indicate that the luminescence intensities of these strains are dependent on temperature, pH, and BOD concentration. In comparison to the standard BOD(5) method, the time needed for BOD measurement can be shortened by 90, 120, and 150 min when V. fischeri, P. phosphoreum, and recombinant E. coli, respectively, are used. Recombinant E. coli can be adapted to measure BOD in domestic wastewater containing a wide range of BOD concentrations, V. fischeri is not suitable for measuring diluted wastewater, and P. phosphoreum has only a limited application in measuring concentrated wastewater. To the best of our knowledge, this is the first report in which V. fischeri, P. phosphoreum, and recombinant luminescent E. coli are compared in terms of their potential in BOD measurement systems.

A rapid method for the detection of representative coliforms in water samples: polymerase chain reaction-enzyme-linked immunosorbent assay (PCR-ELISA).

Methods to detect the presence of coliform bacteria in drinking water usually involve a series of complex cultivating steps that are time-consuming and subject to external influences. For this reason, the new 16S rRNA probe has been developed in this study as an alternative detector PCR-ELISA technique that does not involve the culture of bacteria and that is able to detect, identify, and quantify the representative coliform species present in water samples. Our results indicate that this technique is both rapid (detection time of 4 h) and accurate (1.4% error rate). The limit of detection (LOD) was 5 CFU/100 ml for total coliforms, which meets the standards set by most countries for drinking water. Our comparative study demonstrated that this PCR-ELISA method is superior to current conventional methods in terms of detection time, LOD, and accuracy.

Diversity of the bacterial community in a bioreactor during ammonia gas removal.

Polymerase chain reaction analysis in combination with denaturing gradient gel electrophoresis (DGGE) was used to determine changes in the composition of the bacterial community of a bioreactor during ammonia removal. A minimum of 13 bands were observed in the DGGE profile. Phylogenetic analysis revealed that phylum Proteobacteria was predominantly represented in the bacterial community of the bioreactor, followed by Firmicutes, Actinobacteria, and Flavobacteriaceae. However, the occurrence and predominance of specific bacterial species varied with the concentrations of NH(3) introduced into the bioreactor. The complexity of the bacterial species generally decreased with increasing inlet NH(3) concentration. Based on the characteristics of the identified species, there is a potential for nitrification, denitrification, nitrate reduction, nitrite reduction, and ammonia assimilation to occur simultaneously in the bioreactor. The strains identified in this study are potential candidate strains for the purification of waste gases containing high concentrations of NH(3).

Biological decolorization of dye solution containing malachite green by Pandoraea pulmonicola YC32 using a batch and continuous system.

In our study, we have isolated a relatively newly identified bacteria species, Pandoraea pulmonicola YC32, and first assessed its capability to treat malachite green (MG). The effects of various factors on decolorization efficiency were investigated in a batch system. The decolorization efficiency was found to be optimal within a pH of 7-10 and it increased, with increasing initial MG concentration up to 100 mg/l. The relationship between the decolorization rate and MG concentration agreed with Lineweaver-Burk equation. The apparent kinetic parameters, R(MG,max) and K(m), were 6.23 mg-MG/g-cell/h and 153.4 mg/l, respectively. The initial step in the biodegradation pathway of MG by P. pulmonicola YC32 was a reduction or N-demethylation reaction. We achieved a decolorization efficiency of 85.2% with 50mg/l MG in the immobilized P. pulmonicola YC32 continuous column system. This is the first report on the application of a continuous column system to decolorize MG using a microorganism.

Biodegradation of crystal violet by Pseudomonas putida.

Crystal violet (CV), which has been extensively used as a biological stain and a commercial textile dye, is a recalcitrant molecule. A strain of Pseudomonas putida was isolated that effectively degraded CV: up to 80% of 60 microM CV as the sole carbon source, was degraded in liquid media within 1 week. Nine degradation products were isolated and identified. We propose that CV degradation occurs via a stepwise demethylation process to yield mono-, di-, tri-, tetra-, penta- and hexa-demethylated CV species.