Bioaugmentation of triclocarban and its dechlorinated congeners contaminated soil with functional degraders and the bacterial community response.

Partial removal of haloaromatic antimicrobial triclocarban (TCC) during wastewater treatment caused the final introduction of residual TCC into soils. Bioaugmentation has been proposed for the biodegradation of TCC and its dechlorinated congeners 4,4'-dichlorocarbanilide (DCC) and carbanilide (NCC) in soil. The isolated TCC-degrading strain Ochrobactrum sp. TCC-2 and chloroanilines-degrading strain Diaphorobacter sp. LD72 were used to study the removal efficiency of TCC, DCC and NCC mixture and their chloroanilines intermediates, respectively. The potential degradation competition between TCC and its dechlorinated congeners, and the response of bacterial community during the bioremediation were also investigated. The biodegradation of DCC and TCC was significantly enhanced for soil with inoculums compared with sterilized and natural soils. Chloroanilines products could also be effectively removed. For the degradation of combined substrates in the aqueous medium, NCC had negative effect on the degradation of TCC and DCC, while TCC and DCC negatively influenced each other. The bioaugmentation with two degraders obviously changed the phylogenetic composition and function of indigenous soil microbiome. Importantly, the inoculated degraders could be maintained, suggesting their adaptability and potential application in bioaugmentation for such recalcitrant contaminants. This study offers new insights into the enhanced bioremediation of TCC and its dechlorinated congeners contaminated soils by the bioaugmentation of functional degraders and the structure and function response of the indigenous soil microbiome to the bioremediation process.

Aerobic and anoxic degradation and detoxification of profenofos insecticide by Pseudomonas plecoglossicida strain PF1.

Profenofos insecticide is one of the most broadly used organophosphorus pesticides causing the contamination of soil and groundwater. Since dissolved oxygen concentration in groundwater is limited, this study aimed to investigate profenofos biodegradation and detoxification under aerobic and anoxic conditions using the profenofos-degrading Pseudomonas plecoglossicida strain PF1 (PF1). Anoxic biodegradation under the presence of nitrate was the focus. The results showed that profenofos at 10-150 mg/L was degraded under aerobic and anoxic conditions with removal efficiencies of 38-55% and 27-45%, respectively. Kinetic analysis following the Michaelis-Menten model revealed that the maximum substrate degradation rates and the Michaelis constants were 13.07 and 8.92 mg/L/d and 92.07 and 84.76 mg/L under aerobic and anoxic conditions, respectively. The culture preferred an aerobic environment resulting in better biodegradation performance. During the degradation experiment, 4-bromo-2-chlorophenol and 1,1-dimethylethylphenol were detected as profenofos biodegradation intermediate products. Microbial toxicity, phytotoxicity, and cytogenotoxicity assays showed that the toxicity of the contaminated water significantly decreased after both aerobic and anoxic biodegradation by PF1. The results from this study indicated that PF1 has the potential for bioremediation in a profenofos-contaminated environment under the presence or absence of oxygen.

Engineering of Corynebacterium glutamicum as a prototrophic pyruvate-producing strain: Characterization of a ramA-deficient mutant and its application for metabolic engineering.

To construct a prototrophic Corynebacterium glutamicum strain that efficiently produces pyruvate from glucose, the effects of inactivating RamA, a global regulator responsible for activating the oxidative tricarboxylic acid (TCA) cycle, on glucose metabolism were investigated. ΔramA showed an increased specific glucose consumption rate, decreased growth, comparable pyruvate production, higher formation of lactate and acetate, and lower accumulation of succinate and 2-oxoglutarate compared to the wild type. A significant decrease in pyruvate dehydrogenase complex activity was observed for ΔramA, indicating reduced carbon flow to the TCA cycle in ΔramA. To create an efficient pyruvate producer, the ramA gene was deleted in a strain lacking the genes involved in all known lactate- and acetate-producing pathways. The resulting mutant produced 161 mM pyruvate from 222 mM glucose, which was significantly higher than that of the parent (89.3 mM; 1.80-fold).

Improved heterologous expression of the membrane-bound quinoprotein quinate dehydrogenase from Gluconobacter oxydans.

Gluconobacter oxydans produces 3-dehydroquinate by oxidation of quinate through a reaction catalyzed by the quinate dehydrogenase (QDH), membrane-bound, pyrroloquinoline quinone (PQQ)-dependent dehydrogenase. We previously reported the nucleotide and deduced amino acid sequence of QDH and constructed a heterologous expression system of QDH in Pseudomonas sp. (A.S. Vangnai, W. Promden, W. De-Eknamkul, K. Matsushita, H. Toyama, Biochemistry (Moscow) 75:452-459, 2010). Through this study, we aim to update the sequences of QDH and improve the heterologous expression of QDH in Gluconobacter strains using a broad-host-range plasmid. Expression of QDH using a plasmid containing a long 5'-UTR was higher than that using a plasmid with a short 5'-UTR. In addition, the usage of the putative promoter region of the membrane-bound, alcohol dehydrogenase (ADH) of Gluconobacter resulted in higher expression levels compared to the usage of the lacZ promoter. Base substitution experiments allowed to identify the correct TTG initiation codon between two possibilities, and the result of these experiments were consistent with the N-terminal amino acid sequence of the expressed QDH. However, change of the TTG codon to ATG did not increase QDH expression. Therefore, the optimal plasmid for QDH expression included the structural gene with a long 5'-UTR and the ADH promoter. Cell membrane of the recombinant Gluconobacter strain presented approximately 10-times higher specific QDH activity than that observed in the wild-type strain.

The effects of disruption in membrane lipid biosynthetic genes on 1-butanol tolerance of Bacillus subtilis.

Microbes with enhanced 1-butanol tolerance have the potentials to be utilized in various biotechnological processes. To achieve the rational design of such strains, we previously conducted an untargeted metabolomics analysis of Bacillus subtilis under 1-butanol stress and uncovered a novel type of microbial responses as the alterations in the glycerolipid and phospholipid composition. However, the current knowledge about the relevance of these changes on 1-butanol tolerance remains quite limited. Here, we constructed the B. subtilis mutants with disruption in the pssA, ugtP (U), mprF (M), yfnI, and yfnI/mprF genes in the membrane lipid biosynthetic pathways. The 1-butanol tolerance test indicated markedly increased and decreased 1-butanol resistance in M and U compared to the wild-type strain, respectively, and slight effects in other strains under high stress level. Further examination of the lipid contents of these strains in the presence of 1-butanol by liquid chromatography-mass spectrometry demonstrated an elevated ratio of neutral and anionic to cationic lipids in direct relation with an improved 1-butanol tolerance. Last, cell morphological studies showed the shortening of only the U cells, compared to the wild-type. All strains including U were capable of elongating by 14-24% under 1-butanol stress. Together, the studies indicated the involvement of membrane lipid biosynthetic genes, which regulated glycerolipid and phospholipid composition, on 1-butanol tolerance and allowed for the procurement of M with enhanced 1-butanol tolerance trait, highlighting the usefulness of the overall approaches on discovery of novel biological insights and engineering of microorganisms with desired resistance characteristics.

Membrane Alterations in Pseudomonas putida F1 Exposed to Nanoscale Zerovalent Iron: Effects of Short-Term and Repetitive nZVI Exposure.

In this study, we report the effect of the commercial nanoscale zerovalent iron (nZVI) on environmental bacteria, emphasizing the importance of nZVI-bacterial membrane interaction on nZVI toxicity as well as the adaptability of bacteria to nZVI. Exposure of Pseudomonas putida F1 to 0.1, 1.0, and 5.0 g/L of nZVI caused the reduction in colony forming units (CFUs) substantially for almost 3 orders of magnitude. However, a rebound in the cell number was observed after the prolonged exposure except for 5.0 g/L nZVI at which bacterial viability was completely inhibited. Upon exposure, nZVI accumulated on and penetrated into the bacterial cell membrane. Cell membrane composition analysis revealed the conversion of the cis to trans isomer of unsaturated fatty acid upon short-term nZVI exposure, resulting in a more rigid membrane counteracting the membrane-fluidizing effect of nZVI. Several cycles of repetitive exposure of cells to 0.1 g/L nZVI induced a persistent phenotype of P. putida F1 as indicated by smaller colony morphology, a more rigid membrane, and higher tolerance to nZVI. A low interaction between nZVI particles and the surface of the nZVI-persistent phenotypic cells reduced the nZVI-induced membrane damage. This study unveils the significance of nZVI-membrane interaction on toxicity of nZVI toward bacteria.

Plant-growth promoting Candida sp. AVGB4 with capability of 4-nitroaniline biodegradation under drought stress.

This study focused on rhizospheric yeast capable of degrading a priority pollutant, 4-nitroaniline (4-NA), under drought stress. Candida sp. AVGB4 (AVGB4) inhabiting in soil was isolated and characterized with plant-growth promoting (PGP) traits. 4-NA-dependent growth kinetic and biodegradation kinetics were analyzed and revealed 4-NA complete degradation and tolerance property. AVGB4 proliferation, PGP activities, and 4-NA degradation activity were well maintained under drought stress induced by PEG-6000 incorporation, and could be strengthened in the presence of succinate, an organic compound generally found in plant root exudates. The in vitro experiments proved that AVGB4 significantly enhanced plant growth and increased the shoot and root biomass of Vigna radiata plant in the absence or presence of 4-NA. The overall results including cytogenotoxicity and phytotoxicity test with legumes indicated that not only AVGB4 was capable of 4-NA detoxification facilitating plants to cope with chemical-toxicity stress, but it also has advantageous role in promoting plant growth and sustainable rhizoremediation of 4-NA contaminated sites.

Untargeted metabolomics analysis revealed changes in the composition of glycerolipids and phospholipids in Bacillus subtilis under 1-butanol stress.

1-Butanol has been utilized widely in industry and can be produced or transformed by microbes. However, current knowledge about the mechanisms of 1-butanol tolerance in bacteria remains quite limited. Here, we applied untargeted metabolomics to study Bacillus subtilis cells under 1-butanol stress and identified 55 and 37 ions with significantly increased and decreased levels, respectively. Using accurate mass determination, tandem mass spectra, and synthetic standards, 86 % of these ions were characterized. The levels of phosphatidylethanolamine, diglucosyldiacylglycerol, and phosphatidylserine were found to be upregulated upon 1-butanol treatment, whereas those of diacylglycerol and lysyl phosphatidylglycerol were downregulated. Most lipids contained 15:0/15:0, 16:0/15:0, and 17:0/15:0 acyl chains, and all were mapped to membrane lipid biosynthetic pathways. Subsequent two-stage quantitative real-time reverse transcriptase PCR analyses of genes in the two principal membrane lipid biosynthesis pathways revealed elevated levels of ywiE transcripts in the presence of 1-butanol and reduced expression levels of cdsA, pgsA, mprF, clsA, and yfnI transcripts. Thus, the gene transcript levels showed agreement with the metabolomics data. Lastly, the cell morphology was investigated by scanning electron microscopy, which indicated that cells became almost twofold longer after 1.4 % (v/v) 1-butanol stress for 12 h. Overall, the studies uncovered changes in the composition of glycerolipids and phospholipids in B. subtilis under 1-butanol stress, emphasizing the power of untargeted metabolomics in the discovery of new biological insights.

Removal of Chlorpyrifos by Water Hyacinth (Eichhornia crassipes) and the Role of a Plant-Associated Bacterium.

The objective of this research was to study the efficiency of water hyacinth (Eichhornia crassipes) and the role of any plant-associated bacteria in removing chlorpyrifos from water. The relative growth rate (RGR) of E. crassipes in the presence of 0.1 mg/L chlorpyrifos was not significantly different from that in its absence and only slightly decreased at concentrations of 0.5 and 1.0 mg/L by ∼1.1- and ∼1.2-fold, respectively, with an observed dry weight based RGRDW for E. crassipes of 0.036-0.041 mg/g/d. The removal rate constants of chlorpyrifos in the absence of plants were low at 3.52, 2.29 and 1.84 h(-1) for concentrations of 0.1, 0.5 and 1.0 mg/L, respectively, but were some 3.89- to 4.87-fold higher in the presence of E. crassipes. Chlorpyrifos removal was markedly facilitated by the presence of a root-associated bacterium, preliminarily identified as Acinetobacter sp. strain WHA. The interaction of E. crassipes and Acinetobacter sp. strain WHA provide an efficient and ecological alternative to accelerate the removal and degradation of chlorpyrifos pollution from aquatic systems including wastewater.

Overexpression of a type II 3-dehydroquinate dehydratase enhances the biotransformation of quinate to 3-dehydroshikimate in Gluconobacter oxydans.

Shikimate and 3-dehydroshikimate are useful chemical intermediates for the synthesis of various compounds, including the antiviral drug oseltamivir. Here, we show an almost stoichiometric biotransformation of quinate to 3-dehydroshikimate by an engineered Gluconobacter oxydans strain. Even under pH control, 3-dehydroshikimate was barely detected during the growth of the wild-type G. oxydans strain NBRC3244 on the medium containing quinate, suggesting that the activity of 3-dehydroquinate dehydratase (DHQase) is the rate-limiting step. To identify the gene encoding G. oxydans DHQase, we overexpressed the gox0437 gene from the G. oxydans strain ATCC621H, which is homologous to the aroQ gene for type II DHQase, in Escherichia coli and detected high DHQase activity in cell-free extracts. We identified the aroQ gene in a draft genome sequence of G. oxydans NBRC3244 and constructed G. oxydans NBRC3244 strains harboring plasmids containing aroQ and different types of promoters. All recombinant G. oxydans strains produced a significant amount of 3-dehydroshikimate from quinate, and differences between promoters affected 3-dehydroshikimate production levels with little statistical significance. By using the recombinant NBRC3244 strain harboring aroQ driven by the lac promoter, a sequential pH adjustment for each step of the biotransformation was determined to be crucial because 3-dehydroshikimate production was enhanced. Under optimal conditions with a shift in pH, the strain could efficiently produce a nearly equimolar amount of 3-dehydroshikimate from quinate. In the present study, one of the important steps to convert quinate to shikimate by fermenting G. oxydans cells was investigated.

Enhancement of (R)-1,3-butanediol production by engineered Escherichia coli using a bioreactor system with strict regulation of overall oxygen transfer coefficient and pH.

(R)-1,3-butanediol ((R)-1,3-BD) is an important substrate for the synthesis of industrial chemicals. Despite its large demand, a bioprocess for the efficient production of 1,3-BD from renewable resources has not been developed. We previously reported the construction of recombinant Escherichia coli that could efficiently produce (R)-1,3-BD from glucose. In this study, the fermentation conditions were optimized to further improve 1,3-BD production by the recombinant strain. A batch fermentation was performed with an optimized overall oxygen transfer coefficient (82.3 h(-1)) and pH (5.5); the 1,3-BD concentration reached 98.5 mM after 36 h with high-yield (0.444 mol (mol glucose)(-1)) and a high maximum production rate (3.63 mM h(-1)). In addition, a fed-batch fermentation enabled the recombinant strain to produce 174.8 mM 1,3-BD after 96 h cultivation with a yield of 0.372 mol (mol glucose)(-1), a maximum production rate of 3.90 mM h(-1), and a 98.6% enantiomeric excess (% ee) of (R)-1,3-BD.

Metabolomics responses and tolerance of Pseudomonas aeruginosa under acoustic vibration stress.

Sound has been shown to impact microbial behaviors. However, our understanding of the chemical and molecular mechanisms underlying these microbial responses to acoustic vibration is limited. In this study, we used untargeted metabolomics analysis to investigate the effects of 100-Hz acoustic vibration on the intra- and extracellular hydrophobic metabolites of P. aeruginosa PAO1. Our findings revealed increased levels of fatty acids and their derivatives, quinolones, and N-acylethanolamines upon sound exposure, while rhamnolipids (RLs) showed decreased levels. Further quantitative real-time polymerase chain reaction experiments showed slight downregulation of the rhlA gene (1.3-fold) and upregulation of fabY (1.5-fold), fadE (1.7-fold), and pqsA (1.4-fold) genes, which are associated with RL, fatty acid, and quinolone biosynthesis. However, no alterations in the genes related to the rpoS regulators or quorum-sensing networks were observed. Supplementing sodium oleate to P. aeruginosa cultures to simulate the effects of sound resulted in increased tolerance of P. aeruginosa in the presence of sound at 48 h, suggesting a potential novel response-tolerance correlation. In contrast, adding RL, which went against the response direction, did not affect its growth. Overall, these findings provide potential implications for the control and manipulation of virulence and bacterial characteristics for medical and industrial applications.

Identification of CtpL as a chromosomally encoded chemoreceptor for 4-chloroaniline and catechol in Pseudomonas aeruginosa PAO1.

Bacterial chemotaxis influences the ability of bacteria to survive and thrive in most environments, including polluted ones. Despite numerous reports of the phenotypic characterization of chemotactic bacteria, only a few molecular details of chemoreceptors for aromatic pollutants have been described. In this study, the molecular basis of chemotaxis toward an environmentally toxic chlorinated aromatic pollutant, 4-chloroaniline (4CA), was evaluated. Among the three Pseudomonas spp. tested, Pseudomonas aeruginosa PAO1 exhibited positive chemotaxis both to the nonmetabolizable 4CA, where 4-chloroacetanilide was formed as a dead-end transformation product, and to the metabolizable catechol. Molecular analysis of all 26 mutants with a disrupted methyl-accepting chemotaxis gene revealed that CtpL, a chromosomally encoded chemoreceptor, was responsible for the positive chemotactic response toward 4CA. Since CtpL has previously been described to be a major chemoreceptor for inorganic phosphate at low concentrations in PAO1, this report describes a fortuitous ability of CtpL to function toward aromatic pollutants. In addition, its regulation not only was dependent on the presence of the chemoattractant inducer but also was regulated by conditions of phosphate starvation. These results expand the range of known chemotactic transducers and their function in the environmental bacterium PAO1.

Co-application of citric acid and Nocardiopsis sp. strain RA07 enhances phytoremediation potentiality of Sorghum bicolor L.

This study investigated the combined effects of citric acid (CA) and Nocardiopsis sp. RA07 on the phytoremediation potential of lead (Pb)- and copper (Cu)-contaminated soils by Sorghum bicolor L. The strain RA07 was able to tolerate Pb and Cu, and exhibited plant growth-promoting features like siderophore production, indole-3-acetic acid (IAA) synthesis, 1-aminocyclopropane-1-carboxylate (ACC) deaminase activity and phosphate solubilization. The combined application of CA and strain RA07 significantly increased S. bicolor growth, chlorophyll content and antioxidant enzymatic activity, and decreased oxidative stress (hydrogen peroxide and malondialdehyde content) under Pb and Cu stress circumstances as compared to individual treatments (i.e., CA and strain RA07). Furthermore, the combined application of CA and RA07 significantly enhanced S. bicolor ability to accumulate Pb and Cu by 64.41% and 60.71% in the root and 188.39% and 125.56% in the shoot, respectively, as compared to the corresponding uninoculated plants. Our results indicate that inoculation of Nocardiopsis sp. together with CA could be a useful practical approach to mitigate Pb and Cu stress on plant growth and increase the effectiveness of phytoremediation in Pb- and Cu-polluted soils.

Design of electron-donating group substituted 2-PAM analogs as antidotes for organophosphate insecticide poisoning.

The use of organophosphate (OPs) pesticides is widespread in agriculture and horticulture, but these chemicals can be lethal to humans, causing fatalities and deaths each year. The inhibition of acetylcholinesterase (AChE) by OPs leads to the overstimulation of cholinergic receptors, ultimately resulting in respiratory arrest, seizures, and death. Although 2-pralidoxime (2-PAM) is the FDA-approved drug for treating OP poisoning, there is difficulty in blood-brain barrier permeation. To address this issue, we designed and evaluated a series of 2-PAM analogs by substituting electron-donating groups on the para and/or ortho positions of the pyridinium core using in silico techniques. Our PCM-ONIOM2 (MP2/6-31G*:PM7//B3LYP/6-31G*:UFF) binding energy results demonstrated that 13 compounds exhibited higher binding energy than 2-PAM. The analog with phenyl and methyl groups substituted on the para and ortho positions, respectively, showed the most favorable binding characteristics, with aromatic residues in the active site (Y124, W286, F297, W338, and Y341) and the catalytic residue S203 covalently bonding with paraoxon. The results of DS-MD simulation revealed a highly favorable apical conformation of the potent analog, which has the potential to enhance reactivation of AChE. Importantly, newly designed compound demonstrated appropriate drug-likeness properties and blood-brain barrier penetration. These results provide a rational guide for developing new antidotes to treat organophosphate insecticide toxicity.

Bioengineering of biowaste to recover bioproducts and bioenergy: A circular economy approach towards sustainable zero-waste environment.

The inevitable need for waste valorisation and management has revolutionized the way in which the waste is visualised as a potential biorefinery for various product development rather than offensive trash. Biowaste has emerged as a potential feedstock to produce several value-added products. Bioenergy generation is one of the potential applications originating from the valorisation of biowaste. Bioenergy production requires analysis and optimization of various parameters such as biowaste composition and conversion potential to develop innovative and sustainable technologies for most effective utilization of biowaste with enhanced bioenergy production. In this context, feedstocks, such as food, agriculture, beverage, and municipal solid waste act as promising resources to produce renewable energy. Similarly, the concept of microbial fuel cells employing biowaste has clearly gained research focus in the past few decades. Despite of these potential benefits, the area of bioenergy generation still is in infancy and requires more interdisciplinary research to be sustainable alternatives. This review is aimed at analysing the bioconversion potential of biowaste to renewable energy. The possibility of valorising underutilized biowaste substrates is elaborately presented. In addition, the application and efficiency of microbial fuel cells in utilizing biowaste are described in detail taking into consideration of its great scope. Furthermore, the review addresses the significance bioreactor development for energy production along with major challenges and future prospects in bioenergy production. Based on this review it can be concluded that bioenergy production utilizing biowaste can clearly open new avenues in the field of waste valorisation and energy research. Systematic and strategic developments considering the techno economic feasibilities of this excellent energy generation process will make them a true sustainable alternative for conventional energy sources.

Microbial engineering strategies for synthetic microplastics clean up: A review on recent approaches.

Microplastics are the small fragments of the plastic molecules which find their applications in various routine products such as beauty products. Later, it was realized that it has several toxic effects on marine and terrestrial organisms. This review is an approach in understanding the microplastics, their origin, dispersal in the aquatic system, their biodegradation and factors affecting biodegradation. In addition, the paper discusses the major engineering approaches applied in microbial biotechnology. Specifically, it reviews microbial genetic engineering, such as PET-ase engineering, MHET-ase engineering, and immobilization approaches. Moreover, the major challenges associated with the plastic removal are presented by evaluating the recent reports available.

Bioproduction of vanillin using an organic solvent-tolerant Brevibacillus agri 13.

Nowadays, majority of vanillin supplied to the world market is chemically synthesized from a petroleum-based raw material, raising a concern among the consumers regarding the product safety. In this study, an organic solvent-tolerant Brevibacillus agri 13 previously reported for a strong predilectic property was utilized as a whole-cell biocatalyst for bioproduction of vanillin from isoeugenol (IG). B. agri 13 is the first biocatalyst reported for bioproduction of vanillin at a temperature as high as 45°C. Both pH and temperature were found to affect vanillin production significantly. An extreme level of organic solvent tolerance of B. agri 13 allowed us to utilize it in a biphasic system using organic solvents generally considered as highly toxic to most bacteria. With an addition of butyl acetate at 30% (v/v) as an organic second phase, toxicity of IG exerted onto the biocatalyst was reduced dramatically while faster and more efficient vanillin production was obtained (1.7 g/L after 48 h with 27.8% molar conversion).

Development of a whole-cell biocatalyst co-expressing P450 monooxygenase and glucose dehydrogenase for synthesis of epoxyhexane.

Oxygenases-based Escherichia coli whole-cell biocatalyst can be applied for catalysis of various commercially interesting reactions that are difficult to achieve with traditional chemical catalysts. However, substrates and products of interest are often toxic to E. coli, causing a disruption of cell membrane. Therefore, organic solvent-tolerant bacteria became an important tool for heterologous expression of such oxygenases. In this study, the organic solvent-tolerant Bacillus subtilis 3C5N was developed as a whole-cell biocatalyst for epoxidation of a toxic terminal alkene, 1-hexene. Comparing to other hosts tested, high level of tolerance towards 1-hexene and a moderately hydrophobic cell surface of B. subtilis 3C5N were suggested to contribute to its higher 1,2-epoxyhexane production. A systematic optimization of reaction conditions such as biocatalyst and substrate concentration resulted in a 3.3-fold increase in the specific rate. Co-expression of glucose dehydrogenase could partly restored NADPH-regenerating ability of the biocatalyst (up to 38 % of the wild type), resulting in approximately 53 % increase in specific rate representing approximately 22-fold increase in product concentration comparing to that obtained prior to an optimization.

Construction and application of an Escherichia coli bioreporter for aniline and chloroaniline detection.

Aniline and chlorinated anilines (CAs) are classified as priority pollutants; therefore, an effective method for detection and monitoring is required. In this study, a green-fluorescence protein-based bioreporter for the detection of aniline and CAs was constructed in Escherichia coli DH5α, characterized and tested with soil and wastewater. The sensing capability relied on the regulatory control between a two-component regulatory protein, TodS/TodT, and the P( todX ) promoter of Pseudomonas putida T-57 (PpT57), since the gene expression of todS, todT, and todC2 are positively induced with 4-chloroaniline. The bioreporter system (DH5α/pPXGFP-pTODST) is markedly unique with the two co-existing plasmids. The inducibility of the fluorescence response was culture-medium- and time-dependent. Cells grown in M9G medium exhibited a low background fluorescence level and were readily induced by 4CA after 3-h exposure, reaching the maximum induction level at 9 h. When tested with benzene, toluene, ethyl-benzene and xylene, aniline and CAs, the response data were best fit by a sigmoidal dose-response relationship, from which the K(½) value was determined for the positive effectors. 3CA and 4CA were relatively powerful inducers, while some poly-chlorinated anilines could also induce green fluorescence protein expression. The results indicated a broader recognition range of PpT57'sTodST than previously reported for P. putida. The test results with environmental samples were reliable, indicating the potential application of this bioreporter in the ecotoxicology assessment and bioremediation of areas contaminated with aniline- and/or CAs.