Mechanisms of benzene and benzo[a]pyrene biodegradation in the individually and mixed contaminated soils.

There is a lack of knowledge on the biodegradation mechanisms of benzene and benzo [a]pyrene (BaP), representative compounds of polycyclic aromatic hydrocarbons (PAHs), and benzene, toluene, ethylbenzene, and xylene (BTEX), under individually and mixed contaminated soils. Therefore, a set of microcosm experiments were conducted to explore the influence of benzene and BaP on biodegradation under individual and mixed contaminated condition, and their subsequent influence on native microbial consortium. The results revealed that the total mass loss of benzene was 56.0% under benzene and BaP mixed contamination, which was less than that of individual benzene contamination (78.3%). On the other hand, the mass loss of BaP was slightly boosted to 17.6% under the condition of benzene mixed contamination with BaP from that of individual BaP contamination (14.4%). The significant differences between the microbial and biocide treatments for both benzene and BaP removal demonstrated that microbial degradation played a crucial role in the mass loss for both contaminants. In addition, the microbial analyses revealed that the contamination of benzene played a major role in the fluctuations of microbial compositions under co-contaminated conditions. Rhodococcus, Nocardioides, Gailla, and norank_c_Gitt-GS-136 performed a major role in benzene biodegradation under individual and mixed contaminated conditions while Rhodococcus, Noviherbaspirillum, and Phenylobacterium were highly involved in BaP biodegradation. Moreover, binary benzene and BaP contamination highly reduced the Rhodococcus abundance, indicating the toxic influence of co-contamination on the functional key genus. Enzymatic activities revealed that catalase, lipase, and dehydrogenase activities proliferated while polyphenol oxidase was reduced with contamination compared to the control treatment. These results provided the fundamental information to facilitate the development of more efficient bioremediation strategies, which can be tailored to specific remediation of different contamination scenarios.

Prefeeding of Clarias gariepinus with Spirulina platensis counteracts petroleum hydrocarbons-induced hepato- and nephrotoxicity.

Petroleum aromatic hydrocarbons are considered one of the most dangerous aquatic pollutants due to their widespread across water bodies, persistence, and extension to the food chain. To our knowledge, there hasn't been any research investigating the hepatorenoprotective effects of Spirulina platensis (SP) against toxicity induced by these environmental toxicants in fish. Thus, we decided to explore its potential safeguarding against benzene and toluene exposure in adult Clarias gariepinus. To achieve this objective, fish were divided into five groups (60 per group; 20 per replicate). The first group served as a control. The second and third groups were intoxicated with benzene and toluene at doses of 0.762 and 26.614 ng/L, respectively for 15 days. The fourth and fifth groups (SP + benzene and SP + toluene, respectively) were challenged with benzene and toluene as previously mentioned following dietary inclusion of SP at a dose of 5 g/kg diet for 30 days. The marked increase in liver metabolizing enzymes, glucose, total protein, albumin, globulin, albumin/globulin ratio, and creatinine confirmed the hepato- and nephrotoxic impacts of benzene and toluene. These outcomes were coupled with cytopathological affections and excessive collagen deposition. The incorporation of SP in ration formulation, on the contrary, restored the previously mentioned toxicological profile due to its antioxidant and cytoprotective attributes. Regardless of SP intervention, the renal tissues still displayed histo-architectural lesions, because of insufficient dose and timeframe. Additional research will be required to identify the ideal SP remediation regimen.

Production of Biobased Ethylbenzene by Cascade Biocatalysis with an Engineered Photodecarboxylase.

Production of commodity chemicals, such as benzene, toluene, ethylbenzene, and xylenes (BTEX), from renewable resources is key for a sustainable society. Biocatalysis enables one-pot multistep transformation of bioresources under mild conditions, yet it is often limited to biochemicals. Herein, we developed a non-natural three-enzyme cascade for one-pot conversion of biobased l-phenylalanine into ethylbenzene. The key rate-limiting photodecarboxylase was subjected to structure-guided semirational engineering, and a triple mutant CvFAP(Y466T/P460A/G462I) was obtained with a 6.3-fold higher productivity. With this improved photodecarboxylase, an optimized two-cell sequential process was developed to convert l-phenylalanine into ethylbenzene with 82 % conversion. The cascade reaction was integrated with fermentation to achieve the one-pot bioproduction of ethylbenzene from biobased glycerol, demonstrating the potential of cascade biocatalysis plus enzyme engineering for the production of biobased commodity chemicals.

In Situ Discrimination and Cultivation of Active Degraders in Soils by Genome-Directed Cultivation Assisted by SIP-Raman-Activated Cell Sorting.

The identification and in situ cultivation of functional yet uncultivable microorganisms are important to confirm inferences regarding their ecological functions. Here, we developed a new method that couples Raman-activated cell sorting (RACS), stable-isotope probing (SIP), and genome-directed cultivation (GDC)─namely, RACS-SIP-GDC─to identify, sort, and cultivate the active toluene degraders from a complex microbial community in petroleum-contaminated soil. Using SIP, we successfully identified the active toluene degrader Pigmentiphaga, the single cells of which were subsequently sorted and isolated by RACS. We further successfully assembled the genome of Pigmentiphaga based on the metagenomic sequencing of 13C-DNA and genomic sequencing of sorted cells, which was confirmed by gyrB gene comparison and average nucleotide identity determination. Additionally, the genotypes and phenotypes of this degrader were directly linked at the single-cell level, and its complete toluene metabolic pathways in petroleum-contaminated soil were reconstructed. Based on its unique metabolic properties uncovered by genome sequencing, we modified the traditional cultivation medium with antibiotics, amino acids, carbon sources, and growth factors (e.g., vitamins and metals), achieving the successful cultivation of RACS-sorted active degrader Pigmentiphaga sp. Our results implied that RACS-SIP-GDC is a state-of-the-art approach for the precise identification, targeted isolation, and cultivation of functional microbes from complex communities in natural habitats. RACS-SIP-GDC can be used to explore specific and targeted organic-pollution-degrading microorganisms at the single-cell level and provide new insights into their biodegradation mechanisms.

Novel BTEX-degrading strains from subsurface soil: Isolation, identification and growth evaluation.

Monoaromatic hydrocarbons such as benzene, toluene, ethylbenzene, and o, m, and p-xylenes (BTEX) are high-risk pollutants because of their mutagenic and carcinogenic nature. These pollutants are found with elevated levels in groundwater and soil in Canada at several contaminated sites. The intrinsic microbes present in the subsurface have the potential to degrade pollutants by their metabolic pathways and convert them to non-toxic products. However, the low subsurface temperature (5-10 °C) limits their growth and degradation ability. This study examined the feasibility of subsurface heat augmentation using geothermal heating for BTEX bioremediation. Novel potent BTEX-degrading bacterial strains were isolated from soil at 3.0, 42.6, and 73.2 m depths collected from a geothermal borehole during installation and screened using an enrichment technique. The selected strains were identified with Sanger sequencing and phylogenetic tree analysis, revealing that all the strains except Bacillus subtilis are novel with respective to BTEX degradation. The isolates, Microbacterium esteraromaticum and Bacillus infantis showed the highest degradation with 67.98 and 65.2% for benzene, 72.8 and 71.02% for toluene, 77.52 and 76.44% for ethylbenzene, and 74.58 and 74.04% for xylenes respectively. Further, temperature influence at 15 ± 1 °C, 28 ± 1 °C and 40 ± 1 °C was observed, which showed increased growth by two-fold and on average 35-49% more biodegradation at higher temperatures. Results showed that temperature is a positive stimulant for bioremediation, hence geothermal heating could also be a stimulant for in-situ bioremediation.

Efficiency, mechanism, influencing factors, and integrated technology of biodegradation for aromatic compounds by microalgae: A review.

Aromatic compounds have received widespread attention because of their threat to ecosystem and human health. However, traditional physical and chemical methods are criticized due to secondary pollution and high cost. As a result of ecological security and the ability of carbon sequestration, biodegradation approach based on microalgae has emerged as a promising alternative treatment for aromatic pollutants. In light of the current researches, the degradation efficiency of BTEX (benzene, toluene, ethylbenzene, and xylene), polycyclic aromatic hydrocarbons (PAHs), and phenolic compounds by microalgae was reviewed in this study. We summarized the degradation pathways and metabolites of p-xylene, benzo [a]pyrene, fluorene, phenol, bisphenol A, and nonylphenol by microalgae. The influence factors on the degradation of aromatic compounds by microalgae were also discussed. The integrated technologies based on microalgae for degradation of aromatic compounds were reviewed. Finally, this study discussed the limitations and future research needs of the degradation of these compounds by microalgae.

Influence of growth substrate and contaminant mixtures on the degradation of BTEX and MTBE by Rhodococcus rhodochrous ATCC strain 21198.

The degradation of the prevalent environmental contaminants benzene, toluene, ethylbenzene, and xylenes (BTEX) along with a common co-contaminant methyl tert-butyl ether (MTBE) by Rhodococcus rhodochrous ATCC Strain 21198 was investigated. The ability of 21198 to degrade these contaminants individually and in mixtures was evaluated with resting cells grown on isobutane, 1-butanol, and 2-butanol. Growth of 21198 in the presence of BTEX and MTBE was also studied to determine the growth substrate that best supports simultaneous microbial growth and contaminants degradation. Cells grown on isobutane, 1-butanol, and 2-butanol were all capable of degrading the contaminants, with isobutane grown cells exhibiting the most rapid degradation rates and 1-butanol grown cells exhibiting the slowest. However, in conditions where BTEX and MTBE were present during microbial growth, 1-butanol was determined to be an effective substrate for supporting concurrent growth and contaminant degradation. Contaminant degradation was found to be a combination of metabolic and cometabolic processes. Evidence for growth of 21198 on benzene and toluene is presented along with a possible transformation pathway. MTBE was cometabolically transformed to tertiary butyl alcohol, which was also observed to be transformed by 21198. This work demonstrates the possible utility of primary and secondary alcohols to support biodegradation of monoaromatic hydrocarbons and MTBE. Furthermore, the utility of 21198 for bioremediation applications has been expanded to include BTEX and MTBE.

Low-dose blood BTEX are associated with pulmonary function through changes in inflammatory markers among US adults: NHANES 2007-2012.

The effects of blood benzene, toluene, ethylbenzene, and xylenes (BTEX) on lung function among general adults remain unknown. We enrolled 5519 adults with measured blood BTEX concentrations and lung function from the US National Health and Nutrition Examination Survey 2007-2012. Weighted linear models were fitted to assess the associations of BTEX with lung function and inflammation parameters (white blood cell five-part differential count and C-reactive protein). The mediating effect of inflammation between BTEX and lung function was also examined. Blood BTEX concentrations decreased yearly from 1999 and were extremely low from 2007 to 2012. Benzene and toluene exerted the greatest influence on lung function in terms of forced vital capacity (FVC), forced expiratory volume in the first second (FEV1), calculated FEV1:FVC ratio, peak expiratory flow rate (PEFR), and forced mid expiratory flow (FEF25-75%). Both ethylbenzene and all xylene isomers had no effects on FVC but reduced FEV1, FEV1:FVC ratio, PEFR, and FEF25-75%. Weighted quantile analyses demonstrated that BTEX mixture was associated with decreases in FVC, FEV1, FEV1:FVC ratio, PEFR, and FEF25-75%, with benzene weighted most heavily for all lung function parameters. BTEX also increased the levels of inflammation indicated by white blood cell five-part differential count and C-reactive protein, and increased levels of inflammation also reduced lung function. From multiple mediation analysis, inflammation mediated the effects of benzene on FEV1 and PEFR, the effects of toluene on FEV1, and the effects of ethylbenzene on FEV1 and PEFR. Low-dose exposure to BTEX was associated with reduced pulmonary function both in large and small airways. Inflammation could be involved in this pathogenesis.

Construction and function of a high-efficient synthetic bacterial consortium to degrade aromatic VOCs.

Aromatic volatile organic compounds (VOCs) are a type of common pollution form in chemical contaminated sites. In this study, seven aromatic VOCs such as benzene, toluene, ethylbenzene, chlorobenzene, m-xylene, p-chlorotoluene and p-chlorotrifluorotoluene were used as the only carbon source, and four strains of highly efficient degrading bacteria were screened from the soil of chemical contaminated sites, then the synthetic bacterial consortium was constructed after mixing with an existing functional strain (Bacillus benzoevorans) preserved in the laboratory. After that, the synthetic bacterial consortium was used to explore the degradation effect of simulated aromatic VOCs polluted wastewater. The results showed that the functional bacterium could metabolize with aromatic VOCs as the only carbon source and energy. Meanwhile, the growth of the synthetic bacterial consortium increased with the additional carbon resources and the alternative of organic nitrogen source. Ultimately, the applicability of the synthetic bacterial consortium in organic contaminated sites was explored through the study of broad-spectrum activity.

Theoretical Kinetic Isotope Effects in Establishing the Precise Biodegradation Mechanisms of Organic Pollutants.

Compound-specific isotope analysis (CSIA) for natural isotope ratios has been recognized as a promising tool to elucidate biodegradation pathways of organic pollutants by microbial enzymes by relating reported kinetic isotope effects (KIEs) to apparent KIEs (AKIEs) derived from bulk isotope fractionations (εbulk). However, for many environmental reactions, neither are the reference KIE ranges sufficiently narrow nor are the mechanisms elucidated to the point that rate-determining steps have been identified unequivocally. In this work, besides providing reference KIEs and rationalizing AKIEs, good relationships have been explained by DFT computations for diverse biodegradation pathways with known enzymatic models between the theoretical isotope fractionations (εbulk') from intrinsic KIEs on the rate-determining steps and the observed εbulk. (1) To confirm the mechanistic details of previously reported pathway-dependent CSIA, it includes isotope changes in MTBE biodegradation between hydroxylation by CYP450 and SN2 reaction by cobalamin-dependent methyltransferase, the regioselectivity of toluene biodegradation by CYP450, and the rate-determining step in toluene biodegradation by benzylsuccinate synthase. (2) To yield new fundamental insights into some unclear biodegradation pathways, it consists of the oxidative function of toluene dioxygenase in biodegradation of TCE, the epoxidation mode in biodegradation of TCE by toluene 4-monooxygenase, and the weighted average mechanism in biodegradation of cDCE by CYP450.

Engineering the Substrate Specificity of Toluene Degrading Enzyme XylM Using Biosensor XylS and Machine Learning.

Enzyme engineering using machine learning has been developed in recent years. However, to obtain a large amount of data on enzyme activities for training data, it is necessary to develop a high-throughput and accurate method for evaluating enzyme activities. Here, we examined whether a biosensor-based enzyme engineering method can be applied to machine learning. As a model experiment, we aimed to modify the substrate specificity of XylM, a rate-determining enzyme in a multistep oxidation reaction catalyzed by XylMABC in Pseudomonas putida. XylMABC naturally converts toluene and xylene to benzoic acid and toluic acid, respectively. We aimed to engineer XylM to improve its conversion efficiency to a non-native substrate, 2,6-xylenol. Wild-type XylMABC slightly converted 2,6-xylenol to 3-methylsalicylic acid, which is the ligand of the transcriptional regulator XylS in P. putida. By locating a fluorescent protein gene under the control of the Pm promoter to which XylS binds, a XylS-producing Escherichia coli strain showed higher fluorescence intensity in a 3-methylsalicylic acid concentration-dependent manner. We evaluated the 3-methylsalicylic acid productivity of XylM variants using the fluorescence intensity of the sensor strain as an indicator. The obtained data provided the training data for machine learning for the directed evolution of XylM. Two cycles of machine learning-assisted directed evolution resulted in the acquisition of XylM-D140E-V144K-F243L-N244S with 15 times higher productivity than wild-type XylM. These results demonstrate that an indirect enzyme activity evaluation method using biosensors is sufficiently quantitative and high-throughput to be used as training data for machine learning. The findings expand the versatility of machine learning in enzyme engineering.

Gas-phase degradation of VOCs using supported bacteria biofilms.

Herein we report the use of Pseudomonas putida F1 biofilms grown on carbonized cellulosic fibers to achieve biodegradation of airborne volatile organic compounds (VOCs) in the absence of any bulk aqueous-phase media. It is believed that direct exposure of gaseous VOC substrates to biomass may eliminate aqueous-phase mass transfer resistance and facilitate VOC capture and degradation. When tested with toluene vapor as a model VOC, the supported biofilm could grow optimally at 300 p.p.m. toluene and 80% relative humidity, with a specific growth rate of 0.425 day-1 . During long-term VOC biodegradation tests in a tubular packed bed reactor, biofilms achieved a toluene degradation rate of 2.5 mg gDCW -1 h-1 during the initial growth phase. Interestingly, the P. putida F1 film kept biodegrading activity even at the stationary nongrowth phase. The supported biofilms with a biomass loading of 20% (wt) could degrade toluene at a rate of 1.9 mg gDCW -1 h-1 during the stationary phase, releasing CO2 at a rate of 6.4 mg gDCW -1 h-1 at the same time (indicating 100% conversion of substrate carbon to CO2 ). All of these observations promised a new type of "dry" biofilm reactors for efficient degradation of toxic VOCs without involving a large amount of water.

Evaluation of Bacillus subtilis as a Tool for Biodegrading Diesel Oil and Gasoline in Experimentally Contaminated Water and Soil.

Benzene, toluene, ethylbenzene and xylene (BTEX) are toxic petroleum hydrocarbons pollutants that can affect the central nervous system and even cause cancer. For that reason, studies regarding BTEX degradation are extremely important. Our study aimed evaluate the microorganism Bacillus subtilis as a tool for degrading petroleum hydrocarbons pollutants. Assays were run utilizing water or soil distinctly contaminated with gasoline and diesel oil, with and without B. subtilis. The ability of B. subtilis to degrade hydrophobic compounds was analyzed by Fourier-Transform Infrared Spectroscopy (FTIR) and gas chromatography. The FTIR results indicated, for water assays, that B. subtilis utilized the gasoline and diesel oil to produce the biosurfactant, and, as a consequence, performed a biodegradation process. In the same way, for soil assay, B. subtilis biodegraded the diesel oil. The gas chromatography results indicated, for gasoline in soil assay, the B. subtilis removed BTEX. So, B. subtilis was capable of degrading BTEX, producing biosurfactant and it can also be used for other industrial applications. Bioremediation can be an efficient, economical, and versatile alternative for BTEX contamination.

Lead or cadmium co-contamination alters benzene and toluene degrading bacterial communities.

Co-contamination of hydrocarbons with heavy metals in soils often complicates and hinders bioremediation. A comprehensive characterization of site-specific degraders at contaminated sites can help determine if in situ bioremediation processes are sufficient. This study aimed to identify differences in benzene and toluene degradation rates and the microbial communities enriched under aerobic conditions when different concentrations of Cd and Pb are introduced. Microcosms were used to study the degradation of 0.23 mM benzene or 0.19 mM toluene under various concentrations of Pb (up to 240 µM) and Cd (up to 440 µM). Soil collected from a stormwater retention basin receiving runoff from a large parking lot was utilized to seed the microcosms. The hydrocarbon degradation time and rates were measured. After further rounds of amendment and degradation of benzene and toluene, 16S rRNA gene amplicon sequencing and quantitative PCR were used to ascertain the microbial communities enriched under the various concentrations of the heavy metals. The initial degradation time for toluene and benzene was 7 to 9 days and 10 to 13 days, respectively. Degradation rates were similar for each hydrocarbon despite the concentration and presence of metal co-contaminant, however, the enriched microbial communities under each condition differed. Microcosms without metal co-contaminant contained a diversity of putative benzene and toluene degrading bacteria. Cd strongly reduced the richness of the microbial communities. With higher levels of heavy metals, genera such as Ralstonia, Cupriavidus, Azoarcus, and Rhodococcus became more dominant under various conditions. The study finds that highly efficient benzene- and toluene-degrading consortia can develop under variations of heavy metal co-contamination, but the consortia are dependent on the heavy metal type and concentrations.

Quercetin can affect porcine ovarian cell functions and to mitigate some of the effects of the environmental contaminant toluene.

The present in vitro experiments aimed to examine the effects of the plant polyphenol quercetin and the environmental contaminant toluene on basic ovarian cell functions, including the ability of quercetin to be a natural protector against the adverse effects of toluene. The influence of toluene, quercetin, and their combination on proliferation (accumulation of PCNA), apoptosis (accumulation of bax) and release of progesterone, testosterone and insulin-like growth factor I (IGFI) by cultured porcine ovarian granulosa cells was investigated. Toluene stimulated cell proliferation and inhibited progesterone, IGF-I and testosterone release but did not affect apoptosis. Quercetin, when administered alone, inhibited cell proliferation, apoptosis, IGF-I and testosterone release and stimulated progesterone output. When administered in combination with toluene, quercetin mitigated toluene's effects on proliferation and on progesterone release and induced toluene to exhibit a pro-apoptotic effect. These observations demonstrate the direct effects of both quercetin and toluene on basic ovarian functions and a protective effect of quercetin against the effects of toluene. Therefore, quercetin-containing plants could be regulators of porcine reproduction and natural protectors against the adverse effects of the environmental contaminant toluene.

Anaerobic mineralization of toluene by enriched soil-free consortia with solid-phase humin as a terminal electron acceptor.

The anaerobic biodegradation of toluene proceeds very slowly owing to limited electron acceptors in contaminated aquifer. The liquid reagents traditionally used to enhance this process readily migrate away from the contaminated site, and continuous addition would cause secondary pollution. In our previous study, the reduced solid-phase humic substances (humin), which are redox active, were found to act as electron donors to promote the microbial reactions. Here, we provide new evidence that humin can promote the anaerobic biodegradation of toluene as a terminal electron acceptor. When inoculating nitrate-reducing (NR) and iron-reducing (IR) consortia with toluene degradation activities, the average toluene degradation rates reached 21.20 ± 1.18 µmol/(L·d) and 15.43 ± 0.41 µmol/(L·d) in the presence of a sediment humin (HMcj), and 94.69% ± 4.26% and 93.20% ± 3.73% of the electrons released from toluene oxidation to CO2 could be recovered by the reduction of HMcj, respectively. Spectroscopy analyses revealed that quinone moieties and nitrogen-containing moieties may be the electron-accepting groups of HMcj. Based on 16S rRNA sequencing, Cellulomonas spp. were the possible functional bacteria in the culture with NR consortium as the inoculum, while Azospira spp., Cellulomonas spp. and Bacillus spp. were the possible functional bacteria in the culture with IR consortium as the inoculum. Further Phylogenetic Investigation of Communities by Reconstruction of Unobserved States (PICRUSt) analyses indicated that toluene oxidation and extracellular electron transfer functions were more abundant in HMcj amended cultures, suggesting a possible enhancement mechanism by HMcj. Additionally, experiments using natural groundwater illustrated that toluene degradation was highly dependent on its concentration, HMcj dosage, pH, and salinity. The study of a column filled with HMcj-coated quartz sand demonstrated a desirable level of toluene degradation in a continuous-flow mode without the presence of other electron acceptors. This study provided an effective and green approach for the remediation of the toluene-contaminated groundwater.

Mechanisms of biostimulant-enhanced biodegradation of PAHs and BTEX mixed contaminants in soil by native microbial consortium.

Despite the co-occurrence of polycyclic aromatic hydrocarbons (PAHs) and benzene, toluene, ethylbenzene, and xylene (BTEX) in the field, to date, knowledge on the bioremediation of benzene and benzo[a]pyrene (BaP) mixed contaminants is limited. In this study, the mechanisms underlying the biodegradation of benzene and BaP under individual and co-contaminated conditions followed by the enhanced biodegradation using methanol, ethanol, and vegetable oil as biostimulants were investigated. The results demonstrated that the benzene biodegradation was highly reduced under the co-contaminated condition compared to the individual benzene contamination, whereas the BaP biodegradation was slightly enhanced with the co-contamination of benzene. Moreover, biostimulation significantly improved the biodegradation of both contaminants under co-contaminated conditions. A trend of significant reduction in the bioavailable BaP contents was observed in all biostimulant-enhanced groups, implying that the bioavailable BaP was the preferred biodegradable BaP fraction. Furthermore, the enzymatic activity analysis revealed a significant increase in lipase and dehydrogenase (DHA) activities, as well as a reduction in the catalase and polyphenol oxidase, suggesting that the increased hydrolysis of fats and proton transfer, as well as the reduced oxidative stress, contributed to the enhanced benzene and BaP biodegradation in the vegetable oil treatment. In addition, the microbial composition analysis results demonstrated that the enriched functional genera contributed to the increased biodegradation efficiency, and the functional genera in the microbial consortium responded differently to different biostimulants, and competitive growth was observed in the biostimulant-enhanced treatments. In addition, the enrichment of Pseudomonas and Rhodococcus species was noticed during the biostimulation of benzene and BaP co-contamination soil, and was positively correlated with the DHA enzyme activities, indicating that these species encode DHA genes which contributed to the higher biodegradation. In conclusion, multiple lines of evidence were provided to shed light on the mechanisms of biostimulant-enhanced biodegradation of PAHs and BTEX co-contamination with native microbial consortiums.

Fluoro-recognition: New in vivo fluorescent assay for toluene dioxygenase probing induction by and metabolism of polyfluorinated compounds.

The present study examined the regulatory and metabolic response of the aromatic degrader Pseudomonas putida F1 and its tod operon, controlling toluene degradation, to fluorinated aromatic and aliphatic compounds. The tod operon is upregulated by inducer binding to the TodS sensing domain of a two-component regulator. The induced enzymes include toluene dioxygenase that initiates catabolic assimilation of benzenoid hydrocarbons. Toluene dioxygenase was shown to oxidize 6-fluoroindole to a meta-stable fluorescent product, 6-fluoroindoxyl. The fluorescent output allowed monitoring relative levels of tod operon induction in whole cells using microtiter well plates. Mono- and polyfluorinated aromatic compounds were shown to induce toluene dioxygenase, in some cases to a greater extent than compounds serving as growth substrates. Compounds that are oxidized by toluene dioxygenase and undergoing defluorination were shown to induce their own metabolism. 1,2,4-Trifluorobenzene caused significant induction and computational modelling indicated productive binding to the TodS sensor domain of the TodST regulator. Toluene dioxygenase also showed preferential binding of 1,2,4-trifluorobenzene such that defluorination was favoured. Fluorinated aliphatic compounds were shown to induce toluene dioxygenase. An aliphatic ether with seven fluorine atoms, 1,1,1,2-tetrafluoro-2-trifluoromethoxy-4-iodobutane (TTIB), was an excellent inducer of toluene dioxygenase activity and shown to undergo transformation in cultures of P. putida F1.

Insights into the syntrophic microbial electrochemical oxidation of toluene: a combined chemical, electrochemical, taxonomical, functional gene-based, and metaproteomic approach.

Biodegradation of aromatic hydrocarbons in anoxic contaminated environments is typically limited by the lack of bioavailable electron acceptors. Microbial electrochemical technologies (METs) are able to provide a virtually inexhaustible electron acceptor in the form of a solid electrode. Recently, we provided first experimental evidence for the syntrophic degradation of toluene in a continuous-flow bioelectrochemical reactor known as the "bioelectric well". Herein, we further analyzed the structure and function of the electroactive toluene-degrading microbiome using a suite of chemical, electrochemical, phylogenetic, proteomic, and functional gene-based analyses. The bioelectric well removed 83 ± 7 % of the toluene from the influent with a coulombic efficiency of 84 %. Cyclic voltammetry allowed to identify the formal potentials of four putative electron transfer sites, which ranged from -0.2 V to +0.1 V vs. SHE, consistent with outer membrane c-type cytochromes and pili of electroactive Geobacter species. The biofilm colonizing the surface of the anode was indeed highly enriched in Geobacter species. On the other hand, the planktonic communities thriving in the bulk of the reactor harbored aromatic hydrocarbons degraders and fermentative propionate-producing microorganisms, as revealed by phylogenetic and proteomic analyses. Most likely, propionate, acetate or other VFAs produced in the bulk liquid from the degradation of toluene were utilized as substrates by the electroactive biofilm. Interestingly, key-functional genes related to the degradation of toluene were found both in the biofilm and in the planktonic communities. Taken as a whole, the herein reported results highlight the importance of applying a comprehensive suite of techniques to unravel the complex cooperative metabolisms occurring in METs.

Toxicity Assessment of an Anti-Cancer Drug of p-Toluene Sulfonamide in Zebrafish Larvae Based on Cardiovascular and Locomotion Activities.

p-Toluene sulfonamide (p-TSA), a small molecular drug with antineoplastic activity is widely gaining interest from researchers because of its pharmacological activities. In this study, we explored the potential cardio and neural toxicity of p-TSA in sublethal concentrations by using zebrafish as an in vivo animal model. Based on the acute toxicity assay, the 96hr LC50 was estimated as 204.3 ppm, suggesting the overall toxicity of p-TSA is relatively low in zebrafish larvae. For the cardiotoxicity test, we found that p-TSA caused only a minor alteration in treated larvae after no overall significant alterations were observed in cardiac rhythm and cardiac physiology parameters, as supported by the results from expression level measurements of several cardiac development marker genes. On the other hand, we found that acute p-TSA exposure significantly increased the larval locomotion activity during the photomotor test while prolonged exposure (4 days) reduced the locomotor startle reflex activities in zebrafish. In addition, a higher respiratory rate and blood flow velocity was also observed in the acutely treated fish groups compared to the untreated group. Finally, by molecular docking, we found that p-TSA has a moderate binding affinity to skeletal muscle myosin II subfragment 1 (S1), ATPase activity, actin- and Ca2+-stimulated myosin S1 ATPase, and v-type proton ATPase. These binding interactions between p-TSA and proteins offer insights into the potential molecular mechanism of action of p-TSA on observed altered responses toward photo and vibration stimuli and minor altered vascular performance in the zebrafish larvae.