Benzo(k)fluoranthene and benzo(b)fluoranthene degradation by Selenastrum capricornutum and identification of metabolites using HPLC-FD and HPLC-ESI-QqQ-MS/MS.

Selenastrum capricornutum efficiently degrades high molecular weight polycyclic aromatic hydrocarbons (HMW PAHs). Until now, there are few studies on the benzo(k)fluoranthene (BkF) and benzo(b)fluoranthene (BbF) biodegradation by this microalga. For this reason, in the present work, extracts obtained from cultures of S. capricornutum were incubated with BkF and BbF individually, and analyzed by HPLC with fluorescence and different mass spectrometry detection modes: i) the HPLC-ESI(+)-MS/MS (MRM mode) analysis that confirmed the formation of monohydroxylated and dihydrodiol metabolites indicating that these PAHs could be simultaneously degraded through the monooxygenase and dioxygenase; ii) HPLC-ESI(+)-MS (full scan mode) that showed the formation of key metabolites containing four and two aromatic rings possibly resulting from aromatic ring-opening oxygenases, not known until now in microalgae; iii) HPLC-FD analysis that confirmed the individual BkF and BbF degradation occurring in extra- and intra-cellular extracts, indicating that an oxygenase enzyme complex is released by microalgae cells to the external environment to perform HMW PAHs biodegradation. So, this work presents new insights into the metabolic pathways of BkF and BbF biodegradation by S. capricornutum; likewise, the intra- and extra-cellular extracts of this microalgae have great potential to be applied in environmental procedures.

Electrophoretic characterization of cellular and extracellular proteins from Selenastrum capricornutum cultures degrading benzo(a)pyrene and their identification by UPLC-ESI-TOF mass spectrometry.

Selenastrum capricornutum efficiently degrades benzo(a)pyrene (BaP) but few proteins related to BaP degradation have been identified in this microalgae. So far, it has only been suggested that it could degrade BaP via the monooxygenase and/or dioxygenase pathways. To know more about this fact, in this work, cultures of S. capricornutum incubated with BaP were used to obtain the molecular weights (MWs) of proteins existing in its extra- and cellular extracts by electrophoresis and UPLC-ESI(+)-TOF MS analysis. The results of this proteomic approach indicated that BaP markedly induces the MWs: 6-20, 30, 45, and 65 kDa in cells; 6-20, 30.3, 38-45, and 55 kDa in liquid medium. So, these proteins could be related to BaP biodegradation. An identified protein with monooxygenase activity and rubredoxins (Rds) show to be related to BaP degradation: Rds could participate, together with the monooxygenase in the electron transfer during the formation of monohydroxylated-BaP metabolites. Rds may be also associated with a dioxygenase system that degrades BaP to form dihydrodiol-BaP metabolites. A multi-pass membrane protein was identified too, and it can regulate the transport of molecules like enzymes from inside the cell to the outside environment. At the same time, the presence of a dihydrolipoamide acetyltransferase validated the stress caused by the exposure to BaP. It is noteworthy that these findings provide valuable and original information on the characterization of the proteins of S. capricornutum cultures degrading BaP, whose enzymes have so far not been known. It is important to highlight that the functions of the identified proteins can help in understanding the metabolic and environmental behavior of this microalgae, and the extracts containing the degrading enzymes could be utilized in bioremediation applications.

A review on the enzymes and metabolites identified by mass spectrometry from bacteria and microalgae involved in the degradation of high molecular weight PAHs.

High molecular weight PAHs (HMW PAHs) are dangerous pollutants widely distributed in the environment. The use of microorganisms represents an important tool for HMW PAHs bioremediation, so, the understanding of their biochemical pathways facilitates the development of biodegradation strategies. For this reason, the potential role of species of microalgae, bacteria, and microalga-bacteria consortia in the degradation of HMW PAHs is discussed. The identification of their metabolites, mostly by GC-MS and LC-MS, allows a better approach to the enzymes involved in the key steps of the metabolic pathways of HMW PAHs biodegradation. So, this review intends to address the proteomic research on enzyme activities and their involvement in regulating essential biochemical functions that help bacteria and microalgae in the biodegradation processes of HMW PAHs. It is noteworthy that, given that to the best of our knowledge, this is the first review focused on the mass spectrometry identification of the HMW PAHs metabolites; whereby and due to the great concern of the presence of HMW PAHs in the environment, this material could help the urgency of developing new bioremediation methods. The elucidation of the metabolic pathways of persistent pollutant degrading microorganisms should lead to a better knowledge of the enzymes involved, which could contribute to a very ecological route to the control of environmental contamination in the future.

On-line SPE chromatography with spectrophotometric diode array detection as a simple and advantageous choice for the selective trace analysis of benzo(a)anthracene degradation products from microalgae.

In this work, a methodology based on on-line solid phase extraction (SPE) chromatography with spectrophotometric diode array detection was optimized and validated for the trace analysis of benzo(a)anthracene dihydrodiol degradation products from microalgae cultures 5,6-dihydrodiol, 8,9-dihydrodiol and 10,11-dihydrodiol. The two on-line methods for the constituents of the culture, an SPE/on-line SPE chromatographic method for liquid medium and a matrix solid phase dispersion (MSPD)/on-line SPE chromatographic method for biomass presented good linearity in the ranges of 0.5-47ngmL-1 and 2-80ngmg-1 of samples, respectively, with correlation coefficients r>0.99. The percent relative standard deviation (RSD%) values were ≤4.9%. For the liquid medium and biomass methods, the global recoveries were between 84% and 90% and between 67% and 78%, and the limit of detection LODs were ≤0.3ngmL-1 and ≤0.8ngmg-1 respectively. The methodology was applied to exposure bioassays, and for the first time the three metabolites were detected and quantified individually by their appearance in the biomass and when they were excreted into the liquid medium. The metabolite formed in the greatest amount was 10,11-dihydrodiol, and the maximum production of all metabolites was at 6h of exposure. This work contributes to the study of the degradation route of BaA, which has not been elucidated for microalgae until now.

Monitoring dihydrodiol polyaromatic hydrocarbon metabolites produced by the freshwater microalgae Selenastrum capricornutum.

We found that microalgae exposed to a mixture of polycyclic aromatic hydrocarbons (PAHs) did not show growth inhibition. Thus, we assumed that they could metabolize these compounds. In this study, the dihydrodiol-type PAH metabolites of benzo(a)pyrene (BaP), benzo(a)anthracene (BaA), benzo(b)fluoranthene (BbF) and benzo(k)fluoranthene (BkF) produced by the freshwater microalgae Selenastrum capricornutum were monitored and quantified using high-performance liquid chromatography with fluorescence detection (HPLC-FD) techniques. Exposure bioassays with S. capricornutum were performed using a 266 ng mL(-1) mixture of PAHs at different exposure times (0.75, 1, 3, 8, 16, 24 and 48 h) under controlled temperature (25 °C); the dihydrodiol metabolites formed in the liquid medium and the biomass were quantified. Metabolite identities were confirmed using HPLC-mass spectrometry; most of the metabolites formed were derived from BaA degradation. At 48 h after exposure 5,6-dBaA and 8,9-dBaA/10,11-dBaA were present in the liquid medium at 20% and 67% of the initial mass of BaA, respectively. Three metabolites of BaP were monitored in the liquid medium and biomass and, at 24 h, 4,5-dBaP accounted for 19%; , 7,8-dBaP, 5%; and 9,10-dBaP, 5% relative to the initial BaP mass. Microalgae exposed to BbF showed the presence of 1,2-dBbF and 9,10-dBbF (at 0.3% and 0.1% of the initial BbF mass, respectively) and those exposed to BkF produced 8,9-dBkF (6.5% of the initial BkF mass) in the liquid medium. Seven unknown compounds were formed after exposure; two compounds were identified as the metabolites of BaA and BaP. The results could facilitate the elucidation of the controversial biodegradation mechanism in microalgae.

Biodegradation of benzo(a)pyrene by two freshwater microalgae Selenastrum capricornutum and Scenedesmus acutus: a comparative study useful for bioremediation.

A comparative evaluation of the removal of benzo(a)pyrene (BaP) by sorption and degradation by two microalgal species, Selenastrum capricornutum and Scenedesmus acutus was performed. The monitoring of the amount of BaP remaining in the liquid culture media and the biomass along with the appearance of three metabolites (4,5 dihydrodiol-BaP; 7,8-dihydrodiol-BaP; and 9,10 dihydrodiol-BaP) at short time periods (from 0.25 to 72 h) in cultures exposed to BaP was made by high-performance liquid chromatography (HPLC) with fluorescence and UV detection. Complete removal of BaP was achieved by the two live microalgal species: S. capricornutum at 15 h of exposure (99%) and S. acutus at 72 h of exposure (95%). Sorption is an important phenomenon for BaP removal by S. capricornutum but biodegradation is the principal means of removing BaP in live cells. The formation of metabolites by S. capricornutum is rapid and seems to be proportional to the amount of the BaP added to cultures. In contrast, in these bioassays, most of the BaP removal of S. acutus is due to sorption rather than degradation. The appearance of metabolites in the cultures is very slow and at a low amount compared to cultures of S. capricornutum. The similarities and differences existing between the two microalgae are important for the establishment of the conditions for bioremediation.

Extraction and analysis of polycyclic aromatic hydrocarbons and benzo[a]pyrene metabolites in microalgae cultures by off-line/on-line methodology based on matrix solid-phase dispersion, solid-phase extraction and high-performance liquid chromatography.

This paper describes the development and validation of an analytical methodology to determine the presence of four PAHs: benzo[a]anthracene, benzo[b]fluoranthene, benzo[k]fluoranthene and benzo[a]pyrene in cultures of the green microalgae Selenastrum capricornutum. The metabolites of benzo[a]pyrene (BaP), 4,5-dihydrodiol benzo[a]pyrene, 9,10-dihydrodiol benzo[a]pyrene, 3-hydroxy benzo[a]pyrene and 9-hydroxy benzo[a]pyrene were also included. The methodology consisted of three parts: (1) separation of liquid media from biomass samples by centrifugation of pure cultures, (2) off-line extraction of analytes from biomass by a miniaturized matrix solid phase dispersion (MSPD) method and from liquid media by a solid phase extraction (SPE) method and (3) on-line SPE preconcentration and analysis of the MSPD and SPE extracts, separately, by high performance liquid chromatography with fluorescence detection (HPLC-FD). The off-line/on-line (MSPD/SPE-HPLC-FD) method was validated over a concentration range of 20-200 pg mg(-1) obtaining good linearity (r(2)>0.9912) and precision values measured as relative standard deviation (RSD)<5%, recovery values were in the range of (40-66%) and the limits of detection (LODs) ranged from 2 to 6.5 pg mg(-1). The off-line/on-line (SPE/SPE-HPLC-FD) method was validated over a concentration range of 5-120 pg mL(-1); r(2)>0.9913 and RSD<7.36%, recovery values were in the range of 38-74% and LODs ranged from 0.8 to 2.3 pg mL(-1). This methodology was applied to samples from cultures exposed to BaP at 5 ng mL(-1) with different exposure times (0.75, 1.5, 3, 6, 24 and 48 h). The analytical methodology was suitable for measuring the very low amounts of residual BaP and metabolites produced in bioassays. Results showed that some of the metabolites favored by the microalgae are the dihydrodiols. The microalgae cultures were able to decrease the BaP level in the liquid medium below the United States Environmental Protection Agency (USEPA) limit (<0.2 ng mL(-1)).

A method for the analysis of organophosphorus pesticide residues in Mexican axolotl.

A method based on matrix solid-phase dispersion (MSPD) was developed for quantitative extraction of three organophosphorus pesticides (OPPs) from the Mexican axolotl, Ambystoma mexicanum. The determination was carried out using high- performance liquid chromatography (HPLC) with diode array spectrophotometric UV detection (DAD). The MSPD extraction with octadecylsilyl (C18) sorbent combined with a silica gel clean-up and acetonitrile elution was optimised for chlorpyrifos, fenthion and methyl parathion. The method was validated, yielding recovery values higher than 90%. The precision, expressed as the relative standard deviation (RSD), was less than or equal to 6% in muscle samples at spiking levels of 10 and 5 ppm. Linearity was studied from 15 to 60 ppm for chlorpyrifos and fenthion, and from 7.5 to 30 ppm for methyl parathion. The limits of detection (LODs) were found to be less than or equal to 0.5 ppm. This method was applied to the analysis of samples from a chlorpyrifos-exposed axolotl, demonstrating its use as an analytical tool for toxicological studies.

Binding characteristics of bovine serum albumin encapsulated in sol-gel glasses: an alternative for protein interaction studies.

Silica glasses doped with 500-700 microg of bovine serum albumin were prepared by the sol-gel method; two pH conditions (pH 5 and 7) were assayed for protein encapsulation. Both biomaterials showed a highly porous structure, with pore sizes in the range 5-28 nm. Columns packed with the ground biogels were on-line coupled to a C18 HPLC column for evaluation of the entrapped protein binding properties using propranolol. Binding capacities (at saturation) were approximately 3.7 and 7.1 microg of propranolol (drug-protein molar ratios 1.4 and 2.7) for the biogels prepared at pH 5 and 7, respectively. The significant difference indicates increased albumin denaturation upon encapsulation at pH 5. A frontal analysis study was then performed in cartridges packed with biogel prepared at pH 7 to evaluate the protein interaction with naproxen at low concentrations (