Determination of Total Sulfur Content in Fuels: A Comprehensive and Metrological Review Focusing on Compliance Assessment.

Sulfur-containing compounds are naturally found in crude oil, and they can be partially removed during the refining process. The wide use of fossil fuels has a significant contribution to sulfur emissions into the atmosphere, and Governments are striving to reduce the amount of the fuels by environmental regulations. The reduction of sulfur levels in diesel and other transportation fuels is beneficial from economic and environmental points, but meeting this standard represents a major operational and economic challenge for the oil and gas industry. Quantitative measurement of the sulfur amount must be taken along the oil refining chains guided by standards of measurement and recommended analytical methods such as various American Society for Testing and Materials methods (ASTM D2622, ASTM D5453, ASTM D7039, and ASTM D7220). Advancement in the refining processes and environmental regulations also require reliable measurements and well-defined criteria for compliance assessment. This work presented a brief review of the ASTM Standards used in the laboratories of the Brazilian oil and gas industry to determine the total sulfur content in fuels. We also presented an approach based on the reproducibility of the measurement methods and the guard band concept to evaluate the conformity statement.

Determination of Cu, Ni, Mn and Zn in diesel oil samples using energy dispersive X-ray fluorescence spectrometry after solid phase extraction using sisal fiber.

As a natural adsorbent, sisal (agave sisalana) fibers were used to extract Cu, Ni, Mn, and Zn from diesel oil samples for posterior determination (i.e., direct analytical measurements on the solid support) of the analytes by energy dispersive X-ray fluorescence spectrometry (EDXRF). In the proposed procedure, 0.2 g of sisal fiber was directly added to 5.0 mL of diesel oil contained in a glass tube. After 5 min of contact time, the mixture was filtered, and the collected fibers were oven-dried for 30 min at 70 °C. After drying, the analytes were quantified directly by EDXRF using the sisal fibers as a solid support. The calibration curves showed linear concentration ranges of 0.09-1.00, 0.12-1.00, 0.09-1.00, 0.06-1.0 µg g-1 for Cu, Ni, Mn, and Zn, respectively. The limits of detection (LOD) for Cu, Ni, Mn, and Zn were 0.03, 0.04, 0.03, and 0.02 µg g-1, respectively. The repeatability, evaluated by performing ten measurements at a concentration of 0.50 µg g-1 for each metal, with the results expressed in terms of the relative standard deviation (RSD), was 3.2, 6.5, 6.8, and 6.1% for Cu, Ni, Mn, and Zn, respectively. The results obtained by the proposed method were compared with the results obtained by a comparative method using inductively coupled plasma optical emission spectrometry, and both results showed good agreement. The proposed method was applied for Ni, Cu, Mn, and Zn determination in diesel oil samples collected from different gas stations.

In-depth investigation of Sodium percarbonate as oxidant of PAHs from soil contaminated with diesel oil.

Sodium percarbonate (SPC, 2Na2CO3∙3H2O2), is a compound that can be used under multiple environmental applications. In this work, SPC was employed as oxidant in the treatment of soil contaminated with diesel oil. The soil samples were collected during the earthmoving stage of RNEST Oil Refinery (Petrobras), Brazil. Then, the samples were air-dried, mixed and characterized. Subsequently, raw soil was contaminated with diesel and treated by photo-Fenton reaction (H2O2/Fe2+/UV). SPC played a significant role in the generation of hydroxyl radicals under the catalytic effect of ferrous ions (Fe2+), hydrogen peroxide (H2O2) and radiation. These radicals provoked the photodegradation of polycyclic aromatic hydrocarbons (PAHs), in the soil remediation. A factorial design 33 was carried out to assess the variables which most influenced the decrease in total organic carbon (TOC). The study was performed with the following variables: initial concentration of [H2O2] and [Fe2+], between 190.0 and 950.0 mmol L-1 and 0.0-14.4 mmol L-1, respectively. UV radiation was supplied from sunlight, blacklight lamps, and system without radiation. All experiments were performed with 5.0 g of contaminated soil in 50.0 mL of solution. The initial concentration of Fe2+ showed the statistically most significant effect. The oxidation efficiency evaluated in the best condition showed a decrease from 34,765 mg kg-1 to 15,801 mg kg-1 in TOC and from 85.750 mg kg-1 to 20.770 mg kg-1 in PAHs content. Moreover, the sums of low and high molecular weight polycyclic aromatic hydrocarbons (LMW-PAHs and HMW-PAHs) were 19.537 mg kg-1 and 1.233 mg kg-1, respectively. Both values are within the limits recommended by the United Sates Environmental Protection Agency (USEPA) and evidenced the satisfactory removal of PAHs from contaminated soil, being an alternative to classic oxidation protocols.

Determination of Cu, Ni, Mn, and Pb in diesel oil samples using reversed-phase vortex-assisted liquid-liquid microextraction associated with energy dispersive X-ray fluorescence spectrometry.

A method was developed based on reversed-phase vortex-assisted liquid-liquid microextraction (RP-VALLME) combined with energy dispersive X-ray fluorescence (EDXRF) spectrometry for the determination of Cu, Mn, Ni, and Pb in diesel oil samples. In this procedure, a nitric acid solution was used as the extraction phase to isolate analytes from organic samples. After a centrifugation step, the aqueous phase was added dropwise to a filter paper disc for EDXRF determinations. The following variables were optimized: type of extraction phase solution, concentration of the extraction phase, stirring time, and sample volume. Some instrumental parameters were also evaluated: atmospheric condition, irradiation energy, and irradiation time. Using 100 µL of a 0.075 mol L-1 nitric acid solution as the extraction phase for a sample volume of 5.0 mL and a stirring time of 45 s, the limits of detection were 14, 8, 10, and 7 µg L-1 for Cu, Mn, Ni, and Pb, respectively. The enrichment factors obtained were 34 (Cu), 62 (Mn), 59 (Ni), and 64 (Pb). The precisions, expressed as relative standard deviations (RSDs, %), were calculated from ten replications of the experiment under optimized conditions using standard solutions containing 200 µg L-1 and 400 µg L-1 of all four analytes and ranged between 2.1 and 6.4%. The results of recovery tests ranged from 87 to 112%. The proposed procedure was efficiently applied to the determination of the four analytes in diesel oil samples. The results were compared with those obtained by inductively coupled plasma optical emission spectrometry (ICP-OES) after sample digestion, and no significant differences were found.

Conversion of renewable substrates for biosurfactant production by Rhizopus arrhizus UCP 1607 and enhancing the removal of diesel oil from marine soil

BACKGROUND: The use of agro-industrial wastes to produce high value-added biomolecules such as biosurfactants is a promising approach for lowering the total costs of production. This study aimed to produce biosurfactants using Rhizopus arrhizus UCP 1607, with crude glycerol (CG) and corn steep liquor (CSL) as substrates. In addition, the biomolecule was characterized, and its efficiency in removing petroderivatives from marine soil was investigated. RESULTS: A 22 factorial design was applied, and the best condition for producing the biosurfactant was determined in assay 4 (3% CG and 5% CSL). The biosurfactant reduced the surface tension of water from 72 to 28.8 mN/m and produced a yield of 1.74 g/L. The preliminary biochemical characterization showed that the biosurfactant consisted of proteins (38.0%), carbohydrates (35.4%), and lipids (5.5%). The compounds presented an anionic character, nontoxicity, and great stability for all conditions tested. The biomolecule displayed great ability in dispersing hydrophobic substrates in water, thereby resulting in 53.4 cm2 ODA. The best efficiency of the biosurfactant in removing the pollutant diesel oil from marine soil was 79.4%. CONCLUSIONS: This study demonstrated the ability of R. arrhizus UCP1607 to produce a low-cost biosurfactant characterized as a glycoprotein and its potential use in the bioremediation of the hydrophobic diesel oil pollutant in marine soil

Burning rate, flame geometry and temperature of convection-controlled circular diesel oil pool fire under air crossflow conditions.

Hazardous fires in fuel storage tanks may result in casualties of great impact. Efforts are being done to master the physics of the phenomena, aiming both avoidance and damage minimization. Flame characterization is an important approach studied by many researchers. This article presents the results of an experimental investigation of diesel oil (S500 type) pool fires under air crossflow conditions. A reduced scale model properly instrumented was tested in an aerodynamic channel. Measurement techniques include analysis of infrared images of pool fires, mass burning rates and flame geometry as a function of air crossflow velocity (which ranged from 0 m/s - quiescent air - to 4.0 m/s). Mass burning rates showed an oscillatory behavior as the crossflow velocity was raised. Regarding flame geometry, for an increasing air crossflow, firstly flame tilt angle increased, flame length decreased and flame height decreased until about 1.5-2.0 m/s, while after that velocity, flame geometry became nearly steady. Geometry results were also compared with thirteen correlations from literature for geometric parameters obtaining good agreement for some of them. The IR-measured temperatures showed that the flame region (reacting region) became hotter and the plume region (non-reacting region) became colder as the air crossflow velocity was increased.

Proteomic response of gill microsomes of Crassostrea brasiliana exposed to diesel fuel water-accommodated fraction.

Diesel fuel water-accommodated fraction (diesel-WAF) is a complex mixture of organic compounds that may cause harmful effects to marine invertebrates. Expression of microsomal proteins can be changed by oil exposure, causing functional alterations in endoplasmic reticulum (ER). The aim of this study was to investigate changes in protein expression signatures in microsomes of oysterl Crassostrea brasiliana (=C.gasar) gill after exposure to 10% diesel-WAF for 24 and 72 h. Protein expression signatures of gills of oysters exposed to diesel-WAF were compared to those of unexposed oysters using two-dimensional electrophoresis (2-DE) to identify differentially expressed proteins. A total of 458 protein spots with molecular weights between 30-75 kDa were detected by 2-DE in six replicates of exposed oyster proteomes compared to unexposed ones. Fourteen differentially expressed proteins (six up-regulated and eight down-regulated) were identified. They are: proteins related to xenobiotic biotransformation (cytochrome P450 6 A, NADPH-cytochrome P450 reductase); cytoskeleton (α-tubulin, ß-tubulin, gelsolin); processing and degradation of proteins pathways (thioredoxin domain-containing protein E3 ubiquitin-protein ligase MIB2); involved in the biosynthesis of glycolipids and glycoproteins (beta-1,3-galactosyltransferase 1); associated with stress responses (glutamate receptor 4 and 14-3-3 protein zeta, corticotropin-releasing factor-binding protein); plasmalogen biosynthesis (fatty acyl-CoA reductase 1), and sodium-and chloride-dependent glycine transporter 2 and glyoxylate reductase/hydroxypyruvate reductase. Different patterns of protein responses were observed between 24 and 72 h-exposed groups. Expression pattern of microsomal proteins provided a first insight on the potential diesel-WAF effects at protein level in microsomal fraction of oyster gills and indicated new potential biomarkers of exposure and effect. The present work can be a basis for future ecotoxicological studies in oysters aiming to elucidate the molecular mechanisms behind diesel-WAF toxicity and for environmental monitoring programs.

Effect of tank diameter on thermal behavior of gasoline and diesel storage tanks fires.

Studies on fire behavior are extremely important as they contribute in a firefighting situation or even to avoid such hazard. Experimental studies of fire in real scale are unfeasible, implying that reduced-scale experiments must be performed, and results extrapolated to the range of interest. This research aims to experimentally study the fire behavior in tanks of 0.04m, 0.20m, 0.40m, 0.80m and 4.28m diameter, burning regular gasoline or diesel oil S-500. The following parameters were here obtained: burning rates, burning velocities, heat release rates, flame heights, and temperature distributions adjacent to the tank. Such parameters were obtained for each tank diameter with the purpose of correlating the results and understanding the relationship of each parameter for the different geometrical scale of the tanks. Asymptotic results for larger tanks were found as (regular gasoline and diesel oil S-500, respectively): burning rates 0.050kg/(m2s) and 0.031kg/(m2s), burning velocities 4.0mm/min and 2.5mm/min, heat release rates per unit area 2200kW/m2 and 1500kW/m2, normalized averaged flame heights (Hi/D, where Hi is the average flame height, D is the tank diameter) 0.9 and 0.8. Maximum temperatures for gasoline pools were higher than for diesel oil pools, and temperature gradients close to the tanks were also higher for the former fuel. The behavior of the maximum temperature was correlated as a function of the tank diameter, the heat release rate of each fuel and the dimensionless distance from the tank.

Gladius and its derivatives as potential biosorbents for marine diesel oil.

The demand for low cost and effective materials to remove contaminants such as residues of oil spills has encouraged studies on new biosorbents produced from wastes. Considering the overgeneration of fishing residues and the necessity to provide an alternative purpose for such materials, this study aimed to evaluate squid gladius and its derivatives (ß-chitin and chitosan) as sorbents to remove marine diesel oil (MDO) from fresh and artificial seawater. It was also executed an attempted to improve their performances through a high-intensity ultrasound treatment (UT-gladius and UT-ß-chitin). All sorbents removed MDO at both salinities. Contact surface area, salinity, and water retention seemed to play a key role in the outcomes. UT-ß-chitin's performance was significantly superior to ß-chitin's and chitosan's in MDO removal at salinity 0, as well as at salinity 30, where gladius and UT-gladius also excelled. Ultrasound treatment improved the oil removal performance of UT-ß-chitin by increasing its contact surface area. This is the first report on the efficiency of gladius and UT-ß-chitin for such purpose, and brought up huge possibilities and new questions that can lead to the achievement of biosorbents of great efficiency.

Effects of an in situ diesel oil spill on oxidative stress in the clam Anomalocardia flexuosa.

Intensive exploitation and transport of oil and derivatives are increasing the risk of coastal contamination by either dramatic disasters or diffuse sources. Tools for monitoring diffuse contamination, such as diesel oil that leaks from marine vessels are much needed. We experimentally tested the efficiency of antioxidant biomarkers as indicators of chronic exposure to diesel oil in a mudflat from the subtropical Bay of Paranaguá, in southern Brazil. We examined the effects of three successive diesel oil spills, with two weeks of recovery time between exposures, on the edible clam Anomalocardia flexuosa. Previous studies have highlighted its potential as a bioindicator species for diesel oil contamination in subtropical and tropical ecosystems. Endpoints measured in gill and digestive gland homogenates included the activity of antioxidant enzymes SOD, GPx, GST and levels of lipid peroxides. PAHs concentration in sediments and soft tissue were also quantified. GST and SOD were the most responsive biomarkers to the exposure. There were significant but non-cumulative departures from control levels in organisms from treated samples, which were, in all cases, more common 48 h after each experimental spill. Biomarker responses were more evident in the digestive gland than in gills. This work validated the short-term responsiveness of biomarkers as measures of repeated pulsed in situ exposure to low concentrations of diesel oil. For their routine implementation into monitoring programs for tropical estuaries our general recommendations are 1) to include several reference sites, 2) to analyze biomarker data using a logarithmic-scale and 3) to interpret deviations from "normal" activity as multiplicative interval differences.

Biosurfactants produced by Microbacterium sp, isolated from aquatic macrophytes in hydrocarbon-contaminated area in the Rio Negro, Manaus, Amazonas / Biossurfactantes produzidos por Microbacterium sp, isolada de macrófita aquática em área impactada por hidrocarbonetos no rio Negro, Manaus, Amazonas

Endophytic bacteria isolated from Eichhornia crassipes (Mart) Solms., collected in oil contaminated wastewater of effluent generated by Petrobras refinery in Manaus were investigated to determine their potential for producing biosurfactants. Assay with 2.6-dichlorophenol indophenol (DCPIP) indicator to verify hydrocarbon biodegradation activity; oil emulsification test; drop-collapse method; surface tension and growth curve of biosurfactant production. The M87 Microbacterium sp. strain chosen for this work was identified by the sequencing of the rDNA region and the chemical characterization was performed by FTIR, UFLC/MS and 1H RMN techniques. The selected bacterial isolate provided 3g L-1 of biosurfactant, using diesel oil as sole carbon source, being efficient in biodegrading oil as demonstrated by the DCPIP test. Fractions obtained by column chromatography were efficient in reducing water surface tension around 40 mN m-1, especially fraction 1, which reduced it to 34.17 mN m-1. The different techniques of chemical analysis used for the identification of the biosurfactant isolate indicated that this is probably a long - chain fatty acid lipid type, which may be used in the future as both biosurfactant in decontamination processes of hydrocarbon-polluted areas or as bioemulsifier in countless processes, since it exhibited no toxicity as determined by Alamar Blue assay.

Foram investigadas bactérias endofíticas isoladas de Eichhornia crassipes (Mart.) Solms., coletadas em águas contaminadas com resíduos de petróleo em um afluente da refinaria da Petrobrás/Manaus, para avaliação da produção de biossurfactantes. Para selecionar o micro-organismos à produção e caracterização de biossurfactantes, foram realizados os seguintes testes: a descoloração do indicador 2,6 indofenol (DCPIP), emulsificação do diesel, colapso da gota, tensão superficial e curva de produção. A caracterização química foi realizada por meio das técnicas de FT-IR, UFLC/MS e RMN1H. A bactéria M87 Microbacterium neste estudo, foi identificada pelo sequenciamento da região rDNA e produziu 3g L-1 de biossurfactantes utilizando o diesel como fonte de carbono, mostrando-se eficiente na ação biodegradadora do petróleo, por meio do teste de Indofenol (DCPIP). As frações obtidas, mostraram-se eficazes na redução da tensão superficial da água abaixo de 40 mN m-1, com destaque para a fração 1 que reduziu a tensão superficial para 34,17 mN m-1. Pelas análises química utilizadas, pode-se inferir que, provavelmente, se trata de um ácido graxo de cadeia longa, que pode ser utilizado futuramente tanto como biossurfactante em processos de descontaminação de ambientes impactados por hidrocarbonetos, assim como bioemulsificante em inúmeros processos uma vez que não apresentou toxicidade por meio do teste realizado.

Biosurfactants produced by Microbacterium sp., isolated from aquatic macrophytes in hydrocarbon-contaminated area in the Rio Negro, Manaus, Amazonas / Biossurfactantes produzidos por Microbacterium sp., isolada de macrófita aquática em áreaimpactada por hidrocarbonetos no rio Negro, Manaus, Amazonas

Endophytic bacteria isolated from Eichhornia crassipes (Mart) Solms., collected in oil contaminated wastewater of effluent generated by Petrobras refinery in Manaus were investigated to determine their potential for producing biosurfactants. Assay with 2.6-dichlorophenol indophenol (DCPIP) indicator to verify hydrocarbon biodegradation activity; oil emulsification test; drop-collapse method; surface tension and growth curve of biosurfactant production. The M87 Microbacterium sp. strain chosen for this work was identified by the sequencing of the rDNA region and the chemical characterization was performed by FTIR, UFLC/MS and 1H RMN techniques. The selected bacterial isolate provided 3g L-1 of biosurfactant, using diesel oil as sole carbon source, being efficient in biodegrading oil as demonstrated by the DCPIP test. Fractions obtained by column chromatography were efficient in reducing water surface tension around 40 mN m-1, especially fraction 1, which reduced it to 34.17 mN m-1. The different techniques of chemical analysis used for the identification of the biosurfactant isolate indicated that this is probably a long - chain fatty acid lipid type, which may be used in the future as both biosurfactant in decontamination processes of hydrocarbon-polluted areas or as bioemulsifier in countless processes, since it exhibited no toxicity as determined by Alamar Blue assay. 

Foram investigadas bactérias endofíticas isoladas de Eichhornia crassipes (Mart.) Solms., coletadas em águas contaminadas com resíduos de petróleo em um afluente da refinaria da Petrobrás/Manaus, para valiação da produção de biossurfactantes. Para selecionar o micro-organismos à produção e caracterização de biossurfactantes, foram realizados os seguintes testes: a descoloração do indicador 2,6 indofenol (DCPIP), emulsificação do diesel, colapso da gota, tensão superficial e curva de produção. A caracterização química foi realizada por meio das técnicas de FT-IR, UFLC/MS e RMN1H. A bactéria M87 Microbacterium neste estudo, foi identificada pelo sequenciamento da região rDNA e produziu 3g L-1 de biossurfactantes utilizando o diesel como fonte de carbono, mostrando-se eficiente na ação biodegradadora do petróleo, por meio do teste de Indofenol (DCPIP). As frações obtidas, mostraram-se eficazes na redução da tensão superficial da água abaixo de 40 mN m-1, com destaque para a fração 1 que reduziu a tensão superficial para 34,17 mN m-1. Pelas análises química utilizadas, pode-se inferir que, provavelmente, se trata de um ácido graxo de cadeia longa, que pode ser utilizado futuramente tanto como biossurfactante em processos de descontaminação de ambientes impactados por hidrocarbonetos, assim como bioemulsificante em inúmeros processos uma vez que não apresentou toxicidade por meio do teste realizado.(AU)

Metabolomics of Ulva lactuca Linnaeus (Chlorophyta) exposed to oil fuels: Fourier transform infrared spectroscopy and multivariate analysis as tools for metabolic fingerprint.

Fossil fuels, e.g. gasoline and diesel oil, account for substantial share of the pollution that affects marine ecosystems. Environmental metabolomics is an emerging field that may help unravel the effect of these xenobiotics on seaweeds and provide methodologies for biomonitoring coastal ecosystems. In the present study, FTIR and multivariate analysis were used to discriminate metabolic profiles of Ulva lactuca after in vitro exposure to diesel oil and gasoline, in combinations of concentrations (0.001%, 0.01%, 0.1%, and 1.0% - v/v) and times of exposure (30min, 1h, 12h, and 24h). PCA and HCA performed on entire mid-infrared spectral window were able to discriminate diesel oil-exposed thalli from the gasoline-exposed ones. HCA performed on spectral window related to the protein absorbance (1700-1500cm-1) enabled the best discrimination between gasoline-exposed samples regarding the time of exposure, and between diesel oil-exposed samples according to the concentration. The results indicate that the combination of FTIR with multivariate analysis is a simple and efficient methodology for metabolic profiling with potential use for biomonitoring strategies.

Production of rhamnolipids and diesel oil degradation by bacteria isolated from soil contaminated by petroleum.

Biosurfactants are microbial secondary metabolites. The most studied are rhamnolipids, which decrease the surface tension and have emulsifying capacity. In this study, the production of biosurfactants, with emphasis on rhamnolipids, and diesel oil degradation by 18 strains of bacteria isolated from waste landfill soil contaminated by petroleum was analyzed. Among the studied bacteria, gram-positive endospore forming rods (39%), gram positive rods without endospores (17%), and gram-negative rods (44%) were found. The following methods were used to test for biosurfactant production: oil spreading, emulsification, and hemolytic activity. All strains showed the ability to disperse the diesel oil, while 77% and 44% of the strains showed hemolysis and emulsification of diesel oil, respectively. Rhamnolipids production was observed in four strains that were classified on the basis of the 16S rRNA sequences as Pseudomonas aeruginosa. Only those strains showed the rhlAB gene involved in rhamnolipids synthesis, and antibacterial activity against Escherichia coli, P. aeruginosa, Staphylococcus aureus, Bacillus cereus, Erwinia carotovora, and Ralstonia solanacearum. The highest production of rhamnolipids was 565.7 mg/L observed in mineral medium containing olive oil (pH 8). With regard to the capacity to degrade diesel oil, it was observed that 7 strains were positive in reduction of the dye 2,6-dichlorophenolindophenol (2,6-DCPIP) while 16 had the gene alkane mono-oxygenase (alkB), and the producers of rhamnolipids were positive in both tests. Several bacterial strains have shown high potential to be explored further for bioremediation purposes due to their simultaneous ability to emulsify, disperse, and degrade diesel oil. © 2015 American Institute of Chemical Engineers Biotechnol. Prog., 32:262-270, 2016.

GPo1 alkB gene expression for improvement of the degradation of diesel oil by a bacterial consortium.

To facilitate the biodegradation of diesel oil, an oil biodegradation bacterial consortium was constructed. The alkane hydroxylase (alkB) gene of Pseudomonas putida GPo1 was constructed in a pCom8 expression vector, and the pCom8-GPo1 alkB plasmid was transformed into Escherichia coli DH5α. The AlkB protein was expressed by diesel oil induction and detected through SDS-polyacrylamide gel electrophoresis. The culture of the recombinant (pCom8-GPo1 alkB/E. coli DH5α) with the oil biodegradation bacterial consortium increased the degradation ratio of diesel oil at 24 h from 31% to 50%, and the facilitation rates were increased as the proportion of pCom8-GPo1 alkB/E. coli DH5α to the consortium increased. The results suggested that the expression of the GPo1 gene in E. coli DH5α could enhance the function of diesel oil degradation by the bacterial consortium.

GPo1 alkB gene expression for improvement of the degradation of diesel oil by a bacterial consortium

To facilitate the biodegradation of diesel oil, an oil biodegradation bacterial consortium was constructed. The alkane hydroxylase (alkB) gene of Pseudomonas putida GPo1 was constructed in a pCom8 expression vector, and the pCom8-GPo1 alkB plasmid was transformed into Escherichia coli DH5α. The AlkB protein was expressed by diesel oil induction and detected through SDS-polyacrylamide gel electrophoresis. The culture of the recombinant (pCom8-GPo1 alkB/E. coli DH5α) with the oil biodegradation bacterial consortium increased the degradation ratio of diesel oil at 24 h from 31% to 50%, and the facilitation rates were increased as the proportion of pCom8-GPo1 alkB/E. coli DH5α to the consortium increased. The results suggested that the expression of the GPo1 gene in E. coli DH5α could enhance the function of diesel oil degradation by the bacterial consortium.

GPo1 alkB gene expression for improvement of the degradation of diesel oil by a bacterial consortium

To facilitate the biodegradation of diesel oil, an oil biodegradation bacterial consortium was constructed. The alkane hydroxylase (alkB) gene of Pseudomonas putida GPo1 was constructed in a pCom8 expression vector, and the pCom8-GPo1 alkB plasmid was transformed into Escherichia coli DH5α. The AlkB protein was expressed by diesel oil induction and detected through SDS-polyacrylamide gel electrophoresis. The culture of the recombinant (pCom8-GPo1 alkB/E. coli DH5α) with the oil biodegradation bacterial consortium increased the degradation ratio of diesel oil at 24 h from 31% to 50%, and the facilitation rates were increased as the proportion of pCom8-GPo1 alkB. coli DH5α to the consortium increased. The results suggested that the expression of the GPo1 gene in E. coli DH5α could enhance the function of diesel oil degradation by the bacterial consortium.(AU)

Antioxidant defense responses in Mytella guyanensis (Lamarck, 1819) exposed to an experimental diesel oil spill in Paranaguá Bay (Paraná, Brazil).

We evaluated the effects of diesel oil on the bivalve Mytella guyanensis using biomarkers of oxidative stress (glutathione S-transferase, glutathione peroxidase, and reduced glutathione) after an experimental in situ spill in a mangrove area in southern Brazil. A linear model was developed for the Multiple Before-After Control-Impact (MBACI) experimental design to assess the significance of biological responses. Control and impacted sites were sampled seven and two days before as well as two and seven days after the spill. With the exception of a late response of reduced glutathione (GSH) levels on day seven, none of the biomarkers were significantly altered by the impact. This result was attributed to the high environmental variability of the experimental sites combined with a low sensitivity of Mytella guyanensis to diesel oil at short time-scales. The high resistance of M. guyanensis suggests that its antioxidant response is triggered only after a medium- to long-term exposure to contaminants.

Isolation and characterization of a novel hydrocarbon-degrading bacterium Achromobacter sp. HZ01 from the crude oil-contaminated seawater at the Daya Bay, southern China.

Microorganisms play an important role in the biodegradation of petroleum contaminants, which have attracted great concern due to their persistent toxicity and difficult biodegradation. In this paper, a novel hydrocarbon-degrading bacterium HZ01 was isolated from the crude oil-contaminated seawater at the Daya Bay, South China Sea, and identified as Achromobacter sp. Under the conditions of pH 7.0, NaCl 3% (w/v), temperature 28 °C and rotary speed 150 rpm, its degradability of the total n-alkanes reached up to 96.6% after 10 days of incubation for the evaporated diesel oil. Furthermore, Achromobacter sp. HZ01 could effectively utilize polycyclic aromatic hydrocarbons (PAHs) as its sole carbon source, and could remove anthracene, phenanthrene and pyrence about 29.8%, 50.6% and 38.4% respectively after 30 days of incubation. Therefore, Achromobacter sp. HZ01 may employed as an excellent degrader to develop one cost-effective and eco-friendly method for the bioremediation of marine environments polluted by crude oil.

Biochemical responses in armored catfish (Pterygoplichthys anisitsi) after short-term exposure to diesel oil, pure biodiesel and biodiesel blends.

Biodiesel fuel is gradually replacing petroleum-based diesel oil use. Despite the biodiesel being considered friendlier to the environment, little is known about its effects in aquatic organisms. In this work we evaluated whether biodiesel exposure can affect oxidative stress parameters and biotransformation enzymes in armored catfish (Pterygoplichthys anisitsi, Loricariidae), a South American endemic species. Thus, fish were exposed for 2 and 7d to 0.01mLL(-1) and 0.1mLL(-1) of pure diesel, pure biodiesel (B100) and blends of diesel with 5% (B5) and 20% (B20) biodiesel. Lipid peroxidation (malondialdehyde) levels and the activities of the enzymes glutathione S-transferase, superoxide dismutase, catalase and glutathione peroxidase were measured in liver and gills. Also, DNA damage (8-oxo-7, 8-dihydro-2'-deoxyguanosine) levels in gills and 7-ethoxyresorufin-O-deethylase activity in liver were assessed. Pure diesel, B5 and B20 blends changed most of the enzymes tested and in some cases, B5 and B20 induced a higher enzyme activity than pure diesel. Antioxidant system activation in P. anisitsi was effective to counteract reactive oxygen species effects, since DNA damage and lipid peroxidation levels were maintained at basal levels after all treatments. However, fish gills exposed to B20 and B100 presented increased lipid peroxidation. Despite biodiesel being more biodegradable fuel that emits less greenhouse gases, the increased lipid peroxidation showed that biofuel and its blends also represent hazards to aquatic biota.