Phenanthrene Emulsification and Biodegradation Using Rhamnolipid Biosurfactants and Acinetobacter calcoaceticus In Vitro

The ability of biosurfactants and Acinetobacter calcoaceticus to enhance the emulsification and biodegradation of phenanthrene was investigated. Phenanthrene is a polycyclic aromatic hydrocarbon that may be derived from various sources, for example incomplete combustion of petroleum fuel, and thus it occurs ubiquitously throughout the environment. In order to assess the efficacy of a biosurfactant microparticle system, emulsification assays and in vitro biodegradation studies were conducted. Emulsification assays were carried out to assess the stability of phenanthrene emulsions. Emulsion stability was determined by the height of the emulsion layer (Emulsification Index) and turbidity. In vitro biodegradation tests were done to estimate phenanthrene degradation from an aqueous system by A. calcoaceticus supplemented with encapsulated (ERhBS) and nonencapsulated biosurfactants (NERhBS). Results show that phenanthrene emulsifications were stabilized after 48 h with NERhBS and remained stable for 72 additional hours. Phenanthrene emulsifications were stabilized with ERhBS after 216 h and remained stable for an additional 96 h. A. calcoaceticus alone and supplemented with rhamnolipid biosurfactant were able to biodegrade 10 to 50 mg L^?1 of phenanthrene within 250 h. When supplemented with NERhBS, A. calcoaceticus degraded phenanthrene significantly faster than when nonsupplemented or supplemented with ERhBS. Addition of exogenous biosurfactants was considered to be a major factor driving the direct correlation between decreasing phenanthrene concentration in the system and increasing bacterial biomass.     

Production of Rhamnolipid Biosurfactant by Fed-batch Culture of Pseudomonas aeruginosa Using Glucose as a Sole Carbon Source

The pH-stat fed-batch culture of Pseudomonas aeruginosa YPJ-80 was done to produce a rhamnolipid biosurfactant. With glucose as the sole carbon source, the final concentrations of cells and rhamnolipid biosurfactant obtained in 25 h were 25 g cell dry weight/l and 4.4 g/l, respectively.     

Isolation and Characterization of Biosurfactant-and Bioemulsifier-Producing Bacteria from Petroleum Contaminated Sites in Western Canada

Biosurfactant-producing bacteria were isolated from two petroleum contaminated sites in western Canada. Seven potential biosurfactant/bioemulsifier-producing isolates were screened and characterized. All of the seven isolates were able to form emulsions. Emulsion-stabilizing capacity was also measured up to 48 hrs. Strain C-111-2 and C-203-2 would lead to highly reduced surface tension. For strain C-203-2, the optimum conditions that supported bacteria growth and production were investigated. The influences of carbon sources, medium pH values, and temperature were taken into account. The experimental results indicated that the crude oil and glucose were promising carbon sources for biosurfactants production; the isolated strains produced a maximum concentration of biosurfactant in a neutral pH environment and showed a higher surface activity under the temperature level of 35°C than that under 10°C. To further optimize the carbon and nitrogen source for biosurfactant production, response surface methodology (RSM) was applied to explore the favorable concentration of two carbon sources: glucose, crude oil, and one nitrogen source, NaNO₃. The optimal concentration of 8.1g/L, 4% and 3.9 g/L for glucose, crude oil, and NaNO₃, respectively, which can be obtained through RSM analysis.     

Engineering bacteria for production of rhamnolipid as an agent for enhanced oil recovery

Rhamnolipid as a potent natural biosurfactant has a wide range of potential applications, including enhanced oil recovery (EOR), biodegradation, and bioremediation. Rhamnolipid is composed of rhamnose sugar molecule and beta-hydroxyalkanoic acid. The rhamnosyltransferase 1 complex (RhlAB) is the key enzyme responsible for transferring the rhamnose moiety to the beta-hydroxyalkanoic acid moiety to biosynthesize rhamnolipid. Through transposome-mediated chromosome integration, the RhlAB gene was inserted into the chromosome of the Pseudomonas aeruginosa PAO1-rhlA(-) and Escherichia coli BL21 (DE3), neither of which could produce rhamnolipid. After chromosome integration of the RhlAB gene, the constitute strains P. aeruginosa PEER02 and E. coli TnERAB did produce rhamnolipid. The HPLC/MS spectrum showed that the structure of purified rhamnolipid from P. aeruginosa PEER02 was similar to that from other P. aeruginosa strains, but with different percentage for each of the several congeners. The main congener (near 60%) of purified rhamnolipid from E. coli TnERAB was 3-(3-hydroxydecanoyloxy) decanoate (C(10)-C(10)) with mono-rhamnose. The surfactant performance of rhamnolipid was evaluated by measurement of interfacial tension (IFT) and oil recovery via sand-pack flooding tests. As expected, pH and salt concentration of the rhamnolipid solution significantly affected the IFT properties. With just 250 mg/L rhamnolipid (from P. aeruginosa PEER02 with soybean oil as substrate) in citrate-Na(2)HPO(4), pH 5, 2% NaCl, 42% of oil otherwise trapped was recovered from a sand pack. This result suggests rhamnolipid might be considered for EOR applications.     

Case Study of Petroleum Contaminated Area of Novi Sad After NATO Bombing in Yugoslavia

The 1999 NATO bombing of the oil refinery in Novi Sad (Yugoslavia) has heavily contaminated the Danube River and its sediments, as well as the surrounding soil and groundwater. The destruction of the factories released 73,569 tons of crude oil of which 90% was incinerated, 560 tons reached the Danube River, and the remainder was spilled onto the soil. The contents of oil and oil derivatives in the soil were in the range of 3 to 42,000?mg/kg. The first soil layer contained an average of 67,000?mg/kg of crude oil and oil derivatives. The layers beneath it, above the groundwater table, contained 56?ml/l of free oil derivatives in the drained water. The spreading of this pollution could imperil the groundwater quality in the water supply zone because the refinery is located in the hinterland of the zone. The quality of water and sediment samples was monitored from April 1999 to November 2000 by measuring concentrations of hydrocarbons and polyaromatic hydrocarbons (PAH). The hydrocarbon content in the Danube River water in October 2000 was about 20% of the value measured at the time of the accident. Immediately after the accident the concentration of mineral oil in the surface sediment was in the range of 0.11 to 0.29?g/kg. At the same time PAH concentrations in the river sediment were up to 160?mg/kg, depending on the sampling site location. The values showed a decrease in the course of further monitoring.     

Risk-Based Corrective Action of Hydrocarbon Contamination at a Former Major Urban Petroleum Storage Site in the U.K.

The former site of a major petroleum storage facility adjacent to a major urban watercourse was found to have potentially significant concentrations of hydrocarbons in soil and groundwater that needed to be addressed prior to redevelopment. A series of intrusive investigations were undertaken to collect physical and chemical data for a Quantitative Risk Assessment (QRA) of potential impacts on human health and the wider environment, in order to derive a remedial strategy for redevelopment of the site for light industrial use. A site-specific QRA methodology was devel oped using both U.K. and U.S. guidance to produce Risk-Based Clean-up Levels (RBCLs) for benzene, and other petroleum hydrocarbons. The U.K. has no nationally based guidance on risk assessment and studies are designed by the consultant for submission to the U.K. Environment Agency (EA) for their approval. It is the EA's role to determine whether the work has been undertaken satisfactorily. To achieve these RBCLs, ex situ bioremediation was identified as the best practicable remedial option. This was carried out in windrows using mechanical aeration (to achieve oxygenation with ammonia nitrate granule and woodchip addition) for a total of approximately 5291?m³ of soil. The bioremediation process was successful in achieving the site specific RBCLs for benzene and for other hydrocarbons within an average of 5 to 6 weeks. This article describes the successful implementation of Risk-Based Corrective Action (RBCA) at this petroleum release site as a demonstration of how risk-based remedial standards for contaminated sites can be achieved with regulatory approval.     

Fenton's Reagent-Based In Situ Chemical Oxidation Treatment of Saturated and Unsaturated Soils at a Historic Railroad Site

A targeted treatment program utilizing in situ chemical oxidation was used to remediate diesel fuel-derived petroleum compounds in unsaturated and saturated soils at a historic railroad facility. This program consisted of multiple injections at varying depths within temporary Geoprobe® injection points. The actual treatment time was less than 3 months. Overall concentrations of volatile and semivolatile organic petroleum compounds were reduced by approximately 70%, while the total petroleum hydrocarbon concentration was reduced by nearly 50%. Treatment efficiency in unsaturated soil was similar to that in saturated soil. The results of the remedial program indicate that the effect of grain size of the subsurface materials on treatment efficacy is significant. The project has shown that the use of this technology can be as effective as other in situ treatment technologies used for treating subsurface diesel fuel contamination.     

Bioavailability to Fish of Sediment PAH as an Indicator of the Success of In Situ Remediation Treatments at an Experimental Oil Spill

A weathered medium crude oil was applied to experimental plots of Scirpus pungens (Three-square Bulrush) in a freshwater wetland to determine the efficacy of strategies for shoreline oil spill bioremediation based on nutrient enrichment (bioremediation) and plant growth (phytoremediation). Plots were unoiled, oiled with no added nutrients, or oiled with repeated applications of phosphate and nitrate fertilizers. Following initial treatments, the experimental plots were raked to simulate the activity of wave action on oil penetration, and plants in one fertilized plot were cut repeatedly. The sediments were sampled at regular intervals for 15 months after oiling, and the loss of oil was assessed by 4-day laboratory tests of polynuclear aromatic hydrocarbon (PAH) bioaccumulation by trout, as demonstrated by increases in activity of liver cytochrome P450 (CYP1A) enzymes. Oil alone, oil mixed with sediments in the lab, and oiled sediments from treated plots all induced CYP1A activity relative to untreated controls, indicating the presence and bioavailability of PAH. Induction did not vary with nutrient treatments, but declined by 80% within 15 months of oiling, and chemical analyses indicated equivalent losses of hydrocarbons in sediment. These results demonstrate that bioavailable PAHs persisted in measurable quantities for at least 1.25 years following oiling, and that stimulation of plant growth did not affect the rate of oil disappearance. The controlling factors were likely weathering and sediment movement.     

Kinetic Analysis of Leucine-Enkephalin Cellular Uptake at the Luminal Side of the Blood-Brain Barrier of an In Situ Perfused Guinea-Pig Brain

Abstract: The uptake of enkephalin-(5-L-leucine) (Leu-en-kephalin) at the luminal side of the blood-brain barrier was measured by means of an in situ vascular brain perfusion technique in the anaesthetized guinea pig. This method allows measurements of cerebrovascular peptide uptake over periods of up to 20 min, and excludes the solute under study from the general circulation and systemic metabolic influences. A capillary unidirectional transfer constant, K_in, for [tyrosyl-3,5-³H]Leu-enkephalin was estimated graphically from the multiple-time brain uptake data in the presence of different concentrations of unlabelled peptide, and dose-dependent self-inhibition was demonstrated. Analysis of unidirectional influx of blood-borne Leu-en kephalin into the brain revealed Michaelis-Menten saturation kinetics in the parietal cortex, caudate nucleus, and hippocampus, with V_max between 0.14 and 0.16 nmol min^?1 g^?1 and K_m ranging from 34 to 41 μM, for the saturable component, whereas the estimated diffusion constant, K_d, was not significantly different from zero. Entry of [³H]Leu-enkephalin was not inhibited in the presence of either a 5 mM concentration of unlabelled L-tyrosine, tyro-sylglycine, and tyrosylglycylglycine, or aminopeptidase inhibitor, bestatin (0.5 mM), suggesting that the saturable mechanism of the tracer at the luminal side of the blood-brain barrier does not involve uptake of the peptide's N-terminal amino acid and/or its tyrosine-containing fragments. The specific δ-opioid antagonist, allyl²-Tyr-AIB-Phe-OH, and μ-opioid receptor agonist, Tyr-D-Ala-Gly-Me-Phe-NH(CH₂)20H, at concentrations in the perfusate above the K_m value for the saturable transport of Leu-enkephalin, did not affect significantly uptake of [³H]Leu-enkephalin. The present study provides, for the first time, a characterization of the kinetic parameters of the unidirectional uptake of a peptide from the luminal side of the blood-brain barrier