The effects of biostimulation and bioaugmentation on crude oil biodegradation in two adjacent terrestrial oil spills of different age, in a hyper-arid region.

This study deals with two adjacent terrestrial oil spills, with similar properties, located in a hyper-arid region in Israel, one from 1975 and the other from 2014. It tests the effect of biostimulation on crude oil degradation in both spills and whether biostimulated sediments from the 1975 spill can bioaugment crude oil degradation in the 2014 spill. Soil hydrophobicity, expressed as Water Drop Penetration Time (WDPT), and Gasoline Range Organics (GRO) and Diesel Range Organics (DRO) content in sediments were measured in one-month ex-situ experiments. No significant reduction in hydrophobicity and GRO + DRO content was observed in non-biostimulated controls. A combined treatment of mineral fertilization at t0 and maintaining 50% water saturation, significantly accelerated the decrease in hydrophobicity and GRO + DRO content in sediments of both spills. The addition of biostimulated sediments from the 1975 spill failed to accelerate the reduction of GRO + DRO content and hydrophobicity in the 2014 spill. Surprisingly, the GRO + DRO degradation rate in biostimulated sediments from the 2014 spill was 36% higher than in biostimulated sediments from the 1975 spill. Crude oil composition in both spills changes during its degradation and is characterized by an increase in the GRO fraction. To a certain range, WDPT was found to serve as a reliable indicator for oil content in the soil. We conclude that even in a hyper-arid region, oil bio-degradation capabilities develop in a relatively short time. Moreover, while biostimulation was effective in accelerating biodegradation, bioaugmentation with biostimulated sediments from a nearby older spill was found ineffective.

A secreted aminopeptidase of Pseudomonas aeruginosa. Identification, primary structure, and relationship to other aminopeptidases.

Using leucine-p-nitroanilide (Leu-pNA) as a substrate, we demonstrated aminopeptidase activity in the culture filtrates of several Pseudomonas aeruginosa strains. The aminopeptidase was partially purified by DEAE-cellulose chromatography and found to be heat stable. The apparent molecular mass of the enzyme was approximately 56 kDa; hence, it was designated AP(56). Heating (70 degrees C) of the partially purified aminopeptidase preparations led to the conversion of AP(56) to a approximately 28-kDa protein (AP(28)) that retained enzyme activity, a reaction that depended on elastase (LasB). The pH optimum for Leu-pNA hydrolysis by AP(28) was 8.5. This activity was inhibited by Zn chelators but not by inhibitors of serine- or thiol-proteases, suggesting that AP(28) is a Zn-dependent enzyme. Of several amino acid p-nitroanilide derivatives examined, Leu-pNA was the preferred substrate. The sequences of the first 20 residues of AP(56) and AP(28) were determined. A search of the P. aeruginosa genomic data base revealed a perfect match of these sequences with positions 39-58 and 273-291, respectively, in a 536-amino acid residue open reading frame predicted to encode an aminopeptidase. A search for sequence similarities with other proteins revealed 52% identity with Streptomyces griseus aminopeptidase, approximately 35% identity with Saccharomyces cerevisiae aminopeptidase Y and a hypothetical aminopeptidase from Bacillus subtilis, and 29-32% with Aeromonas caviae, Vibrio proteolyticus, and Vibrio cholerae aminopeptidases. The residues potentially involved in zinc coordination were conserved in all these proteins. Thus, P. aeruginosa aminopeptidase may belong to the same family (M28) of metalloproteases.

A 28,000 mol. wt toxin from Bacillus thuringiensis israelensis induces cation transport in rat muscle cultures.

The mechanism by which the Bacillus thuringiensis israelensis (Bti) 28,000 mol. wt toxin exerts its effect on mature muscle cultures was examined. The toxin inhibited Na+/K(+)-ATPase activity as revealed by 86Rb influx. A 50% inhibition of Na+/K(+)-ATPase activity was obtained with 0.2 microgram/ml of the toxin. The inhibition was time and dose dependent, and it was reversible with low doses of the toxin (up to 0.2 microgram/ml. A considerable release of 86Rb was obtained by doses greater than 0.2 microgram/ml. The 86Rb release was also time and dose dependent. This effect is probably non-specific, since 45Ca influx is also accelerated by toxin-treated cultures. Pre-incubation of the toxin with phosphotidylserine (PS) antagonized the toxin. It is concluded that the toxin is a hydrophobic protein which interacts with the membrane. In low doses this interaction reduces the activity of the sodium pump and in high doses it causes non-specific permeability of the sarcolemma.

Biochemical and morphological changes in rat muscle cultures caused by 28,000 mol. wt toxin of Bacillus thuringiensis israelensis.

The 28,000 mol. wt protein of Bacillus thuringiensis israelensis showed a high degree of toxicity to rat muscle in culture. Application of 1 microgram/ml to the culture medium completely inhibited cell fusion. Reversibility of this effect was demonstrated by replacement of the culture medium with fresh medium, and the consequence was that cell fusion was resumed. When differentiated myotubes were treated with 1 microgram/ml of the toxin, the spontaneous contractile activity was abolished within 20 min. Cytotoxic effects were observed 1 hr after treatment was initiated, as manifested by creatine kinase (CK) release to the medium. Two hours after toxin was applied to the muscle culture, the myotubes were deteriorated whereas the mononucleated cells were not affected. Six or 7-day-old cultures which were treated by 1 microgram/ml of 28,00 mol. wt toxin revealed a change in the levels of Na+ and K+ within the fibres as analysed by X-ray microanalysis (XRMA). Preincubation of the toxin for 20 min with phospholipids before application to the cells reduced the cytotoxic effect. Phosphatidylinositol and phosphatidylserine were the most efficient inhibitors, whereas phosphatidylcholine, sphingomyelin and phosphatidylethanolamine were less effective in protecting cultures from the cytotoxic effects of the 28,000 mol. wt protein.

Inhibition of myogenesis by trifluoperazine and compound 48/80.

When trifluoperazine (TFP), a calmodulin antagonist, was given to chick or rat myoblasts in cultures, formation of multinucleated myotubes was inhibited. The inhibition of cell fusion by TFP in rat cultures prevents the normal increase in the amount of acetylcholine receptors (AChR) and creatine kinase (CK), while the levels of these proteins in chick muscle cultures are hardly affected. Another calmodulin antagonist, compound 48/80, inhibits fusion at doses that correspond closely to its antagonistic effects on calmodulin. Thus, our results suggest a possible role for calmodulin in the regulation of myoblast fusion, but not on the appearance of muscle proteins.