Bioremediation (bioaugmentation/biostimulation) trials of oil polluted seawater: a mesocosm simulation study.

Bioaugmentation (amendment with selected bacterial strains) and/or biostimulation (nutrients addition and/or air supply) are relatively new fields in environmental microbiology for preventing pollution and cleanup contamination. In this study, the efficiency of application of bioaugmentation/biostimulation treatments, for recovery of crude oil-polluted seawater, was evaluated. Three different series of experiments were performed in a "Mesocosm Facility" (10.000 L). Natural seawater was artificially polluted with crude oil (1000 ppm) and was amended with inorganic nutrients (Mesocosm 1, M1), inorganic nutrient and an inoculum of Alcanivorax borkumensis SK2(T) (Mesocosm 2, M2) and inorganic nutrient and an inoculum of A. borkumensis SK2(T) and Thalassolituus oleivorans MIL-1(T) (Mesocosm 3, M3), respectively. During the experimental period (20 days) bacterial abundance (DAPI count), culturable heterotrophic bacteria (CFU count), MPN, microbial metabolic activity [Biochemical Oxygen Demand and enzymatic activity (leucine aminopeptidase LAP, ß-glucosidase BG, alkaline phosphatase AP)] and quali-, quantitative analysis of the composition of total extracted and resolved hydrocarbons and their derivates (TERHCs) were carried out. The microbiological and physiological analysis of marine microbial community found during the three different biostimulation and bioaugmentation assays performed in mesocosms show that the load of crude oil increases total microbial abundance, inhibits the activity of some enzymes such as LAP while stimulates both AP and BG activities. The biodegradation results show that bioaugmentation with A. borkumensis SK2(T) alone is able to produce the highest percentage of degradation (95%) in comparison with the biostimulation treatment (80%) and bioaugmentation using an Alcanivorax-Thalassolituus bacterial consortium (70%). This result highlights the reduced biodegradation capability of the consortium used in this study, suggesting an unfavourable interaction between the two bacterial genera.

Conjugated polyelectrolyte capsules: light-activated antimicrobial micro "Roach Motels".

Microcapsules consisting of alternating layers of oppositely charged poly(phenylene ethynylene)-type conjugated polyelectrolytes (CPEs) were prepared via layer-by-layer deposition onto MnCO3 template particles followed by dissolution of the template particles using an ethylenediaminetetraacetate solution. The resulting microcapsules exhibit bright-green fluorescence emission characteristics of the CPEs. Strong antimicrobial activity was observed upon mixing of polyelectrolyte capsules with Cobetia marina or Pseudomonas aeruginosa followed by white-light irradiation. It was demonstrated that the materials act as highly effective light-activated micro "Roach Motels" with greater than 95% kill after exposure to approximately 1 h of white light.

Light-induced biocidal action of conjugated polyelectrolytes supported on colloids.

A series of water soluble, cationic conjugated polyelectrolytes (CPEs) with backbones based on a poly(phenylene ethynylene) repeat unit structure and tetraakylammonium side groups exhibit a profound light-induced biocidal effect. The present study examines the biocidal activity of the CPEs, correlating this activity with the photophysical properties of the polymers. The photophysical properties of the CPEs are studied in solution, and the results demonstrate that direct excitation produces a triplet excited-state in moderate yield, and the triplet is shown to be effective at sensitizing the production of singlet oxygen. Using the polymers in a format where they are physisorbed or covalently grafted to the surface of colloidal silica particles (5 and 30 microm diameter), we demonstrate that they exhibit light-activated biocidal activity, effectively killing Cobetia marina and Pseudomonas aeruginosa. The light-induced biocidal activity is also correlated with a requirement for oxygen suggesting that interfacial generation of singlet oxygen is the crucial step in the light-induced biocidal activity.

Identification of an operon involved in tyrosinase activity and melanin synthesis in Marinomonas mediterranea.

The genomic region of Marinomonas mediterranea containing the genes required for tyrosinase activity and melanin synthesis has been cloned by marker rescue using the transposon-generated, amelanogenic strain T105. Five ORFs, two incomplete and three complete, have been sequenced in the genomic region where the transposon was inserted. RT-PCR analysis indicates that ORF 3, coding for tyrosinase, and ORF4, coding for a protein of 250 amino acids, are in the same transcriptional unit, constituting an operon whose promoter region has been determined by 5'-RACE. This operon has been sequenced in the wild-type and several mutant strains, indicating that both ORFs are required for expression of tyrosinase activity and melanin synthesis. The nitrosoguanidine generated, amelanogenic mutant ng56, shows a nonsense mutation in ORF3 coding for the tyrosinase. On the other hand, in the strain T105 the transposon is inserted in ORF4. The product of this gene is related to copper metabolism, since the addition of this metal ion to cell extracts or culture media partially restores melanin synthesis and tyrosinase activity in the strain T105. However, it does not show significant sequence similarity to previously characterized metallochaperones and hence may be an example of a new kind of those proteins. The operon has been denoted as ppoB, taking into consideration that ppoA denotes the M. mediterranea gene coding for the previously cloned polyphenol oxidase with laccase activity. This is the first demonstration of the tyrosinase gene forming part of an operon in a Gram-negative bacterium.