MALDI-TOF Mass Spectrometry Discriminates Known Species and Marine Environmental Isolates of Pseudoalteromonas.

The genus Pseudoalteromonas constitutes an ecologically significant group of marine Gammaproteobacteria with potential biotechnological value as producers of bioactive compounds and of enzymes. Understanding their roles in the environment and bioprospecting for novel products depend on efficient ways of identifying environmental isolates. Matrix Assisted Laser Desorption/Ionization-Time of Flight Mass Spectrometry (MALDI-TOF MS) biotyping has promise as a rapid and reliable method of identifying and distinguishing between different types of bacteria, but has had relatively limited application to marine bacteria and has not been applied systematically to Pseudoalteromonas. Therefore, we constructed a MALDI-TOF MS database of 31 known Pseudoalteromonas species, to which new isolates can be compared by MALDI-TOF biotyping. The ability of MALDI-TOF MS to distinguish between species was scrutinized by comparison with 16S rRNA gene sequencing. The patterns of similarity given by the two approaches were broadly but not completely consistent. In general, the resolution of MALDI-TOF MS was greater than that of 16S rRNA gene sequencing. The database was tested with 13 environmental Pseudoalteromonas isolates from UK waters. All of the test strains could be identified to genus level by MALDI-TOF MS biotyping, but most could not be definitely identified to species level. We conclude that several of these isolates, and possibly most, represent new species. Thus, further taxonomic investigation of Pseudoalteromonas is needed before MALDI-TOF MS biotyping can be used reliably for species identification. It is, however, a powerful tool for characterizing and distinguishing among environmental isolates and can make an important contribution to taxonomic studies.

Asymmetric cell division and its role in cell fate determination in the green alga Tetraselmis indica.

The prasinophytes (early diverging Chlorophyta), consisting of simple unicellular green algae, occupy a critical position at the base of the green algal tree of life, with some of its representatives viewed as the cell form most similar to the first green alga, the 'ancestral green flagellate'. Relatively large-celled unicellular eukaryotic phytoflagellates (such as Tetraselmis and Scherffelia), traditionally placed in Prasinophyceae but now considered as members of Chlorodendrophyceae (core Chlorophyta), have retained some primitive characteristics of prasinophytes. These organisms share several ultrastructural features with the other core chlorophytes (Trebouxiophyceae, Ulvophyceae and Chlorophyceae). However, the role of Chlorodendrophycean algae as the evolutionary link between cellular individuality and cellular cooperation has been largely unstudied. Here, we show that clonal populations of a unicellular chlorophyte, Tetraselmis indica, consist of morphologically and ultrastructurally variant cells which arise through asymmetric cell division. These cells also differ in their physiological properties. The structural and physiological differences in the clonal cell population correlate to a certain extent with the longevity and function of cells.

Proteomic-based biotyping reveals hidden diversity within a microalgae culture collection: An example using Dunaliella.

Accurate and defendable taxonomic identification of microalgae strains is vital for culture collections, industry and academia; particularly when addressing issues of intellectual property. We demonstrate the remarkable effectiveness of Matrix Assisted Laser Desorption Ionisation Time of Flight Mass Spectrometry (MALDI-TOF-MS) biotyping to deliver rapid and accurate strain separation, even in situations where standard molecular tools prove ineffective. Highly distinctive MALDI spectra were obtained for thirty two biotechnologically interesting Dunaliella strains plus strains of Arthrospira, Chlorella, Isochrysis, Tetraselmis and a range of culturable co-occurring bacteria. Spectra were directly compared with genomic DNA sequences (internal transcribed spacer, ITS). Within individual Dunaliella isolates MALDI discriminated between strains with identical ITS sequences, thereby emphasising and enhancing knowledge of the diversity within microalgae culture collections. Further, MALDI spectra did not vary with culture age or growth stage during the course of the experiment; therefore MALDI presents stable and accurate strain-specific signature spectra. Bacterial contamination did not affect MALDI's discriminating power. Biotyping by MALDI-TOF-MS will prove effective in situations wherein precise strain identification is vital, for example in cases involving intellectual property disputes and in monitoring and safeguarding biosecurity. MALDI should be accepted as a biotyping tool to complement and enhance standard molecular taxonomy for microalgae.

Emerging risks from ballast water treatment: the run-up to the International Ballast Water Management Convention.

Uptake and discharge of ballast water by ocean-going ships contribute to the worldwide spread of aquatic invasive species, with negative impacts on the environment, economies, and public health. The International Ballast Water Management Convention aims at a global answer. The agreed standards for ballast water discharge will require ballast water treatment. Systems based on various physical and/or chemical methods were developed for on-board installation and approved by the International Maritime Organization. Most common are combinations of high-performance filters with oxidizing chemicals or UV radiation. A well-known problem of oxidative water treatment is the formation of disinfection by-products, many of which show genotoxicity, carcinogenicity, or other long-term toxicity. In natural biota, genetic damages can affect reproductive success and ultimately impact biodiversity. The future exposure towards chemicals from ballast water treatment can only be estimated, based on land-based testing of treatment systems, mathematical models, and exposure scenarios. Systematic studies on the chemistry of oxidants in seawater are lacking, as are data about the background levels of disinfection by-products in the oceans and strategies for monitoring future developments. The international approval procedure of ballast water treatment systems compares the estimated exposure levels of individual substances with their experimental toxicity. While well established in many substance regulations, this approach is also criticised for its simplification, which may disregard critical aspects such as multiple exposures and long-term sub-lethal effects. Moreover, a truly holistic sustainability assessment would need to take into account factors beyond chemical hazards, e.g. energy consumption, air pollution or waste generation.

Difficulties in obtaining representative samples for compliance with the Ballast Water Management Convention.

As implementation of the Ballast Water Convention draws nearer a major challenge is the development of protocols which accurately assess compliance with the D-2 Standard. Many factors affect the accuracy of assessment: e.g. large volume of ballast water, the shape, size and number of ballast tanks and the heterogeneous distribution of organisms within tanks. These factors hinder efforts to obtain samples that truly represent the total ballast water onboard a vessel. A known cell density of Tetraselmis suecica was added to a storage tank and sampled at discharge. The factors holding period, initial cell density and sampling interval affected representativeness. Most samples underestimated cell density, and some tanks with an initial cell density of 100 cells ml(-1) showed <10 cells ml(-1) at discharge, i.e. met the D-2 standard. This highlights difficulties in achieving sample representativeness and when applied to a real ballast tank this will be much harder to achieve.

Characterization of bacteria in ballast water using MALDI-TOF mass spectrometry.

To evaluate a rapid and cost-effective method for monitoring bacteria in ballast water, several marine bacterial isolates were characterized by matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS). Since International Maritime Organization (IMO) regulations are concerned with the unintended transportation of pathogenic bacteria through ballast water, emphasis was placed on detecting species of Vibrio, enterococci and coliforms. Seawater samples collected from the North Sea were incubated in steel ballast tanks and the presence of potentially harmful species of Pseudomonas was also investigated. At the genus-level, the identification of thirty six isolates using MALDI-TOF MS produced similar results to those obtained by 16S rRNA gene sequencing. No pathogenic species were detected either by 16S rRNA gene analysis or by MALDI-TOF MS except for the opportunistically pathogenic bacterium Pseudomonas aeruginosa. In addition, in house software that calculated the correlation coefficient values (CCV) of the mass spectral raw data and their variation was developed and used to allow the rapid and efficient identification of marine bacteria in ballast water for the first time.

On board short-time high temperature heat treatment of ballast water: a field trial under operational conditions.

A ballast water short-time high temperature heat treatment technique was applied on board a car-carrier during a voyage from Egypt to Belgium. Ballast water from three tanks was subjected for a few seconds to temperatures ranging from 55 degrees C to 80 degrees C. The water was heated using the vessel's heat exchanger steam and a second heat exchanger was used to pre-heat and cool down the water. The treatment was effective at causing mortality of bacteria, phytoplankton and zooplankton. The International Maritime Organization (IMO) standard was not agreed before this study was carried out, but comparing our results gives a broad indication that the IMO standard would have been met in some of the tests for the zooplankton, in all the tests for the phytoplankton; and probably on most occasions for the bacteria. Passing the water through the pump increased the kill rate but increasing the temperature above 55 degrees C did not improve the heat treatment's efficacy.

Ship board testing of a deoxygenation ballast water treatment.

A ship board trial of a deoxygenation method for treating ballast water was carried out during a voyage from Southampton (United Kingdom) to Manzanillo (Panama). A nutrient solution added to two ballast tanks encouraged bacterial growth, resulting in a gradual change to an anoxic environment. Samples were taken from two treated tanks and two untreated tanks to assess changes in the abundance and viability of zooplankton, phytoplankton and bacteria. The work was carried out before the International Maritime Organization (IMO) standard was agreed so only a broad indication of whether the results achieved the standard was given. For the zooplankton, the standard would have been achieved within 5 or 7 days but the phytoplankton results were inconclusive. The biological efficacy was the result of the combination of several factors, including the treatment, pump damage and an increase in the water temperature during the voyage.