Ole-Kristian Hess-Erga

Recolonization by heterotrophic bacteria after UV irradiation or ozonation of seawater; a simulation of ballast water treatment.

Transport of ballast water with ships represents a risk for introduction of foreign species. If ballast water is treated during uptake, there will be a recolonization of the ballast water by heterotrophic bacteria during transport. We investigated survival and succession of heterotrophic bacteria after disinfection of seawater in the laboratory, representing a model system of ballast water treatment and transport. The seawater was exposed to ultraviolet (UV) irradiation, ozone (2 doses) or no treatment, incubated for 16 days and examined with culture-dependent and -independent methods. The number of colony-forming units (CFU) was reduced below the detection level after disinfection with UV and high ozone dose (700 mV), and 1% of the initial level for the low ozone dose (400 mV). After less than 3 days, the CFU was back or above the starting point for the control, UV and low ozone treatment, whereas it took slightly more than 6 days for the high ozone treatment. Disinfection increased substrate availability and reduced cell densities. Lack of competition and predation induced the recolonization by opportunistic bacteria (r-strategists), with significant increase in bacterial numbers and a low diversity (based on DGGE band pattern). All cultures stabilized after the initial recolonization phase (except Oz700) where competition due to crowding and nutrient limitation favoured bacteria with high substrate affinity (K-strategists), resulting in higher species richness and diversity (based on DGGE band pattern). The bacterial community was significantly altered qualitatively and quantitatively and may have a higher potential as invaders in the recipient depending on disinfection method and the time of release. These results have implications for the treatment strategy used for ballast water.

Ultraviolet radiation as a ballast water treatment strategy: Inactivation of phytoplankton measured with flow cytometry.

This study investigates different UV doses (mJ/cm(2)) and the effect of dark incubation on the survival of the algae Tetraselmis suecica, to simulate ballast water treatment and subsequent transport. Samples were UV irradiated and analyzed by flow cytometry and standard culturing methods. Doses of ≥400 mJ/cm(2) rendered inactivation after 1 day as measured by all analytical methods, and are recommended for ballast water treatment if immediate impairment is required. Irradiation with lower UV doses (100-200 mJ/cm(2)) gave considerable differences of inactivation between experiments and analytical methods. Nevertheless, inactivation increased with increasing doses and incubation time. We argue that UV doses ≥100 mJ/cm(2) and ≤200 mJ/cm(2) can be sufficient if the water is treated at intake and left in dark ballast tanks. The variable results demonstrate the challenge of giving unambiguous recommendations on duration of dark incubation needed for inactivation when algae are treated with low UV doses.

Flow cytometric applicability to evaluate UV inactivation of phytoplankton in marine water samples.

Disinfection of microbes is of importance to prevent the spread of pathogens and non-indigenous species in the environment. Here we test the applicability of using flow cytometry (FCM) to evaluate inactivation of the phytoplankter Tetraselmis suecica after UV irradiation and labeling with the esterase substrate 5-carboxyfluorescein diacetate acetoxymethyl ester (CFDA-AM). Non-irradiated and UV irradiated samples were analyzed with the plate count technique and FCM for 24 days. The numbers of colony forming units were used as a standard to develop a FCM protocol. Our protocol readily distinguishes live and dead cells, but challenges were encountered when determining whether UV damaged cells are dying or repairable. As damaged cells can represent a risk to aquatic organisms and/or humans, this was taken into account when developing the FCM protocol. In spite of the above mentioned challenges we argue that FCM represents an accurate and rapid method to analyze T. suecica samples.

Comparing flow cytometry and microscopy in the quantification of vital aquatic organisms in ballast water

ABSTRACT The ability to quantify vital aquatic organisms in the 2–50 µm size range was compared between five different flow cytometers and several different microscopes. Counts of calibration beads, algal monocultures of different sizes as well as organisms in a Wadden Sea sample were compared. Flow cytometers and microscopes delivered different bead concentrations. These differences between the instruments became larger for algal monocultures and were even higher for the Wadden Sea sample. It was observed that the concentration differences were significant between flow cytometer and microscope counts, and that this difference increased with the size of the objects counted. Microscope counts were more accurate for larger (50 µm) objects because cytometers struggled with bigger particles that clogged the instruments. Contrary to microscopy, the flow cytometers were capable of accurately enumerating cultured cells in the 2–10 µm size range and cells in the lower size range of the 10–50 µm size class. Flow cytometers were also well-suited to assess low abundance samples due to their ability to process larger volumes than microscopes. The results were used to indicate which tools are suitable for ballast water monitoring: flow cytometry is a suitable technology for an indicative and real time analysis of ballast water samples whilst only microscopy would be robust enough for detailed taxonomical analyses.

Application of flow cytometry in ballast water analysis - biological aspects

Ballast water may, when discharged, cause the spread of nonindigenous and potentially invasive species. International ballast water treatment regulations have accelerated the development of new methods to detect, enumerate and assess the status of organisms in the water to be discharged. Flow cytometry (FCM) is a powerful technique with a broad range of applications with the possibility for multi-parametric analysis and the potential of combining it with other techniques being two strong advantages. This review will discuss whether FCM is suitable for ballast water analysis according to international ballast water regulations, and sum up the advantages and disadvantages. It will also give an overview of available labeling techniques. Finally, a discussion on the knowledge gaps and future potential for FCM within ballast water analysis is presented.