Biodegradation of Terrigenous Organic Matter in a Stratified Large-Volume Water Column: Implications of the Removal of Terrigenous Organic Matter in the Coastal Ocean.

Large amounts of terrigenous organic matter (TOM) are delivered to the ocean every year. However, removal processes of TOM in the ocean are still poorly constrained. Here, we report results from a 339-day dark incubation experiment with a unique system holding a vertically stratified freshwater-seawater column. The quality and quantity of dissolved organic matter (DOM), RNA-based size-fraction microbial communities, and environmental factors were high-frequency-monitored. Microbial processes impacted TOM composition, including an increased DOM photobleaching rate with incubation time. The mixed layer had changed the bacterial community structure, diversity, and higher oxygen consumption rate. A two-end member modeling analysis suggested that estimated nutrient concentrations and prokaryotic abundance were lower, and total dissolved organic carbon was higher than that of the measured values. These results imply that DOM biodegradation was stimulated during freshwater-seawater mixing. In the bottom layer, fluorescent DOM components increased with the incubation time and were significantly positively related to highly unsaturated, oxygenated, and presumably aromatic compound molecular formulas. These results suggest that surfaced-derived TOM sinking leads to increased DOM transformation and likely results in carbon storage in the bottom water. Overall, these results suggest that microbial transforming TOM plays more important biogeochemical roles in estuaries and coastal oceans than what we know before.

Enumerating viable phytoplankton using a culture-based Most Probable Number assay following ultraviolet-C treatment.

Ballast water management systems (BWMS) must be tested to assess their compliance with standards for the discharge of organisms, for example in the ≥ 10- and < 50-µm size category, which is dominated by phytoplankton. Assessment of BWMS performance with the vital stains fluorescein diacetate + 5-chlorofluorescein diacetate, required by regulations in the USA, is problematic in the case of ultraviolet-C (UVC) radiation. This is because UVC targets nucleotides-and thus reproduction, hence viability-rather than membrane integrity, which is assayed by the stains. The Serial Dilution Culture-Most Probable Number (SDC-MPN) method, long used to enumerate fragile phytoplankton from natural communities, is appropriate for counting viable phytoplankton. We developed QA/QC "best practice" criteria for its application as a robust and repeatable assay of viable cells in cultures of phytoplankton before and after experimental treatment, then constructed dose-response curves for UVC-induced loss of viable cells in 12 species of phytoplankton from seven divisions. Sensitivity to UVC, expressed as the dose required to reduce viability by 99%-the criterion for type approval of treatment systems-varied more than 10-fold and was not correlated with cell size. The form of the dose-response curves varied between taxa, with most having a threshold dose below which there was no reduction in viability. Analysis of the patterns of growth indicates that if recovery from treatment occurred, it was complete in 1 or 2 days in > 80% of cases, long before the assays were terminated. We conclude that the SDC-MPN assay as described is robust and adaptable for use on natural phytoplankton.

Classification of phytoplankton cells as live or dead using the vital stains fluorescein diacetate and 5-chloromethylfluorescein diacetate.

Regulations for ballast water treatment specify limits on the concentrations of living cells in discharge water. The vital stains fluorescein diacetate (FDA) and 5-chloromethylfluorescein diacetate (CMFDA) in combination have been recommended for use in verification of ballast water treatment technology. We tested the effectiveness of FDA and CMFDA, singly and in combination, in discriminating between living and heat-killed populations of 24 species of phytoplankton from seven divisions, verifying with quantitative growth assays that uniformly live and dead populations were compared. The diagnostic signal, per-cell fluorescence intensity, was measured by flow cytometry and alternate discriminatory thresholds were defined statistically from the frequency distributions of the dead or living cells. Species were clustered by staining patterns: for four species, the staining of live versus dead cells was distinct, and live-dead classification was essentially error free. But overlap between the frequency distributions of living and heat-killed cells in the other taxa led to unavoidable errors, well in excess of 20% in many. In 4 very weakly staining taxa, the mean fluorescence intensity in the heat-killed cells was higher than that of the living cells, which is inconsistent with the assumptions of the method. Applying the criteria of ≤5% false negative plus ≤5% false positive errors, and no significant loss of cells due to staining, FDA and FDA+CMFDA gave acceptably accurate results for only 8-10 of 24 species (i.e., 33%-42%). CMFDA was the least effective stain and its addition to FDA did not improve the performance of FDA alone.

On the use of the serial dilution culture method to enumerate viable phytoplankton in natural communities of plankton subjected to ballast water treatment.

Discharge standards for ballast water treatment (BWT) systems are based on concentrations of living cells, for example, as determined with vital stains. Ultraviolet radiation (UV) stops the reproduction of microorganisms without killing them outright; they are living, but not viable, and ecologically as good as dead. Consequently, UV-treated discharge can be compliant with the intent of regulation while failing a live/dead test. An alternative evaluation of BWT can be proposed based on the assessment of viable, rather than living, cells in discharge water. In principle, the serial dilution culture-most probable number (SDC-MPN) method provides the appropriate measure for phytoplankton. But, the method has been criticized, particularly because it is thought that many phytoplankton species cannot be cultured. A review of the literature shows that although SDC-MPN has been used for more than 50 years-generally to identify and count phytoplankton species that cannot be preserved-its application to enumerate total viable phytoplankton seems to be new, putting past criticisms of the method in a different light. Importantly, viable cells need to grow only enough to be detected, not to be brought into sustained culture, and competition between species in a dilution tube is irrelevant as long as the winner is detectable. Thorough consideration of sources of error leads to recommendations for minimizing and quantifying uncertainties by optimizing growth conditions and conducting systematic comparisons. We conclude that with careful evaluation, SDC-MPN is potentially an effective method for assessing the viability of phytoplankton after BWT.

Curvature in models of the photosynthesis-irradiance response.

An equation for the rate of photosynthesis as a function of irradiance introduced by T. T. Bannister included an empirical parameter b to account for observed variations in curvature between the initial slope and the maximum rate of photosynthesis. Yet researchers have generally favored equations with fixed curvature, possibly because b was viewed as having no physiological meaning. We developed an analytic photosynthesis-irradiance equation relating variations in curvature to changes in the degree of connectivity between photosystems, and also considered a recently published alternative, based on changes in the size of the plastoquinone pool. When fitted to a set of 185 observed photosynthesis-irradiance curves, it was found that the Bannister equation provided the best fit more frequently compared to either of the analytic equations. While Bannister's curvature parameter engendered negligible improvement in the statistical fit to the study data, we argued that the parameter is nevertheless quite useful because it allows for consistent estimates of initial slope and saturation irradiance for observations exhibiting a range of curvatures, which would otherwise have to be fitted to different fixed-curvature equations. Using theoretical models, we also found that intra- and intercellular self-shading can result in biased estimates of both curvature and the saturation irradiance parameter. We concluded that Bannister's is the best currently available equation accounting for variations in curvature precisely because it does not assign inappropriate physiological meaning to its curvature parameter, and we proposed that b should be thought of as the expression of the integration of all factors impacting curvature.

Spectral fluorometric characterization of phytoplankton community composition using the Algae Online Analyser.

The utility of a multiple-fixed-wavelength spectral fluorometer, the Algae Online Analyser (AOA), as a means of quantifying phytoplankton biomass and community composition was tested using natural communities from two southeastern United States estuaries, North Inlet, South Carolina, and the Neuse River Estuary, North Carolina. Estimates of biomass (as chlorophyll a) were correlated with HPLC values and variations (usually over-estimates) were consistent with effects of light intensity and nutrient availability on fluorescence quenching. AOA estimates of taxonomic structure were consistent with those from HPLC-derived marker pigments by ChemTax, with both methods indicating domination by chromophytes and green algae in North Inlet and chromophytes and cyanobacteria in the Neuse. We recommend frequent calibration by discrete sample collection, and calibration with species representative of the region of interest. Overall, the AOA appears to be a useful tool for monitoring of phytoplankton community composition, especially as an early warning system for the detection of harmful algal blooms.