Description: Coomassie stainable particles (CSP) are protein-containing transparent particles that can be stained with Coomassie brilliant blue (CBB) and are found abundantly in aquatic systems; however, their distribution and role remain poorly known, in part due to the lack of an efficient method to study them. We developed a new, simple, and low cost semi-quantitative spectrophotometric method for determination of CSP in aquatic systems. The method is analogous to that used to quantify polysaccharide-rich gel particles called transparent exopolymeric particles (TEP). CSP concentration is determined relative to bovine serum albumin (BSA) standard aggregates (in a manner similar to how TEP is quantified relative to xanthan gum). The method is based on the linear relationship between CSP concentration and the absorbance of the eluted dye from a CBB-protein complex, which has an absorbance maximum (λmax) at 615 nm. The limit of detection and the precision (%RSD) for the proposed method are 6 μg BSA equivalent and 11%, respectively. The new spectrophotometric method was validated with the existing microscopic method. This new method to quantify CSP is simple, enables rapid measurements, and allows a more efficient comparison with TEP concentrations than the present microscopic method. The spectrophotometric analyses will further the investigation of the abundance, distribution, and role of CSP in the biogeochemistry of the ocean.

Description: Highlights: • Coagulation efficiency of the coccolithophorid Emiliania huxleyi was determined with Couette flow devices. • Higher coagulation efficiencies of cells were observed at lower growth rates. • Coagulation efficiency increases with the extracellular polysaccharides fraction. Abstract: Coagulation of small particles results in the formation of larger aggregates that play an important role in the biological pump, moving carbon and other elements from the surface to the deep ocean and seafloor. In this study, we estimated the efficiency of particle coagulation of the coccolithophore Emiliania huxleyi at different growth rates using Couette flow devices at a natural shear rate. To determine the impacts of chemical and biological factors involved in aggregate formation, we investigated how variance in organic matter composition, and in particular the presence of extracellular polysaccharides (EP), including transparent exopolymer particles (TEP) and acidic polysaccharides attached to the coccolith surface, affect the coagulation efficiency (α). When E. huxleyi was grown in a chemostat at different growth rates, coagulation efficiency increased from ~ 0.40 to 1 as cell growth rates declined and nutrients became more limited. With declining growth rate the concentration of EP and the number of detached coccoliths increased. Overall a close correlation between coagulation efficiency of E. huxleyi and the ratio of EP to total particle volume was observed. The minimum value of α of ~ 0.4 determined during this study is higher than estimates published for other phytoplankton cells, and may be related to the presence of EP attached to coccoliths. Based on our findings, we suggest that E. huxleyi is more prone to form aggregates, particularly during the decline of blooms, when increased production of EP and enhanced shedding of coccoliths coincide. This may be one explanation for why blooms of E. huxleyi play an important role in the biological carbon pump, efficiently enhancing the vertical flux of particles, as has been suggested by sediment trap studies.

Description: We measured the vertical distribution of TEP and CSP at a site on the Bermuda Rise by staining parallel samples with Alcian Blue and Coomassie Brilliant Blue, during five research cruises in 2012-2013. We used a new spectrophotometric method, analogous to that for TEP, to measure CSP concentration relative to bovine serum albumin (BSA) standard aggregates. The method is based on the linear relationship between CSP concentration and the absorbance of the eluted dye from a CBB-protein complex. TEP concentrations ranged from 23-53 µg XG eqL-1, and decreased with depth. CSP concentration ranged from 2-24 µg BSA eqL-1, and values decreased with depth, but the CSP max was slightly below that of TEP. The CSP and TEP profile shapes and loss rates were different. These differences support the idea that the two particle types have different origins. Either CSP is more labile than TEP, or TEP and CSP are produced at the same depth, but TEP ascends to the surface due to the combination of its buoyancy, and the high TEP to solid ratio found in low productivity regions.

Description: Highlights: • TEPs and CSPs showed different production patterns and particle-association behaviors. • TEPs and CSPs had different vertical distributions in the Sargasso Sea. • CSP as well as TEP gels are linked by cation bridging. • FlowCAM can be used for in-situ visualization and imaging of TEPs and CSPs in parallel-stained samples. • In-situ visualization of TEPs and CSPs led to new insights about particle interaction and their role in aggregation. Abstract: The discovery of ubiquitous, abundant and transparent gel-like particles, such as the polysaccharide-containing transparent exopolymer particles (TEP) and protein-containing Coomassie stainable particles (CSP) has changed our conception of particle–organism interaction and created new questions about the origin, composition, and role of these particles in aquatic systems. Using both standard and novel staining methods, we studied these gel-like particles to determine whether CSP and TEP are sub-units of the same particle, are distinct particles with different characteristics and behaviors, or are both. Our seawater mesocosm results show that phytoplankton produce both TEP and CSP; however, their highest abundances occur at differences phases in the phytoplankton bloom. We developed a new technique for visualizing stained transparent material in unfiltered aqueous samples with the FlowCAM; this technique allows in-situ visualization and imaging of TEP and CSP in parallel stained samples. Visual examination of stained and unstained TEP and CSP from seawater microcosms, marine algal cultures, and freshwater showed that TEP and CSP have different shape, size and particle-association behavior. In a diatom-dominated microcosm, TEP concentrations were higher than CSP concentrations and unlike CSP, TEP were usually associated with diatom cells or aggregates. The cyanobacteria culture, however, showed higher CSP than TEP concentrations and aggregates of those cells appeared to be CSP-rich. Vertical and seasonal distributions of TEP and CSP in the Sargasso Sea were different. Even though both types of particles were most abundant in the upper 100 m of the water column, CSP closely followed fluorescence and total particle concentration, while the highest TEP concentration was always in the shallowest sample collected. Thus, we conclude that TEP and CSP are different particles, produced by different species at different growth phases and rates. They have different roles and are affected by different processes according to the community composition and environmental conditions.

Description: Melanoidins, condensation products of sugars and amino acids, are thought to represent a key link in the transformation of polysaccharides and proteins to humic material in the marine environment. We investigated adsorption behaviour of melanoidins prepared in equimolar solutions of glucose and amino acids of choice (glutamic acid, valine and lysine) and pseudomelanoidins which were prepared from glucose only. Melanoidins were prepared using different condensation times (2, 4, 16 and 32 days). Synthesized melanoidins were separated into different molecular mass fractions. Fractionation of melanoidins by sorption on the macroreticular resin XAD-8 separated melanoidins into hydrophobic neutral, hydrophobic acid and hydrophilic fractions. Adsorption of melanoidins and their different fractions was studied at a mercury electrode by directly measuring the change of the double layer capacitance caused by the adsorption of organic molecules on the electrode surface through phase sensitive alternating current voltammetry. The hydrophobic acid fraction of melanoidins accounted for most of the adsorption behaviour of melanoidins. Consequently, the higher molecular mass fraction of melanoidins (〉10 kDa) exhibits a stronger adsorption in comparison to the lower molecular mass fraction (〈3 kDa) of the same melanoidin. The good fit of adsorption data of melanoidins and pseudomelanoidins to the same adsorption isotherm supports the idea that melanoidins are comprised of a sugar derived “backbone” that is responsible for the adsorption behaviour of melanoidin, while the presence of nitrogen atoms is responsible for the complexation of copper ions. Adsorption characteristics and complexation ability of melanoidins and natural organic matter were similar. Our results suggest that in the process of humification, selective adsorption of condensation products on aqueous surfaces may lead to a progressive immobilization of certain fractions, i.e., it is probable that higher molecular mass components accumulate at aquatic surfaces, while lower mass components remain in solution.

Description: The quantitative relationship between organic carbon and mineral contents of particles sinking below 1800 m in the ocean indicates that organisms with mineral shells such as coccolithophores are of special importance for transporting carbon into the deep sea. Several hypotheses about the mechanism behind this relationship between minerals and organic matter have been raised, such as mineral protection of organic matter or enhanced sinking rates through ballast addition. We examined organic matter decomposition of calcifying and non-calcifying Emiliania huxleyi cultures in an experiment that allowed aggregation and settling in rotating tanks. Biogenic components such as particulate carbon, particulate nitrogen, particulate volume, pigments, transparent exopolymer particles (TEP), and particulate amino acids in suspended particles and aggregates were followed over a period of 30 d. The overall pattern of decrease in organic matter, the amount of recalcitrant organic matter left after 30 d, and the compositional changes within particulate organic matter indicated that cells without a shell are more subject to loss than calcified cells. It is suggested that biogenic calcite helps in the preservation of particulate organic matter (POM) by offering structural support for organic molecules. Over the course of the experiment, half the particulate organic carbon in both calcifying and non-calcifying cultures was partitioned into aggregates and remained so until the end of the experiment. The partial protection of particulate organic matter from solubilization by biominerals and by aggregation that was observed in our experiment may help explain the robustness of the relationship between organic and mineral matter fluxes in the deep ocean.

Description: The MedFlux project was devised to determine and model relationships between organic matter and mineral ballasts of sinking particulate matter in the ocean. Specifically we investigated the ballast ratio hypothesis, tested various commonly used sampling and modeling techniques, and developed new technologies that would allow better characterization of particle biogeochemistry. Here we describe the rationale for the project, the biogeochemical provenance of the DYFAMED site, the international support structure, and highlights from the papers published here. Additional MedFlux papers can be accessed at the MedFlux web site (http://msrc.sunysb.edu/MedFlux/).

Description: To investigate the role of ballasting by biogenic minerals in the export of organic matter in the ocean, a laboratory experiment was conducted comparing aggregate formation and settling velocity of non-calcifying and calcifying strains of the coccolithophore Emiliania huxleyi. Experiments were conducted by making aggregates using a roller table and following aggregate properties during incubation for a period of 40 days. Size, shape, and settling velocities of aggregates were described by image analysis of video pictures recorded during the roller tank incubation. Our results show that biogenic calcite has a strong effect on the formation rate and abundance of aggregates and on aggregate properties such as size, excess density, porosity, and settling velocity. Aggregates of calcifying cells (AGGCAL) formed faster, were smaller and had higher settling velocities, excess densities, and mass than those of non-calcifying cells (AGGNCAL). AGGCAL showed no loss during the duration of the experiment, whereas AGGNCAL decreased in size after 1 month of incubation. Potential mechanisms that can explain the different patterns in aggregate formation are discussed. Comparison of settling velocities of AGGCAL and AGGNCAL with aggregates formed by diatoms furthermore indicated that the ballast effect of calcite is greater than that of opal. Together these results help to better understand why calcite is of major importance for organic matter fluxes to the deep ocean.