Marine Aggregate Formation Research Articles

Secretion of sulfated fucans by diatoms may contribute to marine aggregate formation

AbstractMicroalgae produce copious amounts of structurally diverse polysaccharides, some are bound within cells and cell walls, while others are secreted into the surrounding seawater. A fraction of the secreted polysaccharides assembles into particles promoting aggregation and in turn formation of aggregates increases the export of carbon into the deep ocean via sinking. However, specific polysaccharides contributing to particle formation and carbon export remain unknown. Here, we studied microalgae polysaccharide composition in a system of reduced complexity consisting of lab grown monospecific cultures of the centric diatom species Thalassiosira weissflogii and Chaetoceros socialis. We followed the abundance and dynamics of five specific polysaccharide types in the dissolved and particulate organic matter (DOM and POM) for two weeks. Polysaccharides were detected using monoclonal antibodies (mAbs) specific for β‐1,4‐mannan, β‐1,4‐xylan, arabinogalactan, and two fucose‐containing sulfated polysaccharide (FCSP) epitopes. Additionally, glycan composition of all samples was analyzed by monosaccharide analysis. The time series revealed polysaccharides partition differently between the dissolved and particulate carbon pools. β‐1,4‐xylan and β‐1,4‐mannan were mainly present in POM, possibly as cell wall polymers, while FCSPs were found in both DOM and POM. The data showed that the main glycan component secreted by diatoms was fucose‐containing polysaccharide, which accumulated in DOM over time. Roller tank experiments were used to induce aggregate formation finding FCSP transitioned from DOM to POM under aggregating conditions. These results suggest that diatom‐secreted FCSPs are involved in the formation of aggregates, which promote the formation of particles, and potentially carbon export.

Turbulence mediates marine aggregate formation and destruction in the upper ocean

Marine aggregates formed through particle coagulation, large ones (>0.05 cm) also called marine snow, make a significant contribution to the global carbon flux by sinking from the euphotic zone, impacting the Earth’s climate. Since aggregate sinking velocity and carbon content are size-dependent, understanding the physical mechanisms controlling aggregate size distribution is fundamental to determining the biological carbon pump efficiency. Theoretical, laboratory and in-situ studies of flocculation have suggested that turbulence in the benthic boundary layer is important for aggregate formation and destruction, but the small number of field observations has limited our understanding of the role of turbulence on aggregation processes in the ocean surface layer away from energetic boundaries. Using simultaneous field observations of turbulence and aggregates, we show how aggregate formation, destruction, morphology and size distribution in the ocean surface layer (10–100 m) are mediated by interactions between turbulence and aggregate concentration. Our findings suggest that turbulence enhances aggregate formation up to a critical turbulent kinetic energy dissipation rate of 10−6 (W kg−1), above which the smallest turbulent eddies limit aggregate size.

Heterotrophic Bacteria Enhance the Aggregation of the Marine Picocyanobacteria Prochlorococcus and Synechococcus.

Marine picocyanobacteria are ubiquitous primary producers across the world’s oceans, and play a key role in the global carbon cycle. Recent evidence stemming from in situ investigations have shown that picocyanobacteria are able to sink out of the euphotic zone to depth, which has traditionally been associated with larger, mineral ballasted cells. The mechanisms behind the sinking of picocyanobacteria remain a point of contention, given that they are too small to sink on their own. To gain a mechanistic understanding of the potential role of picocyanobacteria in carbon export, we tested their ability to form “suspended” (5–60 μm) and “visible” (ca. > 0.1 mm) aggregates, as well as their production of transparent exopolymer particles (TEP)—which are a key component in the formation of marine aggregates. Additionally, we investigated if interactions with heterotrophic bacteria play a role in TEP production and aggregation in Prochlorococcus and Synechococcus by comparing xenic and axenic cultures. We observed TEP production and aggregation in batch cultures of axenic Synechococcus, but not in axenic Prochlorococcus. Heterotrophic bacteria enhanced TEP production as well as suspended and visible aggregate formation in Prochlorococcus, while in Synechococcus, aggregation was enhanced with no changes in TEP. Aggregation experiments using a natural plankton community dominated by picocyanobacteria resulted in aggregation only in the presence of the ballasting mineral kaolinite, and only when Synechococcus were in their highest seasonal abundance. Our results point to a different export potential between the two picocyanobacteria, which may be mediated by interactions with heterotrophic bacteria and presence of ballasting minerals. Further studies are needed to clarify the mechanistic role of bacteria in TEP production and aggregation of these picocyanobacteria.

Effects of Higher CO2 and Temperature on Exopolymer Particle Content and Physical Properties of Marine Aggregates

We investigated how future ocean conditions, and specifically the interaction between temperature and CO2, might affect marine aggregate formation and physical properties. Initially, mesocosms filled with coastal seawater were subjected to three different treatments of CO2 concentration and temperature: (1) 750 ppm CO2, 16°C, (2) 750 ppm CO2, 20°C, and (3) 390 ppm CO2, 16°C. Diatom-dominated phytoplankton blooms were induced in the mesocosms by addition of nutrients. In aggregates produced in roller tanks using seawater taken from the mesocosms during different stages of the bloom, we measured sinking velocity, size, chlorophyll a, particulate organic carbon and nitrogen, and exopolymer particle content; excess density and mass were calculated from the sinking velocity and size of the aggregates. As has been seen in previous experiments, no discernable differences in overall nutrient uptake, chlorophyll-a concentration, or exopolymer particle concentrations could be related to the acidification treatment in the mesocosms. In addition, in the aggregates formed during the roller tank experiments (RTEs), we observed no statistically significant differences in chemical composition among the treatments during Pre-Bloom, Bloom, and Post-Bloom periods. However, physical characteristics were different and showed a synergistic effect of warmer temperature and higher CO2 during the Pre-Bloom period; at this time, temperature had a larger effect than CO2 on aggregate sinking velocity. In RTEs with warmer and acidified treatment (future conditions), aggregates were larger, heavier, and settled faster than aggregates formed at present-day or only acidified conditions. During the Post-Bloom, however, aggregates formed under present and future conditions had similar physical properties. In acidified tanks at ambient temperature, aggregates were slower, smaller and less dense than those formed at the same temperature but under present CO2 or under warmer and acidified conditions. Thus, the sinking velocity of aggregates formed in acidified tanks at ambient temperature was slower than the other two cases. Our findings point out the potential of ocean acidification and warming to modify physical properties of sinking aggregates but also emphasize the need of future experiments investigating multiple environmental stressors to clarify the importance of each factor.

Identification of Bacterial Genes Expressed During Diatom-Bacteria Interactions Using an in Vivo Expression Technology Approach

Diverse microalgae-bacteria interactions play important roles for nutrient exchange processes and marine aggregate formation leading to the cycling, mineralization, or sedimentation of organic carbon in the Oceans. The main goal of this study is to report an alternative way to assess a distinct diatom-bacteria interaction. To study this at the cellular scale, an in vitro interaction model system consisting of the diatom, Thalassiosira weissflogii, and the gamma-proteobacterium, Marinobacter adhaerens HP15, had previously been established. HP15 is able to attach to T. weissflogii cells, to induce transparent exopolymeric particle formation, and to increase marine aggregation formation. Several bacterial genes important during this interaction have been studied thus far, but genes specifically expressed in co-culture remained unknown. To identify such bacterial genes, an in vivo Expression Technology (IVET) screen was employed. For this, a promoter-trapping vector containing a fusion between a promoter-less selection marker gene and a promoterless reporter gene was constructed. Construction of a library of plasmids carrying genomic fragments upstream of the fusion and its subsequent transformation into a selection marker mutant allowed detection of 30 bacterial promoters specifically expressed during the interactions with T. weissflogii. Their sequence analyses revealed that the corresponding genes could be involved in many processes such as biochemical detection of diatom cells, bacterial attachment, metabolic exchange of nitrogen compounds, and resistance towards heavy metals. Identification of genes potentially involved in branched-chain amino acid uptake and utilization confirmed our previous results of a proteomics analysis. Since our current approach identified several additional genes to be induced in co-culture, use of IVET might be a valuable complementing strategy to proteomic or transcriptomic analysis of the diatom-bacteria crossplay. The current IVET approach demonstrated that the interaction between M. adhaerens HP15 and T. weissflogii is multifactorial and is likely to involve a complex network of physiological processes.

Prochlorococcus as a Possible Source for Transparent Exopolymer Particles (TEP).

Transparent exopolymer particles (TEP), usually associated with phytoplankton blooms, promote the formation of marine aggregates. Their exportation to deep waters is considered a key component of the biological carbon pump. Here, we explored the role of solar radiation and picocyanobacteria in the formation of TEP in oligotrophic surface waters of the Atlantic and Pacific Oceans in ten on-deck incubation experiments during the Malaspina 2010 Expedition. TEP concentrations were low on the ocean’s surface although these concentrations were significantly higher on the surface of the Pacific (24.45 ± 2.3 μg XG Eq. L-1) than on the surface of the Atlantic Ocean (8.18 ± 4.56 μg XG Eq. L-1). Solar radiation induced a significant production of TEP in the on-deck experiments from the surface water of the Pacific Ocean, reaching values up to 187.3 μg XG Eq. L-1 compared with the low production observed in the dark controls. By contrast, TEP production in the Atlantic Ocean experiments was lower, and its formation was not related to the light treatments. Prochlorococcus sp. from the surface ocean was very sensitive to solar radiation and experienced a high cell decay in the Pacific Ocean experiments. TEP production in the on-deck incubation experiments was closely related to the observed cell decay rates of Prochlorococcus sp., suggesting that this picocyanobacteria genus is a potential source of TEP. The evidence to propose such potential role was derived experimentally, using natural communities including the presence of several species and a variety of processes. Laboratory experiments with cultures of a non-axenic strain of Prochlorococcus marinus were then used to test TEP production by this genus. TEP concentrations in the culture increased with increasing cell abundance during the exponential phase, reaching the highest TEP concentration at the beginning of the stationary phase. The average TEP concentration of 1474 ± 226 μg XG Eq. L-1 (mean ± SE) observed at the stationary phase of P. marinus cultures is comparable with the values reported in the literature for diatom cultures, also growing in non-axenic as well as axenic cultures. Our results identify Prochlorococcus sp. as a possible relevant source of TEP in the oligotrophic ocean.

Development of a genetic system for Marinobacter adhaerens HP15 involved in marine aggregate formation by interacting with diatom cells

Diatom aggregation is substantial for organic carbon flux from the photic zone to deeper waters. Many heterotrophic bacteria ubiquitously found in diverse marine environments interact with marine algae and thus impact organic matter and energy cycling in the ocean. In particular, Marinobacter adhaerens HP15 induces aggregate formation while interacting with the diatom, Thalassiosira weissflogii. To study this effect at the molecular level, a genetic tool system was developed for strain HP15. The antibiotic susceptibility spectrum of this organism was determined and electroporation and conjugation protocols were established. Among various plasmids of different incompatibility groups, only two were shown to replicate in M. adhaerens. 1.4×10−3 transconjugants per recipient were obtained for a broad-host-range vector. Electroporation efficiency corresponded to 1.1×105CFU per μg of DNA. Transposon and gene-specific mutageneses were conducted for flagellum biosynthetic genes. Mutant phenotypes were confirmed by swimming assay and microscopy. Successful expression of two reporter genes in strain HP15 revealed useful tools for gene expression analyses, which will allow studying diverse bacteria–algae interactions at the molecular level and hence to gain a mechanistic understanding of micro-scale processes underlying ocean basin-scale processes. This study is the first report for the genetic manipulation of a Marinobacter species which specifically interacts with marine diatoms and serves as model to additionally analyze various previously reported Marinobacter–algae interactions in depth.

Complete genome sequence of Marinobacter adhaerens type strain (HP15), a diatom-interacting marine microorganism.

Marinobacter adhaerens HP15 is the type strain of a newly identified marine species, which is phylogenetically related to M. flavimaris, M. algicola, and M. aquaeolei. It is of special interest for research on marine aggregate formation because it showed specific attachment to diatom cells. In vitro it led to exopolymer formation and aggregation of these algal cells to form marine snow particles. M. adhaerens HP15 is a free-living, motile, rod-shaped, Gram-negative gammaproteobacterium, which was originally isolated from marine particles sampled in the German Wadden Sea. M. adhaerens HP15 grows heterotrophically on various media, is easy to access genetically, and serves as a model organism to investigate the cellular and molecular interactions with the diatom Thalassiosira weissflogii. Here we describe the complete and annotated genome sequence of M. adhaerens HP15 as well as some details on flagella-associated genes. M. adhaerens HP15 possesses three replicons; the chromosome comprises 4,422,725 bp and codes for 4,180 protein-coding genes, 51 tRNAs and three rRNA operons, while the two circular plasmids are ~187 kb and ~42 kb in size and contain 178 and 52 protein-coding genes, respectively.

Transparent exopolymer particles (TEP) from marine suspension feeders enhance particle aggregation

Transparent exopolymer particles (TEP) are known to form from dissolved precursors released by phytoplankton and bacteria. Many benthic suspension feeders also have been shown to produce significant amounts of TEP under field and laboratory conditions. Although TEP from phyto- plankton have been implicated in enhancing aggregation and sedimentation of suspended material, the role of TEP from suspension feeders in aggregate formation is still unknown. In order to deter- mine if TEP produced by suspension feeders enhance the formation of marine aggregates, organic matter from actively feeding blue mussels Mytilus edulis and sea vases Ciona intestinalis held in recirculating chambers was collected. Control water was collected from chambers without animals. Animal-conditioned and control water were then mixed with 1 of 2 non-sticky particles (silica-amino beads or diatomaceous earth). The suspensions were transferred to 250 ml bottles, which were placed on a roller table to generate aggregates. Particle aggregation was quantified by examining the size distribution of particles over time by means of an electronic particle counter (particles between 6 and 90 μm), and by counting large (greater than ca. 100 μm) aggregates under a microscope at the end of the experiment. We found that significantly more aggregates formed in the animal-conditioned water than in control water after 48 h, for both species and both kinds of added particles. The number of large aggregates that formed was related to TEP concentration, and not related to total or dissolved organic carbon. Our results suggest that suspension feeders produce significant quantities of TEP that can enhance particle aggregation.

The effect of inhomogeneous stickiness on polymer aggregation

The aggregation of polymers is important in the formation of marine aggregates and the vertical transport of material in the ocean. A polymer may be inhomogeneous along its length, with associating groups at some points along its length where bonds are more likely to form. In this paper we investigate the effects of inhomogeneous ‘stickiness’ along the polymer length. We describe the results of three-dimensional off-lattice simulations of polymer–polymer aggregation for four different types of polymer: polymers which are sticky along their entire length, polymers which are sticky at the ends only and two types of polymer which are slightly sticky along their entire length. We examine the mean radius of gyration and the fractal dimension of the resulting aggregates and the dynamics of aggregation. The slightly sticky polymers and the polymers which are sticky only at the ends form aggregates with a higher fractal dimension than the polymers which are sticky along their entire length. However, the mean radius of gyration of the aggregates formed by polymers which are sticky only at the ends is significantly larger than that of the aggregates formed from slightly sticky polymers. The aggregation dynamics are also different for the polymers which are sticky only at the ends compared to the slightly sticky polymers. A single ‘stickiness value’ is therefore likely to be inadequate to describe a polymer. We also examine the effect of polymer rigidity; it seems that the effect of inhomogeneous stickiness is greater for almost-straight polymers than for coiled chains.

Different parameterizations of marine snow in a 1D-model and their influence on representation of marine snow, nitrogen budget and sedimentation

A model is presented that simulates the formation of marine aggregates from particles of different origin inside a model of pelagic biological processes. Experiments are carried out with parameterizations appropriate for different types of aggregates, using different kinds of physical forcing, and compared to observations of dissolved inorganic nitrogen (DIN), particulate organic nitrogen (PON), marine snow concentration, and sedimentation. The occurrence of large, macroscopically visible aggregates (marine snow) can best be simulated with parameterizations that have been derived from in situ observations of marine snow, but not with a parameterization sufficient for dense particles. The parameterization strongly determines the amount and timing of deep export, as well as the post-bloom development of the food web in the upper layers. Detritus in aggregates plays a role mainly during times when zooplankton are abundant, as e.g. in the western Arabian Sea during Southwest Monsoon. Then the large aggregates as fast sinking vehicles may remove detritus quickly from shallow and mid-water depth, preventing the accumulation of nutrients that are produced via detritus decomposition. In this region, detritus contributes strongly to deep sedimentation. The nitrogen budget at this location with regard to the observations cannot be closed: depending on model type, either the model simulates too high sedimentation, or too high DIN. Possible causes for this mismatch include undercollection by sediment traps, inaccurate representation of physical processes in the model and the neglect of biological processes, such as production of dissolved organic matter or denitrification.

Scales and processes in the aggregation of diatom blooms: high time resolution and wide size range records in a mesocosm study

Diatoms and the large, fast-sinking aggregates they form during blooms play an important role in downward flux of particles in the ocean. To study how the aggregation process operates on particle dynamics, diatom blooms were generated and followed under controlled conditions in nutrient-enriched laboratory mesocosm where a homogeneous mixed surface layer was emulated. The size spectrum of particles (from 12 μm to several mm) was recorded each hour during the 1 month span of the experiment by a non-intrusive image analysis system with two CCD cameras. Beam attenuation was continuously recorded as an additional estimator for particle abundance. The high time resolution and wide size range of the records obtained with this design were able to resolve the time scale for coagulation as well as to determine the lowest time resolution needed to sample any experiment aimed to study aggregation of diatoms. Our results narrow previous theoretical time scales to the order of hours to days for the process of mass transfer from small particles to marine snow. Also, daily analyses of a broad range of biological and chemical variables permitted to link phytoplankton succession to the aggregation process. Finally, the evaluated role of different copious exopolymers suggested a lower implication of Coomassie stained particles (CSP) than transparent exopolymeric particles (TEP) in the formation of marine aggregates.

The pKNOCK series of broad-host-range mobilizable suicide vectors for gene knockout and targeted DNA insertion into the chromosome of gram-negative bacteria.

The pKNOCK series of broad-host-range mobilizable suicide vectors for gene knockout and targeted DNA insertion into the chromosome of gram-negative bacteria.

Microbial growth in turbulent suspension and its relation to marine aggregate formation

Microbial growth and respiration responses to seston of differing organic composition revealed two different mechanisms of marine aggregate formation: an almost immediate physicochemical aggregation of fine (<8 μm) particles and a longer-term (48 hrs) production of large, robust aggregates, mediated by bacterial activity. Suspensions of fine Spartina alterniflora fragments and a glacial till from Nova Scotia were used to stimulate bacterial and protozoan growth while maintained in turbulent suspension. Under high (6 to 11 mg·dm −3) DOC concentrations bacterial responses were rapid and, after 48 hrs, resulted in large, robust flocculated aggregates. With only the till in suspension, DOC levels were low (<3 mg·dm −3) yet the bacterial reponse was still rapid. Initial flocculation due to physical factors was evident in the till-only experiment and was either masked or not important in the high DOC experiments. Bacterial respiration per cell was 2 to 3 times as high under low DOC conditions as under high DOC and initial bacterial attachment to particle surfaces was significantly higher. Significant aggregate formation occurred after 48 hrs and appeared to be due to relict exudates from attached bacteria, in spite of low numbers of attached bacteria at the time of flocculation.